Mitochondria as targeted delivery platform

Abstract

The present invention focuses on methods of delivering various payloads, including nucleic acid molecules (e.g., oligonucleotides), polypeptides (e.g., proteins), drugs, or combinations thereof. Thus, the present invention relates inter alia to a mitochondrial comprising one or more payloads attached to the outer membrane of the mitochondrial wherein the payload is electrostatically attached indirectly or directly to the outer membrane of the mitochondrial. The invention also relates to combining a mitochondrial comprising one or more payloads attached to the outer membrane of the mitochondrial with a protective layer that wraps / encapsulates and / or coats the mitochondrial and the payloads to provide a further delivery platform. The method is particularly effective for increasing the uptake and efficiency of one or more payloads for therapeutic purposes.

Description

[0001] The present invention focuses on methods for delivering various payloads, including nucleic acid molecules (e.g., oligonucleotides), polypeptides (e.g., proteins), drugs, or combinations thereof. Thus, the present invention relates particularly to a mitochondria comprising one or more payloads attached to the outer membrane of the mitochondria, wherein the payload is indirectly or directly electrostatically attached to the outer membrane of the mitochondria. The present invention also relates to combining mitochondria comprising one or more payloads attached to the outer membrane of the mitochondria with a protective layer that wraps / encapsulates and / or coats the mitochondria and the payload to provide a further delivery platform. This method is particularly effective for increasing the uptake and efficiency of one or more payloads for therapeutic purposes.

[0002] The delivery of nucleic acid molecules (e.g., DNA and RNA), polypeptides (e.g., proteins), drugs, or combinations thereof into cells and tissues remains a major challenge in biotechnology. Direct injection of naked DNA and RNA has been shown to have low transfection efficiency in vitro, ex vivo, and in vivo (NPL1). DNA and RNA molecules have large sizes and poor stability in biological media, making them susceptible to degradation by nucleases. Combinations of viral vectors with synthetic lipids or nanoparticles have been used as delivery platforms, but most of these combination products typically trigger undesirable immune responses, have low transfection efficiency, and may have long-term toxicity (NPL2, NPL3). In addition, when interacting with blood, the formation of a protein corona can lead to abnormal biodistribution, mistargeting, unexpected toxicity, and low therapeutic efficacy (NPL4).

[0003] Isolated mitochondria have been found to be biocompatible and non-toxic materials that can be efficiently taken up by cells through endocytosis, as reported in a study by Pacak et al. (NPL 5). These organelles also have a specific distribution, targeting specific organs such as, but not limited to, the heart, lungs, or kidneys (NPL 6). Mitochondria are also immunologically silent (NPL 7) and therefore may be an attractive delivery platform. However, mitochondria have not yet been successfully used as a carrier for the delivery of various payloads.

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

[0005] This technical problem is solved by the embodiments provided herein and set forth in the claims.

[0006] Therefore, the present invention relates particularly to the following items. 1. A mitochondria comprising one or more payloads attached to the outer membrane of the mitochondria, wherein the payloads are indirectly or directly electrostatically attached to the outer membrane of the mitochondria. 2. The mitochondria of item 1, wherein the payload is one or more of: i) nucleic acid molecules; ii) polypeptide; iii) medicines; or iv) A combination of one or more of (i) to (iii). 3. The mitochondria of item 1 or 2, wherein the payload is charged. 4. The mitochondria of any one of items 1 to 3, wherein the payload has a net charge that is the same as the net charge of the mitochondria. 5. The mitochondria of claim 4, wherein both the payload and the mitochondria have a net negative charge, and wherein the payload is attached to the mitochondria via a positively charged species. 6. The mitochondria according to item 5, wherein the positively charged substance is a polycationic substance. 7. The mitochondria according to item 6, wherein the polycationic substance is a linear or branched polycationic polymer. 8. The mitochondria of item 7, 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 dendrimers, 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. 9. The mitochondria according to item 5, wherein the positively charged substance is a positively charged nanoparticle. 10. The mitochondria according to item 5, wherein the positively charged substance is a positively charged particle. 11. The mitochondria according to item 9, wherein the one or more nucleic acid molecules are attached to the surface of the positively charged nanoparticles or are encapsulated in the positively charged nanoparticles. 12. The mitochondria according to item 10, wherein the one or more nucleic acid molecules are attached to the surface of the positively charged particles or are encapsulated in the positively charged particles. 13. The mitochondria according to any one of items 9 to 12, wherein the positively charged nanoparticles and / or particles are lipid nanoparticles / particles, dendrimer nanoparticles / particles, micellar nanoparticles / particles, protein nanoparticles / particles, liposomes, non-porous silica nanoparticles / particles, mesoporous silica nanoparticles / particles, silicon nanoparticles / particles, gold nanoparticles / particles, gold nanowires, silver nanoparticles / particles, platinum nanoparticles / particles, palladium nanoparticles / particles, titanium dioxide nanoparticles / particles, carbon nanotubes, carbon dot nanoparticles / particles, polymeric nanoparticles / particles, zeolite nanoparticles / particles, aluminum oxide nanoparticles / particles, hydroxyapatite nanoparticles / particles, quantum dot nanoparticles / particles, zinc oxide nanoparticles / particles, zirconium oxide nanoparticles / particles, graphene or graphene oxide nanoparticles / particles. 14. The mitochondria of any one of items 1 to 3, wherein the payload has a net charge that is different from the net charge of the mitochondria. 15. The mitochondria of item 14, wherein the payload and the mitochondria are attached via a zwitterionic substance. 16. The mitochondria of item 14, wherein the payload is uncharged, and wherein the payload is attached to a positively charged substance. 17. The mitochondria according to item 16, wherein the positively charged substance is as defined in any one of items 6 to 13. 18. The mitochondria according to item 2, wherein the one or more nucleic acid molecules are electrostatically linked to an antibody, optionally wherein the antibody is a modified antibody, optionally wherein the modified antibody has one or more positive charges. 19. The mitochondria of item 2, wherein the one or more nucleic acid molecules are encapsulated in nanoparticles, wherein the nanoparticles are electrostatically linked to antibodies, optionally wherein the antibodies are modified antibodies, optionally wherein the modified antibodies have one or more positive charges. 20. The mitochondria according to item 18 or 19, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria, wherein the antigen is OPA1, TOM70, TOMM20, Mitofusin 1, Mitofusin 2 or VDAC1. 21. Mitochondria according to any one of items 1 to 20, wherein the mitochondria are connected to and / or encased in a protective layer. 22. The mitochondria of item 21, wherein the protective layer is a protective polymer. 23. The mitochondria of item 22, wherein the protective polymer is a linear or branched cationic polymer, optionally wherein the linear or branched cationic polymer is electrostatically linked to the one or more payloads. 24. The mitochondria of item 22, wherein the protective polymer is a linear or branched cationic block copolymer, optionally wherein the linear or branched cationic block copolymer is electrostatically linked to the one or more payloads. 25. The mitochondria of item 22, wherein the protective polymer is a cationic graft (g) copolymer, optionally wherein the cationic graft (g) copolymer is electrostatically linked to the one or more payloads. 26. The mitochondria of claim 22, wherein the protective polymer is a linear or branched PEGylated (PEG) cationic polymer, optionally wherein the linear or branched PEGylated (PEG) cationic polymer is electrostatically linked to the one or more payloads. 27. The mitochondria of item 21, wherein the protective layer is a lipid formulation, optionally wherein the lipid formulation is a cationic lipid formulation, further optionally wherein the cationic lipid formulation is electrostatically linked to the one or more payloads. 28. The mitochondria of any one of items 21 to 27, wherein the protective layer is linked to a targeting moiety. 29. The mitochondria of any one of items 21 to 28, wherein the protective layer is attached to an antibody, optionally wherein the protective layer attached to the antibody is electrostatically attached to the one or more payloads, or wherein the protective layer attached to the antibody is covalently attached to the one or more payloads 30. The mitochondria of any one of items 21 to 28, wherein the protective layer is attached to a carbohydrate, optionally wherein the protective layer attached to the carbohydrate is electrostatically attached to the one or more payloads, or wherein the protective layer attached to the carbohydrate is covalently attached to the one or more payloads 31. The mitochondria of 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. 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-poly )-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. 33. The mitochondria according to item 25, wherein the cationic grafted (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-polyarginine, alkyl)-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. 34. The mitochondria according to item 26, wherein the PEGylated 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, amine, 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. 35. The mitochondria according to item 27, wherein the lipid preparation comprises DC-cholesterol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), DLinDMA (1,2-dilinoleoyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleoylmethyl-4-dimethylaminobutyrate), DODMA (1,2-dioleoyloxy-3-dimethylaminopropane), DOGS (dioctadecyl) amide The present invention can be used to prepare 2,3-dioleoyl-N-[2-sperminecarboxamido]ethyl]-N,N-dimethyl-1-propylamine, 2,3-dioleoyl-3-trimethylammonium-propane chloride ... 36. Mitochondria according to item 35, wherein the lipid preparation further comprises another lipid, preferably wherein the lipid is cholesterol, substituted or unsubstituted cholesterol, a cholesterol derivative such as a hydroxylated cholesterol derivative (e.g., hydroxycholesterol), PEG-lipid, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DODAP (1,2-dioleoyl-3-dimethylammoniumpropane), DDA (dimethyldioctadecyl ammonium), 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, bis(monooleoylglycerol)phosphate, or a combination thereof. 37. The mitochondria of item 22, wherein the mitochondria are attached to and / or encapsulated in a zwitterionic protective polymer, optionally wherein the zwitterionic protective polymer is electrostatically attached to the one or more payloads. 38. The mitochondria of item 37, wherein the zwitterionic protective polymer is selected from the group consisting of: poly(2-methacryloyloxyethylphosphorylcholine) (PMPC), polyethyleneimine-g-poly(2-methacryloyloxyethylphosphorylcholine) (PEI-g-PMPC), co-assembly of cationic (carboxyl functionalized) and anionic (amino functionalized) copolyesters based on poly(ε-caprolactone)-block-poly(butylene fumarate)-block-poly(ε-caprolactone) (PCL-b-PBF-b-PCL), poly(lactic-co-glycolic acid) (PLGA)-PCB block copolymer (PLGA-b-PCB). 39. A composition comprising a plurality of mitochondria according to any one of items 1 to 38. 40. A pharmaceutical composition comprising a plurality of mitochondria according to any one of items 1 to 38 and a pharmaceutically acceptable carrier. 41. The pharmaceutical composition according to item 40, wherein the pharmaceutical composition is formulated as a solution. 42. The pharmaceutical composition according to item 40, wherein the pharmaceutical composition is formulated as an aerosol. 43. 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. 44. 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. 45. 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 the treatment of cardiovascular diseases, in particular for the treatment of ischemic heart disease, ischemia-reperfusion injury or atherosclerosis. 46. ​​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 treating age-related diseases, in particular for treating sarcopenia, Parkinson's disease or Hutchinson-Gilford progeria syndrome. 47. 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 the treatment of a kidney disease, in particular for the treatment of autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis or Fabry disease. 48. 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 treating cancer. 49. 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 in vitro, ex vivo or in vivo genome editing. 50. 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 43 for use in radiotherapy. 51. A method for delivering a payload to a target organ, the method 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. 52. A method for delivering a payload to the lung, the method comprising the step of administering the pharmaceutical composition according to item 42 to a subject in need thereof, wherein the pharmaceutical composition is administered by inhalation. 53. A method for attaching a payload to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload in the presence of a positively charged species; and c) attaching the at least one payload to the mitochondria via the positively charged species. 54. The method according to item 53, wherein a) contacting the at least one payload simultaneously with the positively charged species and the mitochondria; b) contacting the at least one payload with the positively charged species to form a positively charged complex, and then contacting the positively charged complex with the mitochondria; or c) contacting said mitochondria with said positively charged species and subsequently with said at least one payload. 55. The method of item 52 or 54, wherein the mitochondria are contacted with the at least one payload and the positively charged substance in a suitable buffer. 56. The method according to item 55, wherein the buffer comprises or consists of HEPES, EGTA, trehalose, CHES and disodium hydrogen phosphate dihydrate, preferably, wherein the buffer comprises a mixture of solution X and solution Y, solution X comprises or consists of HEPES, EGTA and trehalose, and solution Y comprises or consists of CHES and disodium hydrogen phosphate dihydrate, more preferably, wherein the buffer comprises a 4:1 mixture of solution X and solution Y, solution X comprises or consists of 20 mM HEPES, 1 mM EGTA and 300 mM trehalose (pH 7.2), and solution Y comprises or consists of 0.1 M CHES (pH 10) and 0.2 M disodium hydrogen phosphate dihydrate. 57. A method according to any one of items 53 to 56, wherein the mitochondria are contacted with the at least one payload and the positively charged substance for at least 5 minutes, such as at least 10 minutes, 20, 30, 40, 50, 60 or 120 minutes at room temperature. 58. The method of any one of items 53 to 57, wherein the mitochondria are contacted with the at least one payload and the positively charged substance in the dark. 59. The method of any one of items 53 to 58, wherein the payload is a nucleic acid molecule, which is DNA or RNA. 60. The method of any one of items 53 to 59, wherein the positively charged species is a polycationic species, wherein the polycationic species is a linear or branched polycationic polymer, optionally wherein the linear or branched polycationic polymer is electrostatically attached to the at least one payload. 61. The method of claim 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 dendrimers, 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. 62. The method according to any one of items 53 to 59, wherein the positively charged species is a positively charged nanoparticle. 63. The method according to item 62, wherein the method comprises the further steps of: a) attaching the at least one payload to the surface of the positively charged nanoparticles; or b) encapsulating the at least one payload within the positively charged nanoparticles. 64. The method according to item 62 or 63, wherein the positively charged nanoparticles are lipid nanoparticles, dendrimer nanoparticles, micellar 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. 65. A method for preparing mitochondria containing a payload, wherein the method comprises the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria with: i) contacting with a positively charged species if both the payload and the mitochondria have a net negative charge; ii) if the payload has a net charge different from the net charge of the mitochondria, contacting the payload, optionally further contacting with a zwitterionic substance; or iii) if the payload is uncharged, contacting with a payload attached to a positively charged species; c) Obtaining the mitochondria according to any one of items 1 to 20. 66. The method according to item 65, further comprising, after step c), a step of contacting the mitochondria with a component forming a protective layer, and a step of obtaining the mitochondria according to any one of items 22 to 38. 67. The method according to any one of items 53 to 66, wherein an amount of 50 μg to 200 μg of the mitochondria is contacted with 0.1 to 50 pmol of the payload and 0.02 to 10 μg, preferably 0.02 to 5 μg, of the positively charged substance. 68. The method of any one of items 53 to 66, wherein the mitochondria comprise a positively charged substance, wherein the positively charged substance is a polycationic polymer according to any one of the preceding items, and wherein the ratio of the polycationic polymer to the protective layer is about 1:2. 69. The method according to any of the preceding method items, wherein 50 μg to 200 μg of mitochondria are contacted with 0.1 to 50 pmol of payload and 0.2 to 10 μg of the protective layer. 70. A method for delivering a payload to the kidney, the method comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the renal artery of a subject in need thereof. 71. A method for delivering a payload to the heart, the method comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the coronary artery of a subject in need thereof. 72. A method for delivering a payload to the liver, the method 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. 73. A method for delivering a payload to the pancreas, the method comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the hepatic artery of a subject in need thereof. 74. A method for delivering a payload to the duodenum, the method comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the hepatic artery of a subject in need thereof. 75. A method for delivering a payload to the spleen, the method comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the splenic artery of a subject in need thereof. 76. A method for delivering a payload to the lung, the method comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the pulmonary artery of a subject in need thereof. 77. A method for delivering a payload to the intestine, the method 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. 78. A method for delivering a payload to the bladder, the method 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. 79. A method for delivering a payload to a target organ, the method comprising the step of administering the pharmaceutical composition according to items 40 to 50 into the body of a subject in need thereof, wherein the pharmaceutical composition is administered by direct injection into the kidney or bladder or intestine or pancreas or duodenum or liver or lung or spleen.

[0007] Thus, in its broadest aspect, the present invention relates to a mitochondria to which one or more payloads (eg one or more nucleic acid molecules, one or more polypeptides and / or one or more drugs) are attached by an innovative method.

[0008] More specifically, the present invention relates to and / or utilizes mitochondria complexed with 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 ( Figure 1-2 ). The process for producing such a mitochondrial complex includes, for example, functionalizing the mitochondria with a cationic substance (such as a cationic polymer) and then with oligonucleotides, nucleic acid molecules, polypeptides, proteins and / or drugs. The mitochondrial complex of the present invention may comprise one or more additional protective layers, such as a protective polymer layer consisting of a cationic copolymer, which is attached to or encapsulates the mitochondria to protect the attached payload (e.g., nucleic acid molecules, such as oligonucleotides, polypeptides, such as proteins and / or drugs) from degradation and to enable, for example, mitochondria-oligonucleotide complexes or mitochondria-protein complexes to be effectively internalized. The mitochondria-payload complex (e.g., mitochondria-oligonucleotide complex) of the present invention can escape digestive organelles (i.e., lysosomes) upon internalization. The isolated mitochondria of the mitochondrial-based system of the present invention are very suitable for transporting different nucleic acids (e.g., DNA / RNA molecules) or proteins and allow DNA / RNA to have high biological activity (e.g., translation, transcription, protein expression, knockdown) when released within the cell while maintaining low cytotoxicity. It was demonstrated that mRNA translation efficiency exceeded 70% in various cell types, including human epithelial lung cells (A549; 79%) and human cardiac fibroblasts (HCF, 70%), compared to commonly used liposomes (100%) as a control. In addition, it was shown that mitochondrial delivery of siRNA containing a protective layer resulted in greater protein knockdown compared to liposome-siRNA and the previous generation products described in European Patent Application Nos. 22178524.9 and 22211826.7. In addition, there is provided the use of mitochondria to simultaneously deliver one or more (e.g., two or more) different oligonucleotides (e.g., mRNA and siRNA) or oligonucleotides and drugs (e.g., anionic drugs) or one or more (e.g., two or more) oligonucleotides and one or more drugs (e.g., two or more anionic drugs (e.g., siRNA and PX-12) for oncology applications.

[0009] Therefore, the mitochondrial delivery platform of the present invention has several advantages: 1. It is a natural and safe method for delivering payloads such as nucleic acid molecules (eg DNA, RNA), polypeptides or drugs. 2. It has been shown to be successful for in vivo, ex vivo or in vitro delivery as demonstrated by high levels of transcription, translation and protein expression / knockdown. 3. The payload-mitochondria complex has a stabilizing effect on the payload (particularly DNA or RNA) compared to naked nucleic acids, and this is further improved by adding a protective layer to the mitochondria-oligonucleotide complex. 4. Compared with commonly used delivery systems such as viral vectors, this platform does not induce immune responses or cytotoxicity when internalized into cells. 5. It can be administered to cells, tissues or the whole body through different routes (e.g. injection or aerosol). In addition, the mitochondria can be administered as a single dose or at least 2 or more doses. 6. It delivers payloads with high colloidal stability, such as nucleic acid molecules, peptides, or drugs, through mitochondria. 7. A new generation of mitochondrial delivery platforms including a protective layer effectively delivers payloads, such as nucleic acid molecules, peptides, and drugs, achieving higher mRNA transcription and / or higher siRNA protein knockdown compared to previously available methods. 8. It allows combination therapy, where various payloads, such as at least two different nucleic acid molecules, peptides, drugs, oligonucleotides can be delivered simultaneously by a single mitochondria. 9. It allows combination therapy, where at least two or more different payloads, such as nucleic acid molecules, peptides, drugs can be delivered simultaneously by a single mitochondria. 10. It allows combination therapy, where at least one or more different payloads, such as nucleic acid molecules, peptides, drugs, and in particular combinations thereof, can be delivered simultaneously from a single mitochondria. 11. It allows for combination therapy, wherein at least one or more different nucleic acid molecules and one or more drugs can be delivered simultaneously by a single mitochondria. 12. It allows for combination therapy, where at least two or more different polypeptides can be delivered simultaneously from a single mitochondria. 13. It allows for combination therapy where at least two or more different proteins can be delivered simultaneously by a single mitochondria. 14. It allows for combination therapy, wherein at least one or more different polypeptides and one or more drugs can be delivered simultaneously from a single mitochondria. 15. It allows for combination therapy, where at least one or more different proteins and one or more drugs can be delivered simultaneously by a single mitochondria. 16. It allows for combination therapy, wherein at least one or more different nucleic acid molecules (eg oligonucleotides) and at least one or more polypeptides (eg proteins) can be delivered simultaneously from a single mitochondria. 17. It allows combination therapy, wherein at least one or more different nucleic acid molecules (eg oligonucleotides) together with at least one or more polypeptides (eg proteins) and at least one or more drugs can be delivered simultaneously from a single mitochondria. 18. It allows for combination therapy, wherein at least one or more siRNAs and at least one or more anionic drugs can be delivered simultaneously from a single mitochondria. 19. It allows combination therapy, where at least two or more drugs (e.g. anionic drugs) can be delivered simultaneously by a single mitochondria.

[0010] Disclosures in the context of the invention described herein apply to the corresponding uses and vice versa.

[0011] In one aspect, the present invention provides a mitochondria comprising one or more nucleic acid molecules attached to the outer membrane of the mitochondria, wherein the one or more nucleic acid molecules are a) electrostatically attached to the outer membrane of the mitochondria through positively charged species; or b) covalently linked 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 small molecule that targets the mitochondria.

[0012] Mitochondria have a negatively charged surface. According to one aspect of the present invention, the surface can be functionalized with cationic molecules to convert the surface charge of the mitochondrial outer membrane into a completely positive value (i.e., a net positive value) or a partially positive value. In other words, although mitochondria generally have a negative surface charge, part of the surface or the entire surface can be covered / attached by the positively charged molecules provided herein. Therefore, the surface charge of the mitochondria identified by another molecule may be positive. Positively charged mitochondria (i.e., mitochondria having a positive net surface charge or mitochondria having a positively charged surface area) can be combined with negatively charged payload molecules (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0013] Mitochondria are double-membrane-bound organelles found in most eukaryotic organisms. Therefore, the mitochondria of the present invention can be mitochondria from any eukaryotic organism. The mitochondria can be mitochondria from animals, plants, yeast, or fungi. The mitochondria can be human mitochondria. The mitochondria of the present invention can be obtained by any means, such as cell culture. Therefore, 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 tissue. Mitochondria obtained from tissue can be obtained from any tissue of a eukaryotic organism. Therefore, the mitochondria can be obtained from cells or tissues of eukaryotic organisms 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 tissue or cell culture. More preferably, the mitochondria are obtained from human in vitro cell culture. In a preferred embodiment, the mitochondria are obtained from animal tissue or cell culture, in particular from mouse tissue or cell culture. Preferably, the mitochondria are obtained from mouse in vitro cell culture. The mitochondria can be obtained from mouse embryonic fibroblasts (MEFs). The mitochondria can be obtained from MEFs maintained in in vitro cell culture. Mitochondria can be obtained from MEFs maintained in an in vitro cell culture comprising Dulbecco's Modified Eagle's Medium (DMEM) culture medium. In some embodiments, mitochondria are obtained from, for example, human cardiac fibroblasts (HCF). Mitochondria can be obtained from HCFs maintained in an in vitro cell culture. Mitochondria can be obtained from HCFs maintained in an in vitro cell culture comprising fibroblast culture medium-2. In a further embodiment, mitochondria can be obtained from HepG2 cells. Mitochondria can be obtained from HepG2 maintained in an in vitro cell culture. Mitochondria can be obtained from HepG2 maintained in an in vitro cell culture comprising Roswell Park Memorial Institute (RPMI) culture medium.

[0014] The mitochondria of the present invention can also be freshly obtained by isolating mitochondria from cell culture or tissue (e.g., eukaryotic cell culture or tissue). Mitochondria obtained from tissue can be from placenta, liver, muscle, or porcine tissue. Thus, mitochondria can be freshly isolated from animal, plant, yeast, or fungal cell culture or tissue. Preferably, mitochondria can be freshly isolated from human cell culture or tissue. Mitochondria can be freshly isolated from HCF or HepG2. Mitochondria can be freshly isolated from HCF or HepG2 maintained in in vitro cell culture. Mitochondria can be freshly isolated from HCF maintained in in vitro cell culture containing Fibroblast Medium-2. Mitochondria can be freshly isolated from HepG2 maintained in in vitro cell culture containing RPMI medium. In addition, mitochondria can be freshly isolated from mouse cell culture or tissue. Mitochondria can be freshly isolated from mouse in vitro cell culture. Mitochondria can be freshly isolated from MEFs. Mitochondria can be freshly isolated from MEFs maintained in in vitro cell culture. Mitochondria can be obtained by fresh isolation from MEFs maintained in in vitro cell culture containing DMEM medium.

[0015] Mitochondria can be autologous (i.e., self- or autogenous). In some embodiments, mitochondria are autogenous or autologous mitochondria with genetic modification. In some other embodiments, mitochondria are autologous and are connected to imaging agents, diagnostic agents or pharmaceutical agents (e.g., nucleic acid molecules, polypeptides and / or drugs). In some other embodiments, the reagent is embedded in or incorporated into autologous mitochondria. In some other embodiments, mitochondria are allogenic. In some embodiments, mitochondria are allogenic mitochondria with genetic modification. In some other embodiments, mitochondria are allogenic mitochondria that are connected to imaging agents, diagnostic agents or pharmaceutical agents. In some other embodiments, the reagent is embedded in or incorporated into allogenic mitochondria. In some other embodiments, mitochondria are xenogeneic mitochondria. In some embodiments, mitochondria are xenogeneic mitochondria with genetic modification. In some other embodiments, mitochondria are xenogeneic mitochondria that are connected to imaging agents, diagnostic agents or pharmaceutical agents. In some other embodiments, the reagent is embedded in or incorporated into xenogeneic mitochondria. In certain aspects, contemplated herein are methods of obtaining / isolating mitochondria from a subject (patient), particularly in a therapeutic context, e.g., as an ex vivo method, modifying the mitochondria by attaching one or more payloads (e.g., 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 in the methods provided herein), and subsequently administering to the same subject (patient).

[0016] The mitochondria of the present invention can also be obtained from a frozen stock of mitochondria. Therefore, mitochondria obtained from a frozen stock of mitochondria must be thawed before being used in the means and methods of the present invention. Mitochondria can be obtained from a frozen stock containing mitochondria from any eukaryotic organism (e.g., animal, plant, yeast, or fungus). Mitochondria can be obtained from a frozen stock containing human mitochondria. Mitochondria can be obtained from a frozen stock containing mitochondria obtained by fresh isolation or cell culture or tissue culture. Mitochondria can be obtained from a frozen stock containing human mitochondria obtained by fresh isolation or cell culture or tissue culture. Mitochondria can be obtained from a frozen stock containing human mitochondria obtained from HCF by fresh isolation or cell culture or tissue culture. Preferably, mitochondria can be obtained from a frozen stock containing human mitochondria obtained from HCF in vitro cell culture. More preferably, mitochondria can be obtained from a frozen stock containing human mitochondria obtained from HCF in vitro cell culture including Fibroblast Medium-2.

[0017] The mitochondria of the present invention can be labeled or unlabeled. Labeled mitochondria allow for later detection, while unlabeled mitochondria reflect their natural properties. Mitochondria can be labeled by any method known to those skilled in the art. Thus, mitochondria can be labeled with dyes. Mitochondria can be labeled with dyes including rosamine, tetramethylrosamine, X-rosamine, dihydrotetramethylrosamine, dihydro-X-rosamine, carbocyanine, or derivatives thereof. Mitochondria can also be labeled with small molecules or small particles. Thus, the mitochondria of the present invention can be labeled with dyes including rosamine, tetramethylrosamine, X-rosamine, dihydrotetramethylrosamine, dihydro-X-rosamine, carbocyanine, or derivatives thereof. 18 F-rhodamine 6G or iron oxide nanoparticles or gold nanoparticles or gold nanostars or silver nanoparticles labeling.

[0018] " Mitochondria " used herein refers to living mitochondria that are (substantially) free of eukaryotic cell material (e.g., foreign eukaryotic cell material) separated / purified from cells or cell cultures. Therefore, there are only a very small amount of cellular components (other than mitochondria) in the mitochondria (composition) used herein. Preferably, there are no other cellular components other than mitochondria in the mitochondria (composition) used herein. In this sense, "mitochondria" used herein are "isolated mitochondria", and the terms "mitochondria" and "isolated mitochondria" can be used interchangeably. Any current technology known in the art can be used to isolate mitochondria, for example, by repeated differential centrifugation (DC) or density gradient centrifugation (DGC) or differential filtration (McCully, WO2015192020A1) for subcellular fractionation. The mitochondria of the present invention can be used for nucleic acid (e.g., oligonucleotides), polypeptide (e.g., protein) and / or drug delivery to cells. Therefore, the mitochondria of the present invention are preferably alive or viable and have a negative membrane potential. In the present invention, "alive" refers to having or maintaining metabolism or other biological functions or structures.

[0019] As used herein, the term "viable mitochondria" is used to describe living mitochondria, i.e., mitochondria that are complete, active, functioning normally and capable of respiration. According to some embodiments, "viable mitochondria" refers to mitochondria that exhibit biological functions (e.g., respiration and ATP and / or protein synthesis).

[0020] As used herein, the term "intact mitochondria" is used throughout the specification to describe mitochondria that include intact outer and inner membranes, intact intermembrane spaces, intact cristae (formed by the inner membrane), and intact matrix. Alternatively, intact mitochondria are mitochondria that retain their structure and ultrastructure. In another aspect, intact mitochondria contain active respiratory chain complex IV embedded in the inner membrane, maintaining membrane potential and the ability to synthesize ATP.

[0021] The mitochondria of the present invention can be functionalized with targeting molecules (e.g., small targeting molecules, targeting aptamers, targeting peptides, carbohydrates, sugars, and targeting antibodies), drugs, reporter molecules / nanoparticles (e.g., fluorescent molecules, metal nanoparticles, magnetic nanoparticles, etc.) or contact agents, imaging agents, diagnostic agents, or pharmaceutical agents.

[0022] In the sense of the present invention, the terms "nanoparticle", "nanoformulation" and "nanobody" can be used interchangeably. In some embodiments, the nanoparticle is a lipid nanoparticle. Exemplary nanoparticles of the present invention are lipid nanoparticles, dendrimer nanoparticles, micellar 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. It is known to those skilled in the art that nanoparticles can contain different charges or can be functionalized to have a certain charge. In the sense of the present invention, nanoparticles can be functionalized with positively charged substances (e.g., positively charged functional groups (e.g., quaternary ammonium groups) or polycationic substances) to produce positively charged nanoparticles. Furthermore, in some embodiments, the nanoparticles can be chemically modified to be positively charged, such as by protonation of chemical groups contained in the nanoparticles. In the sense of the present invention, "functionalization" can mean "attachment to certain moieties or compounds that have a function (e.g., a biological function, such as a targeting function, a protective function, or a regulatory function). Thus, mitochondria can be functionalized by attaching different agents that impart a desired function (e.g., changing the charge of the nanoparticle).

[0023] In the context of the present invention, the term "particle" or "positively charged particle" preferably refers to lipid particles, dendrimer particles, micellar 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 oxide particles, graphene or graphene oxide particles.

[0024] The mitochondria of the present invention are particularly useful because they can be stored for extended periods without degradation and / or disintegration, i.e., remain stable. Accordingly, the present invention provides a mitochondria comprising one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, attached to the outer membrane of the mitochondria, wherein the one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof: a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) linked to small molecules targeting mitochondria, Preferably, wherein the mitochondria are stored in binding buffer at low temperature (e.g., -80°C or -20°C). The mitochondria of the present invention can be stored in binding buffer at low temperature (e.g., -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 longer, without disintegration or decomposition. Mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane can be stored in binding buffer to maintain high colloidal stability (e.g., no agglomeration / aggregation or disintegration). Mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane are preferably stored in binding buffer at low temperature (e.g., -20°C, preferably -80°C) in the dark to preserve, for example, for at least two months after complex formation.

[0025] The mitochondria of the present invention can be encapsulated in alginate / hydrogel capsules. Encapsulation in alginate / hydrogel capsules can increase the storage time of the mitochondria of the present invention, that is, avoid disintegration and increase stability.

[0026] The mitochondria of the present invention can be contacted with a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a solution (e.g., a buffer). The buffer of the present invention is preferably an aqueous solution of any compound that can be used to bind a payload to the mitochondria. The buffer used in the contacting step is preferably a binding buffer. The binding 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 a buffer, including water, deionized water, double-distilled water, DNAse- and RNAse-free water, DNAse- and RNAse-free deionized water, or DNAse- and RNAse-free double-distilled water. The binding buffer of the present invention can comprise a mixture of solution X and solution Y. Solution X can comprise 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 comprise or consist of N-cyclohexyl-2-aminoethanesulfonic acid (i.e., CHES) and disodium hydrogen phosphate dihydrate. Solution X and solution Y can be aqueous solutions.

[0027] In the sense of the present invention, the composition of solutions X and Y can be used in any amount and at any pH, as long as successful binding of mitochondria to their payload (e.g., nucleic acid or polypeptide) is achieved. 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 or consists of 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 HEPES, 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 EGTA, and 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 trehalose (pH 6, 6.5, 7, 7.5, 8, 8.5 or 9) and optionally an aqueous solvent.Preferably, solution X comprises or consists of 20 mM HEPES, 1 mM EGTA and 300 mM trehalose (pH 7.2) and optionally an aqueous solvent.

[0028] In some embodiments, solution Y comprises or consists of 0.01 to 0.2 M CHES (pH 8 to 12) and 0.02 to 0.6 M sodium 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. Solution Y preferably comprises or consists of 0.1 M CHES (pH 10) and 0.2 M disodium hydrogen phosphate dihydrate and optionally an aqueous solvent.

[0029] In the sense of the present invention, solutions X and Y can be mixed in any feasible ratio, as long as successful binding of mitochondria to their payload (e.g., nucleic acid or polypeptide) is achieved. A mixture of solutions X and Y can produce a binding buffer according to the present invention. Thus, in some embodiments, the binding buffer comprises a 2:1 to 10:1 mixture of solution X and solution Y. In some embodiments, the binding 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 binding buffer comprises a 4:1 mixture of solution X and solution Y.

[0030] In some embodiments, the pH of the binding buffer of the invention is between 7.5 and 11. In some embodiments, the pH of the binding buffer of the invention is 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11.

[0031] In a preferred embodiment, the binding buffer of the present invention comprises a mixture of solution X and solution Y, wherein solution X comprises or consists of 20 mM HEPES + 1 mM EGTA + 300 mM trehalose (pH 7.2), and solution Y comprises or consists of 0.1 M CHES (pH 10) + 0.2 M sodium dihydrogen phosphate dihydrate and optionally an aqueous solvent. In a further preferred embodiment, the binding buffer of the present invention comprises a 4:1 mixture of solution X and solution Y, wherein solution X comprises or consists of 20 mM HEPES, 1 mM EGTA and 300 mM trehalose (pH 7.2), and solution Y comprises or consists of 0.1 M CHES (pH 10) and 0.2 M sodium dihydrogen phosphate dihydrate and optionally an aqueous solvent.

[0032] The binding buffer of the present invention can be used to store the mitochondria, compositions thereof, and pharmaceutical compositions of the present invention. The binding buffer used for storage is referred to as a storage buffer and comprises the components as defined above. Thus, in a preferred embodiment, the storage buffer of the present invention comprises a 4:1 mixture of solution X and solution Y, wherein solution X comprises or consists of 20 mM HEPES, 1 mM EGTA, and 300 mM trehalose (pH 7.2), and solution Y comprises or consists of 0.1 M CHES (pH 10) and 0.2 M sodium dihydrogen phosphate dihydrate.

[0033] The nucleic acid molecules of the present invention can be stored in a buffer comprising or consisting of an aqueous solvent and solution X. The nucleic acid molecules of the present invention can be stored in a DNA / RNA buffer comprising DNase / RNase-free water, PBS, and solution X comprising or consisting of 20 mM HEPES + 1 mM EGTA + 300 mM trehalose (pH 7.2).

[0034] The mitochondria of the present invention can be complexed with one or more payloads (eg, nucleic acid molecules, polypeptides, drugs, or combinations thereof) attached to the outer membrane of the mitochondria.

[0035] In some embodiments, the present invention provides mitochondria comprising one or more nucleic acid molecules, wherein the nucleic acid molecule is DNA or RNA.

[0036] In general, the nucleic acid of the present invention can be any nucleic acid, such as a naturally occurring nucleic acid or a synthetic nucleic acid. The nucleic acid can be endogenous or exogenous nucleic acid. Nucleic acid is a polymer composed of nucleotides, which are monomers comprising a 5-carbon sugar, a phosphate group, and a nitrogenous base (e.g., adenine, cytosine, guanine, thymine, and uracil). It is also contemplated herein that the nucleic acid can be a modified nucleic acid. The nucleic acid of the present invention can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Therefore, the nucleic acid of the present invention can be an oligonucleotide comprising DNA or RNA of any length. Therefore, the term "nucleic acid molecule" or any grammatical variant thereof used herein can be used interchangeably with the term "oligonucleotide". In some embodiments, the oligonucleotide of the present invention can comprise 10 to 15,000 base pairs. The nucleic acid can be single-stranded DNA (ssDNA). In some embodiments, the single-stranded DNA can comprise 10 to 15,000 nucleotides. The nucleic acid can be double-stranded (dsDNA). The nucleic acid of the present invention can be linear. The nucleic acid can be circular. Therefore, 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 acids 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 comprise different fragments. Thus, the nucleic acids can be DNA comprising a sense fragment carrying a translatable sequence. The nucleic acids can be DNA comprising an antisense fragment complementary to the sense fragment. The nucleic acids of the present invention can be of natural origin, or can be synthetic. Thus, the DNA can be derived from any natural origin, such as an organism, such as a eukaryote. The DNA can be derived from animals, plants, bacteria or yeast. Preferably, the DNA is human or substantially similar to human DNA.

[0037] The nucleic acid of the present invention may also be RNA. Therefore, the nucleic acid of the present invention may be an oligonucleotide comprising RNA of any length. In some embodiments, the RNA of the present invention may comprise 10 to 10,000 nucleotides. The nucleic acid may be single-stranded RNA (ssRNA). The nucleic acid may be double-stranded (dsRNA). The RNA of the present invention may be linear. The RNA may be circular. The RNA molecule may include protein-coding RNA (e.g., mRNA) or non-coding RNA (e.g., siRNA). Therefore, the RNA of the present invention may be messenger RNA (mRNA). The RNA of the present invention may be non-coding RNA involved in RNA interference (RNAi), such as small interfering RNA (siRNA) and microRNA (miRNA). The RNA may also be other small RNAs selected from the following groups: small nucleolar RNA (snoRNA), small nuclear RNA (snRNA) (including U1 spliceosomal RNA, U2 spliceosomal RNA, U4 spliceosomal RNA, U5 spliceosomal RNA, and U6 spliceosomal RNA), exRNA, scaRNA, and long ncRNA (e.g., Xist and HOTAIR). The RNA may also be non-coding RNA (ncRNA), such as transfer RNA (tRNA) or ribosomal RNA. The RNA of the present invention may be complementary to a DNA sequence in an animal, plant, bacteria, or yeast. Preferably, the RNA may be complementary to a DNA sequence in a human. Preferably, the RNA may be complementary to a DNA sequence in a human gene. The RNA of the present invention may also be complementary to an RNA sequence in an animal, plant, bacteria, or yeast. Preferably, the RNA may be complementary to an RNA sequence in a human. Preferably, the RNA may be complementary to a human mRNA sequence.

[0038] The RNA of the present invention can be of natural origin, or can be synthetic. Therefore, RNA can be artificial, such as short hairpin RNA (shRNA). RNA can be derived from any natural source. RNA can be endogenous or exogenous. RNA can be derived from animals, plants, bacteria or yeast. RNA can be human RNA or substantially similar to human RNA. RNA can be human mRNA. RNA can be siRNA complementary to human mRNA. Preferably, RNA is siRNA complementary to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (optionally human GAPDH mRNA). Preferably, RNA is siRNA complementary to MDM2 mRNA (optionally human MDM2 proto-oncogene (MDM2) mRNA). In one embodiment, RNA can be siRNA complementary to mRNA of hexokinase 1 mRNA (optionally human hexokinase 1 mRNA). Preferably, RNA of the present invention is mRNA encoding human peptides (e.g., human polypeptides and / or proteins).

[0039] The nucleic acid molecules of the present invention can be functionalized with targeting molecules (e.g., small targeting molecules, targeting aptamers, targeting peptides, carbohydrates, sugars, and targeting antibodies), drugs, reporter molecules / nanoparticles (e.g., fluorescent molecules, metal nanoparticles, magnetic nanoparticles, etc.), or contrast agents.

[0040] In some embodiments, the payload of the present invention (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) is formulated into nanoparticles, particles, cationic lipid formulations (e.g., lipid nanoformulations), block copolymers, cationic lipids, or cationic polymers. The payload (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be attached to the surface of a nanoparticle or particle, or encapsulated in a nanoparticle or particle.

[0041] The present invention provides mitochondria-payload complexes that can be used for delivery into cells, particularly complexes of nucleic acid molecules, polypeptides, drugs, or combinations thereof. The present invention also provides methods for attaching payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) to mitochondria. One or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be electrostatically attached to the outer membrane of mitochondria, particularly when the payload (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) has an overall positive surface charge. When the payload (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) has an overall negative surface charge, electrostatic attachment can be promoted by positively charged substances. In one embodiment, the positively charged substance is a polycationic substance. In another embodiment, the positively charged substance is a positively charged nanoparticle or particle. Electrostatic interactions include attractive or repulsive interactions between charged molecules and / or, for example, the surfaces of subcellular organelles (e.g., the membrane surfaces of mitochondria). In the sense of the present invention, mitochondria can electrostatically interact with payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) to form a complex comprising mitochondria and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof). Therefore, electrostatic interactions can be used to attach positively charged entities to negatively charged entities. In this regard, it is known that isolated mitochondria have a negative net surface charge. In the sense of the present invention, mitochondria can be positively charged, negatively charged, or neutral, depending on the complex of the mitochondria with the various reagents provided herein. In the sense of the present invention, nucleic acids can be positively charged or negatively charged. As long as the mitochondria and the payload (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) have opposite charges or do not have the same charge, any of the above combinations can be successfully attached by electrostatic interactions. In this regard, it is understood that the charge may depend on pH. Technicians know how to handle pH-dependent charges. Typically, the mitochondrial surface has a negative surface charge distribution. Similarly, DNA and RNA are typically negatively charged molecules. According to the present invention, the mitochondrial surface can also be functionalized with positively charged substances to establish electrostatic attachment of payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof). Therefore, one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) can be electrostatically attached to the outer membrane of the mitochondria by positively charged substances. One or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be electrostatically attached to the outer membrane of mitochondria via polycationic substances, wherein the polycationic substances are linear or branched polycationic polymers. As used herein, the term "polycation" refers to a moiety that is positively charged at multiple sites and has an overall positive charge.Payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be electrostatically attached to the outer membrane of mitochondria via linear or branched polycationic polymers, wherein the linear or branched cationic polymers are polylysine, histidylated polylysine, polyornithine, polyarginine, high mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimers, 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.

[0042] In the context of the present invention, the negative surface charge distribution of mitochondria can also be used to electrostatically attach one or more payloads (e.g., 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 be electrostatically attached 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 known to those skilled in the art, the distinction between nanoparticles and particles is generally related to size differences, with nanoparticles typically ranging in size from 1 to 100 nm, while particles typically range in size from 100 nm to 2.5 μm. However, as known to those skilled in the art, the distinction between nanoparticles and particles based on their size is not consistent in the art, and different size distinctions may be made depending on the type of particle. In some embodiments, the particles of the present invention can be microparticles or microspheres. The payload (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be attached to the surface of positively charged nanoparticles or, for example, positively charged particles, or be encapsulated by positively charged nanoparticles or, for example, positively charged particles. Thus, one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be electrostatically attached to the outer membrane of mitochondria via positively charged nanoparticles, wherein one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are attached to the surface of the positively charged nanoparticles or are encapsulated within the positively charged nanoparticles. One or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be electrostatically attached to the outer membrane of mitochondria via positively charged particles, wherein one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are attached to the surface of the positively charged particles or are encapsulated within the positively charged particles.

[0043] In general, the present invention is not limited to the use of any particular nanoparticle or particles for attachment to mitochondria and attachment or encapsulation of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof). Thus, one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) can be attached to the surface of or encapsulated within lipid nanoparticles, dendrimer nanoparticles, micellar 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. In addition, one or more nucleic acid molecules can be attached to the surface of or encapsulated in 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 oxide particles, graphene, or graphene oxide particles.

[0044] Those skilled in the art will appreciate that the above-described methods of electrostatic attachment or encapsulation of nanoparticles or particles are applicable to all products, methods, devices or uses described herein.

[0045] In the sense of the present invention, payload (such as nucleic acid molecule, polypeptide, drug or its combination) can also be covalently attached to the outer membrane of mitochondria. Covalent bond or covalent attachment or covalent interaction is formed by the chemical bond involving the sharing of electron pairs between atoms. Payload (such as nucleic acid molecule, polypeptide, drug or its combination, particularly polypeptide, such as protein) can be attached to mitochondria by peptide bond (such as amide bond (such as, carboxamide bond or urea bond)). The mitochondria with amino groups of mitochondrial membrane-associated protein / peptide of the present invention can be covalently attached to nanoparticles / particles functionalized with N-hydroxysuccinimide ester (NHS), nucleic acid molecules modified with NHS or molecules modified with NHS to form covalently attached ligands and more stable conjugates. Alternatively, the mitochondria with carboxyl groups as part of mitochondrial-associated protein of the present invention can be covalently attached to the amine groups included on nanoparticles / particles or nucleic acid molecules. In general, mitochondria can be covalently attached by any chemical group that can form a chemical bond (preferably with primary amine, such as 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 isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, ethylene oxides, carbonates, aryl halides, imide esters, carbodiimides, anhydrides, and fluorophenyl esters. Thus, in some embodiments, the mitochondria of the present invention can be covalently linked to isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, ethylene oxides, carbonates, aryl halides, imide esters, carbodiimides, anhydrides, or fluorophenyl esters. Thus, in some embodiments, in mitochondria comprising a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), the payload can be covalently linked to an isothiocyanate, isocyanate, acyl azide, sulfonyl chlorides, aldehydes, glyoxals, epoxides, ethylene oxides, carbonates, aryl halides, imide esters, carbodiimides, anhydrides, or fluorophenyl esters. Thus, in some embodiments, the polypeptides of the invention can be covalently linked to isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, or fluorophenyl esters. It will be appreciated by those skilled in the art that the selection of chemical groups (i.e., functional groups) for attachment of the payload (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof), nanoparticles, or particles contained therein to which the payload (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof), nanoparticles, or particles may be determined by the available chemical groups on the mitochondrial surface (e.g., on a polypeptide or protein contained in the outer mitochondrial membrane), or vice versa.

[0046] Therefore, one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) can be covalently attached to the outer membrane of mitochondria.Preferably, one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) can be attached to the polypeptide in the outer membrane of mitochondria by amide bonds.One or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) can be attached to the polypeptide in the outer membrane of mitochondria by amide bonds, wherein one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) have been modified to form amide bonds with the amine functional group included in the polypeptide in the outer membrane of mitochondria.One or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) can be attached to the polypeptide in the outer membrane of mitochondria by ester bonds, wherein one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) have been modified to form ester bonds with the carboxyl functional group (such as included in the polypeptide in the outer membrane of mitochondria).Payload (such as nucleic acid molecules, polypeptides, drugs or its combination) can also be attached to mitochondria by covalently attaching the nanoparticle comprising payload (such as nucleic acid molecules, polypeptides, drugs or its combination) to mitochondria. Nanoparticle can be any nanoparticle well known by persons skilled in the art, and can be charged (i.e. positively charged or negatively charged) or not charged (i.e. having neutral charge).In preferred embodiments, described nanoparticle is positively charged nanoparticle, as described above.Therefore, one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) can be connected to the polypeptide in the mitochondrial outer membrane by amide bond, wherein one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) are encapsulated in nanoparticle, and wherein nanoparticle comprises the functional group that allows nanoparticle to be covalently connected to the polypeptide in the outer membrane of mitochondria.Therefore, one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) can be connected to the polypeptide in the mitochondrial outer membrane by ester bond, wherein one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) are encapsulated in nanoparticle, and wherein nanoparticle comprises the functional group that allows nanoparticle to be covalently connected to the polypeptide in the outer membrane of mitochondria.Payload (such as nucleic acid molecules, polypeptide, drug or its combination) can also be attached to or be encapsulated in positively charged nanoparticle, such as polycationic nanoparticle. Nanoparticles (e.g., positively charged nanoparticles) comprising a payload (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) can be covalently linked to antibodies that specifically bind the antigen contained in the outer mitochondrial membrane. Nanoparticles (e.g., positively charged nanoparticles) comprising a payload (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) can include a phospholipid with a reactive group that can be covalently linked to antibodies that specifically bind the antigen contained in the outer mitochondrial membrane. Therefore, one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) can be encapsulated in nanoparticles, wherein the nanoparticles are covalently linked to antibodies.

[0047] In the sense of the present invention, payload (such as nucleic acid molecules, polypeptides, drugs or its combination) can also be connected to antibodies that specifically bind to the antigen contained in the outer membrane of mitochondria. Such antibodies comprising payload (such as nucleic acid molecules, polypeptides, drugs or its combination) preferably bind to the antigen contained in the outer membrane of mitochondria, thereby promoting the formation of a delivery platform. The present invention is not limited to any specific antigen. In general, the present invention can be implemented with antibodies that specifically bind to any antigen contained in the outer membrane of mitochondria, thereby promoting the formation of mitochondria-nucleic acid complexes (that is, mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination)). Antibodies used herein (plural forms can be used interchangeably) are immunoglobulin molecules that can specifically bind to targets (such as carbohydrates, polynucleotides, lipids, polypeptides, etc.) by at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Preferred targets herein are mitochondria, particularly antigens contained in the outer membrane of human mitochondria. The term "antibody" as used herein 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 fragments (scFv)), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, single-domain antibodies (e.g., camel or llama VHH antibodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of immunoglobulin molecules comprising antigen recognition sites of desired 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 their subclasses), and antibodies do not necessarily belong to any particular class. Immunoglobulins can be classified into different classes based on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0048] "Specific binding" (used interchangeably herein) to an antibody of a target or epitope is a well-known term in the art, and methods for determining such specific binding are also well-known in the art. A molecule is said to exhibit "specific binding" if its reaction or binding to a particular target antigen is more frequent, more rapid, longer lasting, and / or greater affinity than its reaction or binding to an alternative target. An antibody "specifically binds" to a target antigen if its binding to the target antigen is greater affinity, avidity, more readily, and / or longer lasting than its binding to other substances. For example, an antibody that specifically (or preferentially) binds to an epitope is an antibody that binds to the epitope with greater affinity, avidity, more readily, and / or longer lasting than its binding to other targets. By reading this definition, it can also be 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. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally speaking, but not necessarily, reference to binding refers to preferential binding.

[0049] In general, one or more payloads (e.g., 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, Mitofusin1, SDHB, UQCRC1, COX5b, Mitofusin 2, SSBP1, and VDAC1.

[0050] Preferably, one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be linked to any antibody that specifically binds to an antigen contained in the outer mitochondrial membrane. Thus, one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane, wherein the preferred antigen is any one of OPA1, TOM70, TOMM20, Mitofusin 1, Mitofusin 2, and VDAC1.

[0051] Payload (such as nucleic acid molecule, polypeptide, drug or its combination) can be covalently linked to antibody, forming a payload-antibody complex that can bind to mitochondrial antigens. Thus, payload (such as nucleic acid molecule, polypeptide, drug or its combination) can be covalently linked to antibody, forming a payload-antibody complex that can bind to antigens contained in mitochondrial outer membrane. In some embodiments, DNA or RNA molecules can be covalently linked to antibody, forming a payload-antibody complex that can bind to antigens contained in mitochondrial outer membrane. In some embodiments, DNA or RNA molecules can be covalently linked to antibody, forming a payload-antibody complex that can bind to antigens contained in mitochondrial outer membrane.

[0052] Payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can also be electrostatically attached to antibodies that specifically bind to antigens contained in the outer mitochondrial membrane. Thus, one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be attached to antibodies that specifically bind to antigens contained in the outer mitochondrial membrane, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are electrostatically attached to a modified antibody, wherein the modified antibody has one or more positive charges.

[0053] Payload (such as nucleic acid molecule, polypeptide, drug or its combination) can be electrostatically connected to the antibody of modification, such as comprising positive charge antibody, forms the payload-antibody complex that can bind to mitochondrial antigen.Therefore, payload (such as nucleic acid molecule, polypeptide, drug or its combination) can be electrostatically connected to the antibody of modification, such as comprising positive charge antibody, forms the payload-antibody complex that can bind to antigen contained in mitochondrial outer membrane.In some embodiments, DNA or RNA molecule can be electrostatically connected to the antibody of modification, such as comprising positive charge antibody, forms the payload-antibody complex that can bind to antigen contained in mitochondrial outer membrane.In some embodiments, DNA or RNA molecule can be electrostatically connected to the antibody of modification, such as comprising positive charge antibody, forms the nucleic acid-antibody complex that can bind to mitochondrial antigen.

[0054] The antibody of the antigen comprised in the specific binding mitochondrial outer membrane can be used for attachment comprising the nanoparticle of useful load (such as nucleic acid molecule, polypeptide, drug or its combination), such as lipid nanoparticle, thereby helps to attach useful load (such as nucleic acid molecule, polypeptide, drug or its combination) to mitochondria.Therefore, one or more useful loads (such as nucleic acid molecule, polypeptide, drug or its combination) can be connected to the antibody of the antigen comprised in the specific binding mitochondrial outer membrane, wherein one or more useful loads (such as nucleic acid molecule, polypeptide, drug or its combination) are encapsulated in nanoparticle, and wherein nanoparticle is covalently linked to antibody.Described nanoparticle can be any nanoparticle well known to persons skilled in the art, and can be charged (i.e. positively charged or negatively charged) or not charged (i.e. having overall neutral charge).One or more useful loads (such as nucleic acid molecule, polypeptide, drug or its combination) can be connected to the antibody of the antigen comprised in the specific binding mitochondrial outer membrane, wherein nanoparticle is electrostatically linked to the antibody of modification, and wherein the antibody of modification has one or more positive charges.When nanoparticle is electrostatically linked to the antibody of modification with one or more positive charges, described nanoparticle preferably has negative charge. In some embodiments, one or more payloads (such as nucleic acid molecules, polypeptide, medicine or its combination) can be connected to the antibody of the antigen included in the specific binding mitochondrial outer membrane, wherein the nanoparticle is electrostatically connected to the antibody of modification, and wherein the antibody of modification has one or more negative charges.When the nanoparticle is electrostatically connected to the antibody with one or more negative charges, the nanoparticle preferably has a positive charge.The nanoparticle with positive charge is preferably a positively charged nanoparticle as described above.

[0055] Payload (such as nucleic acid molecules, polypeptide, drug or its combination) can also be connected to entity, and then this entity is connected to antibody.Such entity can be biotin, and it is connected to avidin binding antibody.Therefore, one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) can be connected to the antibody of the antigen comprised in specific binding mitochondrial outer membrane, wherein one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) are covalently linked to biotin, and wherein biotin is connected to avidin binding antibody.In addition, payload (such as nucleic acid molecules, polypeptide, drug or its combination) can also be connected to entity, and then it is connected to antibody when being encapsulated in nanoparticle.Therefore, one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) can be connected to the antibody of the antigen comprised in specific binding mitochondrial outer membrane, wherein one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) are encapsulated in nanoparticle, wherein nanoparticle is covalently linked to biotin, and wherein biotin is connected to avidin binding antibody.The present invention is not limited to avidin binding antibody, as known to those skilled in the art, avidin can be replaced with structural analogs such as streptavidin or neutravidin. Streptavidin typically shares approximately 30% sequence identity with avidin, but possesses nearly identical secondary, tertiary, and quaternary structures. Neutravidin is a deglycosylated analog of avidin.

[0056] Payload (such as nucleic acid molecules, polypeptide, drug or its combination) also can be connected to entity, and then this entity is connected to antibody.Such entity can be activated ester, and it is connected to antibody.Therefore, one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) can be connected to the antibody of the antigen comprised in specific binding mitochondrial outer membrane, wherein one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) are covalently linked to activated ester, and wherein activated ester is connected to antibody by amide bond.In addition, payload (such as nucleic acid molecules, polypeptide, drug or its combination) also can be connected to entity, and then it is connected to antibody when being encapsulated in nanoparticle.Therefore, one or more payloads (such as nucleic acid molecules, polypeptide, drug or its combination) can be connected to the antibody of the antigen comprised in specific binding mitochondrial outer membrane, wherein nanoparticle is covalently linked to activated ester, and wherein activated ester is connected to antibody by amide bond.

[0057] A single-stranded nucleic acid molecule (e.g., ssDNA or ssRNA) can hybridize with one or more complementary single-stranded nucleic acid molecules that are attached to or to an antibody that specifically binds to mitochondria, thereby facilitating the attachment of nucleic acids and the formation of a delivery platform. Thus, one or more nucleic acid molecules can be linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane, wherein the nucleic acid molecule is a single-stranded nucleic acid molecule (ssDNA or ssRNA), wherein the single-stranded nucleic acid molecule hybridizes with one or more complementary single-stranded nucleic acid molecules that are attached to or to an antibody modified with one or more complementary single-stranded nucleic acid molecules.

[0058] In the sense 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 that can hybridize to another single-stranded nucleic acid molecule, i.e. a nucleic acid can be attached to the antibody by hybridization.

[0059] In the sense of the present invention, a "modified antibody" also refers to an antibody that has been modified to have one or more positive charges, for example, to attach negatively charged nucleic acids.

[0060] In another aspect of the present invention, a payload (such as a nucleic acid molecule, a polypeptide, a drug or a combination thereof) can be connected to a small molecule targeting mitochondria to promote the attachment of a payload (such as a nucleic acid molecule, a polypeptide, a drug or a combination thereof) and the formation of a delivery platform. Therefore, one or more payloads (such as a nucleic acid molecule, a polypeptide, a drug or a combination thereof) can be attached to the outer membrane of the mitochondria, wherein one or more payloads (such as a nucleic acid molecule, a polypeptide, a drug or a combination thereof) are connected to a small molecule targeting mitochondria. In the sense of the present invention, any small molecule targeting mitochondria can be used to promote attachment. Exemplary small molecules targeting mitochondria are selected from: triphenylphosphine (TPP), dequalinium chloride (DQA), E-4-(1H-indol-3-yl vinyl)-N-methylpyridinium iodide (F16), rhodamine 19, biguanide and guanidine. Thus, one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be linked to a mitochondrial-targeting small molecule, wherein the mitochondrial-targeting small molecule is selected from triphenylphosphine (TPP), dequalinium chloride (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 (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be linked to triphenylphosphine (TPP).

[0061] As used herein, a "targeting moiety" refers to a moiety that is capable of specifically binding to (i) a molecule on the surface of a target cell or (ii) a molecule that is capable of specifically binding to a molecule on the surface of a target cell (e.g., 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.

[0062] The present invention is particularly based on electrostatic interactions. The charge of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, or a mitochondria) can be modified with, for example, a cationic molecule or a polymer. Therefore, the charge of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, or a mitochondria) can, for example, be reversed. Taking the foregoing into account, it will be appreciated by those skilled in the art that when the charge of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, and a mitochondria) is adjusted (e.g., reversed), the products, methods, devices, and uses provided herein can also be performed. Therefore, the present invention also provides a mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) are electrostatically attached to the outer membrane of the mitochondria, wherein: (a) polycations or positively charged substances are attached to the outer surface of mitochondria, resulting in a positive charge on the mitochondrial surface; and (b) one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are electrostatically attached to the positively charged surface of a mitochondrion via a positively charged substance. A mitochondrion comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are electrostatically attached to the outer membrane of the mitochondria, wherein the surface of the mitochondria is positively charged, wherein: (a) one or more payloads (e.g., nucleic acid molecules, peptides, drugs, or combinations thereof) are attached to or encapsulated in positively charged nanoparticles; and (b) Positively charged nanoparticles comprising one or more payloads (eg, nucleic acid molecules, peptides, drugs, or combinations thereof) are electrostatically attached to mitochondria.

[0063] The present invention is not particularly limited to any nanoparticle, and may be any nanoparticle as described above.

[0064] In some embodiments, mitochondria according to the present invention as described above comprising one or more payloads (eg, nucleic acid molecules, polypeptides, drugs, or combinations thereof) may be attached to and / or encapsulated in a protective layer.

[0065] The term "protective layer" as used herein refers to a layer that partially or completely covers, coats and / or encapsulates (i.e., wraps) mitochondria according to the present invention. The protective layer of the present invention is used to modify mitochondria to improve the pharmacokinetic and pharmacodynamic properties of mitochondria. Specifically, the protective layer can especially increase the plasma half-life of mitochondria, protecting mitochondrial payloads (e.g., one or more nucleic acid molecules) from degradation, that is, payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) are decomposed into their components when administered in vivo. In addition, 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 (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof). The protective layer also prevents or reduces the immune response or cytotoxicity when mitochondria are internalized into cells. As can be seen from the accompanying examples (see, e.g., Examples 24 to 29), the mitochondrial delivery platform comprising a protective layer is more effective in delivering, for example, payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof), achieving higher transcription of mRNA and higher protein knockdown of siRNA compared to previous methods.

[0066] The protective layer of the present invention preferably comprises a polymer or lipid component or molecule. In some embodiments, the protective layer encapsulates the mitochondria according to the present invention to form a mitochondrial encapsulated particle. In a further embodiment, the protective layer partially covers or encapsulates the mitochondria according to the present invention to form a mitochondria having a protective layer surface coating. The protective layer can be electrostatically or covalently attached to the mitochondria directly (e.g., directly attached to the outer membrane of the mitochondria) or indirectly (e.g., attached to the outer membrane of the mitochondria via another entity). In a preferred embodiment, the protective layer encapsulates the mitochondria, which contains one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) attached to the outer membrane of the mitochondria, preferably, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are electrostatically attached to the outer membrane of the mitochondria via a polycationic substance, particularly a linear or branched polycationic polymer according to the present invention. The protective layer encapsulating the mitochondria can also be directly attached to the mitochondria, for example, by electrostatic interaction, or by covalent attachment to the outer membrane of the mitochondria, wherein the covalent attachment can be, for example, by amide bonds to polypeptides in the mitochondrial outer membrane. The protective layer can also be covalently attached to antibodies that specifically bind to antigens contained in the mitochondrial outer membrane. In further embodiments, the protective layer is attached to the outer membrane of the mitochondria without encapsulating the mitochondria. The protective layer is attached to the outer membrane of the mitochondria, particularly when the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) attached to the surface of or encapsulated within a nanoparticle, particle, positively charged particle, or positively charged nanoparticle. In embodiments where the mitochondria comprise one or more positively charged particles, positively charged nanoparticles, particles, or nanoparticles, the skilled artisan will recognize that the surface of the mitochondrial outer membrane may not be fully accessible to the molecules comprising the protective layer, thereby preventing complete encapsulation (i.e., encapsulation) of the mitochondria.

[0067] In some embodiments, mitochondria according to the present invention as described above comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) may be attached to and / or encapsulated in a protective layer, wherein the protective layer is a protective polymer.

[0068] As used herein and as defined by FW Billmeyer, JR. in Textbook of Polymer Science, second edition, 1971, the term "polymer" refers to a relatively large molecule composed of smaller chemical repeating units that undergo polymerization to provide a polymer product. The chemical substances that react with each other to form the repeating units of a polymer are referred to herein as "monomers," and a polymer is referred to herein as consisting of "polymerized units" of the monomers that react to form the repeating units. The chemical reaction in which monomers react to form polymerized units of a polymer is referred to herein as "polymerization" or "polymerization reaction." Typically, a polymer contains 11 or more monomers. The structure of a polymer can be linear, branched, star-shaped, cyclic, hyperbranched, cross-linked, or a combination thereof; polymers can have a single type of repeating monomer unit ("homopolymer"), or they can have more than one type of repeating monomer unit ("copolymer"). Copolymers can have various types of repeating monomer units that can be arranged randomly, sequentially, in blocks, in other ways, or in any mixture or combination thereof. Typically, the weight average molecular weight (Mw) of a polymer is 1,000 or higher. Polymer molecular weight can be measured by standard methods, such as size exclusion chromatography or intrinsic viscosity. The widest range of values ​​for MW is between 1,000 (one thousand) Daltons and 2,000,000 (two million) Daltons, preferably between 1,000 and 500,000 Daltons. The preferred range for polycationic materials is a molecular weight between 10,000 and 70,000 Daltons. For protective polymers, a preferred molecular weight is about 15,000 Daltons.

[0069] In some embodiments, the protective polymer is a linear or branched cationic polymer, optionally, the linear or branched cationic polymer is electrostatically linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof). In some embodiments, the protective polymer is a linear or branched cationic polymer, optionally, the linear or branched cationic polymer is covalently linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0070] 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 comprising repeating monomer units linked to each other to form a linear structure, while a "branched polymer" comprises a linear polymer chain substituted with one or more polymer chains (short or long polymer chains). As defined herein, a cationic homopolymer is a polymer comprising one or more cationic monomers as polymerized units. In some embodiments, one or more cationic monomers are used that comprise a cation that exists in a cationic form in solution within a certain pH range that can be used for the applications of the present invention, while the cation may be in a neutral form at certain other pH values. In some embodiments, at least one cationic monomer is used that is in a neutral form during the polymerization process; in such embodiments, after polymerization, the conditions surrounding the polymer (e.g., pH) are changed so that the polymerized units produced by the cationic monomer acquire a positive charge. Independently, in some embodiments, one or more cationic monomers are used that comprise a cationic group that is permanently in a cationic form (i.e., a cation that remains in a cationic form at all pH values ​​below 9). Cations that are permanently in a cationic form include, for example, quaternary ammonium salts. In some embodiments, one or more cationic polymers are used in which each cationic group is permanently in a cationic form. In some embodiments, each cationic group in every kind of cationic polymer used is permanently in cationic form.Corresponding to the anion of the cation can be present in solution, in the complex with the cation (such as nucleic acid-cationic polymer complex or mitochondria-polymer complex), other positions or its combination on the polymer.Corresponding to the anion of the cation of suitable cationic monomer can be any type of anion.Suitable anions include but are not limited to halogen ions (including such as chloride, bromide or iodide), hydroxide, phosphate, sulfate, bisulfate, ethyl sulfate, methyl sulfate, formate, acetate or any mixture thereof.In addition, anion can be replaced in the process of forming mitochondria and protective layer, that is, the polymer constituting the protective layer can have a kind of anion before contacting with mitochondria, and then it is replaced by another anion, such as payload (such as nucleic acid molecule, polypeptide, drug or its combination) or mitochondria of the present invention.

[0071] In some embodiments, the linear or branched cationic polymer can be electrostatically linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof), such that one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be bound to the inner surface of the protective cationic polymer layer. In some embodiments, the linear or branched cationic polymer can be covalently linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof), such that one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be bound to the inner surface of the protective cationic polymer layer.

[0072] The linear or branched cationic polymer is not particularly limited and can be any suitable linear or branched cationic polymer. In a preferred embodiment, 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.

[0073] In some embodiments, the protective polymer is a linear or branched cationic copolymer, optionally electrostatically linked to one or more payloads (eg, nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0074] The term "copolymer" refers to a copolymer as described above. The copolymers of the present invention may be linear (eg, block copolymers, alternating copolymers, periodic copolymers, statistical copolymers, stereoblock copolymers or gradient copolymers) or branched (eg, graft or star copolymers).

[0075] In some embodiments, the protective polymer is a linear or branched cationic block copolymer, optionally wherein the linear or branched cationic block copolymer is electrostatically linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof). In some embodiments, the protective polymer is a linear or branched cationic block copolymer, optionally wherein the linear or branched cationic block copolymer is covalently linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0076] As used herein, the term "block copolymer" is a copolymer comprising more than one monomer, wherein the monomers are present in blocks. Each block of monomers comprises a repeating sequence of monomers. Furthermore, a block is a portion of a polymer comprising repeating monomer units having at least one characteristic that is not present in the adjacent blocks. A representative formula for 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 is not intended to limit the structure of the block copolymer used in the present invention. The block copolymer of the present invention can be a diblock, triblock, tetrablock copolymer, etc. In addition, the block copolymer can also be a linear or branched block copolymer.

[0077] 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-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(amidoamine), RGD-modified poly(ethylene glycol)-block-poly(amidoamine), or a combination thereof.

[0078] In some embodiments, the protective polymer is a cationic graft (g) copolymer, optionally, the cationic graft (g) copolymer is electrostatically attached to one or more nucleic acid molecules. In some embodiments, the protective polymer is a cationic graft (g) copolymer, optionally, the cationic graft (g) copolymer is covalently attached to one or more nucleic acid molecules.

[0079] As used herein, the term "graft copolymer" refers to a branched polymer formed when a polymer or copolymer chain is chemically attached as a side chain to a polymer backbone. Typically, the side chain has a different polymer composition than the backbone. Graft copolymers have unique properties, such as mechanical membrane properties, resulting from thermodynamically driven microphase separation of the polymer.

[0080] 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.

[0081] In a further embodiment, the protective polymer is a linear or branched PEGylated (PEG) cationic polymer, optionally, the linear or branched PEGylated (PEG) cationic polymer is electrostatically linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof). In a further embodiment, the protective polymer is a linear or branched PEGylated (PEG) cationic polymer, optionally, the linear or branched PEGylated (PEG) cationic polymer is covalently linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0082] The term "PEGylated cationic polymer" as used herein refers to a cationic polymer modified with polyethylene glycol (PEG) or its derivatives by covalent or non-covalent forces (e.g., ionic interactions or hydrogen bonds). Modification of a material with groups from PEG (also known as polyethylene oxide) is referred to as PEGylation. PEGylation of a biologically active entity can prevent degradation of the entity, particularly by proteolytic enzymes. Other advantages of PEGylation include, but are not limited to, increased water solubility, increased bioavailability, increased blood circulation, reduced aggregation, reduced immunogenicity, reduced toxicity, and reduced frequency of administration.

[0083] In some embodiments, the PEGylated cationic polymer is PEGylated polyethyleneimine, RGD-modified 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 poly 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.

[0084] In some embodiments, the protective layer is a lipid formulation, optionally wherein the lipid formulation is a cationic lipid formulation, further optionally wherein the cationic lipid formulation is electrostatically linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0085] Lipid preparation comprises the lipid molecule that forms lipid granule (for example liposome) or lipid layer.Lipid preparation of the present invention can be connected to and / or wrap up mitochondria of the present invention.Lipid preparation can partly cover or be coated with mitochondria of the present invention or wrap up mitochondria of the present invention.In some embodiments, the lipid preparation of wrapping up mitochondria of the present invention is liposome.

[0086] The term "liposome" as used herein is a structure with one or more lipid membranes, which are particularly enclosed within the aqueous interior of the mitochondria of the present invention. The present invention includes both unilamellar liposomes (called unilamellar liposomes) and multilamellar liposomes (called multilamellar liposomes). The selection of lipid preparations and the lipids therein comprises depends on a variety of considerations, including stability, physicochemical properties, payload loading efficiency, payload release efficiency, and toxicity. The lipids comprised in the lipid preparations of the present invention can be any lipid that can be connected to and / or encapsulate the mitochondria of the present invention, including but not limited to fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and sterols. The lipids comprised in the lipid preparations can be amphipathic lipids comprising hydrophilic (polar) and hydrophobic (non-polar) groups. Amphipathic lipids include but are not limited to phospholipids, amino lipids, and sphingolipids. The lipids comprised in the lipid preparations of the present invention can include one or more saturated or unsaturated acyl groups with various carbon chain lengths. In a preferred embodiment, the one or more lipids comprised in the lipid preparations include saturated, monounsaturated, or diunsaturated fatty acids with one or more carbon chain lengths between C14 and C22. The lipids of the present invention can also include a mixture of saturated and unsaturated fatty acid chains.

[0087] As used herein, the term "cationic lipid" contained in a cationic lipid formulation refers to a lipid having one or more fatty acids or fatty alkyl chains and a cation or a cation ionizable group (i.e., a functional group) (e.g., an amino group (including alkylamino, dialkylamino, trialkylamino, and tetraalkylamino groups)). A cationic group refers to a group that is positively charged at physiological pH (e.g., at about pH 7.4). A cationic ionizable group refers to a group that can be protonated to form a cationic lipid at or below physiological pH (e.g., at a pH below about 6.5, which is a typical pH within an endosome). An advantage of protonating a cationic ionizable group within an endosome is that it promotes membrane fusion and subsequent cytosolic release. In certain embodiments, the pKa of the protonatable group of a cationic ionizable lipid is 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 different pKa that can be changed by modifying its ionizable group. Therefore, one strategy for adjusting the overall pKa of a lipid formulation is to chemically modify the lipids. Another strategy is to use a mixture of two or more lipids with different pKas and adjust their ratio to achieve the desired apparent pKa. Cationic lipid formulations can also be electrostatically linked to one or more nucleic acid molecules. Cationic lipids include, but are not limited to, DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine), 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-trimethylammoniumpropane chloride), UGG (unsaturated guanidinoglycoside), DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine), and lipofectamine. Lipofectamine (also known as lipofectamine 2000) typically contains a 3:1 mixture of DOSPA and DOPE.

[0088] Lipid formulations can also include one or more neutral lipids, wherein neutral lipid molecules are uncharged or are neutral zwitterionic forms at physiological pH.Neutral lipids include but are not limited to DLinDMA (1,2-dilinoleoyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleoyl methyl-4-dimethylaminobutyrate), DODMA (1,2-dioleoyloxy-3-dimethylaminopropane) and DOGS (dioctadecyl amido glycyl spermine). As understood by those skilled in the art, under the conditions of neutral lipid protonation, neutral lipids can also be ionizable cationic lipids.

[0089] Lipid preparation of the present invention can also comprise one or more anionic lipids.The anionic lipid that is applicable to lipid preparation of the present invention includes but is not limited to phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-acylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine and lysine phosphatidylglycerol.

[0090] In some embodiments, the lipid formulation comprises DC-cholesterol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), DLinDMA (1,2-dilinoleoyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleoylmethyl-4-dimethylaminobutyrate), DODMA (1,2-dioleoyloxy-3-dimethylaminopropane), DOGS (dioctadecylamidoglycylspermine), DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane chloride), DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane chloride)), UGG (unsaturated guanidinoglycoside), DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine), lipofectamine, or a combination thereof.

[0091] In addition, the lipid formulations of the present invention may further comprise one or more additional lipids, preferably wherein the lipid is cholesterol, substituted or unsubstituted cholesterol, a cholesterol derivative such as a hydroxylated cholesterol derivative (e.g., hydroxycholesterol), 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 (dimethyldioctadecyl ammonium), 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, bis(monooleoylglycerol)phosphate, or a combination thereof.

[0092] Lipid formulations of the present invention may also include one or more additional lipids. Additional lipids may be included in lipid formulations for various purposes, such as preventing lipid oxidation, attaching a ligand to the lipid formulation surface, stabilizing lipid formulations, or improving payload delivery. The additional lipids included in the lipid formulations may be any lipid, including but not limited to amphipathic, neutral, cationic and anionic lipids. In the context of the present invention, stabilizing lipids may refer to lipids that make lipid formulations resistant to chemical changes. Stabilizing lipids include but are not limited to sterols, such as cholesterol, substituted or unsubstituted cholesterol, cholesterol derivatives such as hydroxylated cholesterol derivatives (such as hydroxycholesterol), PEG-lipids (such as PEG coupled with phosphatidylethanolamine, PEG combined with ceramide), and the lipids selected to reduce lipid molecule aggregation in the formation process, which may be due to the spatial stabilization of the particle, thereby preventing the charge-induced aggregation in the formation process. The example of the molecule that can be combined with lipid to reduce particle aggregation in the formation process includes PEG, monosialoganglioside (Gm1), polyamide oligomers (PAO), such as ATTA. It should be noted that the compound preventing aggregation does not necessarily need lipid to combine to function normally. Free PEG or free ATTA in solution may be sufficient to prevent aggregation. If the lipid formulation is stable after formation, the PEG or ATTA can be dialyzed away prior to administration to a subject.

[0093] In some embodiments, the lipid formulations of the present invention comprise a mixture of any of the above-mentioned lipids. Exemplary lipid formulations may comprise cationic lipids, neutral lipids (other than cationic lipids), sterols (eg, cholesterol), and PEG-modified lipids.

[0094] In some embodiments, mitochondria of the invention are linked to and / or encapsulated in a zwitterionic protective polymer, optionally wherein the zwitterionic protective polymer is electrostatically linked to one or more payloads (eg, nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0095] The zwitterionic protective polymer can be a homopolymer or copolymer as described above. The zwitterionic polymer comprises one or more positive charges and one or more negative charges, wherein the total (i.e., net) charge of the polymer is substantially neutral. In one embodiment, the zwitterionic polymer is a zwitterionic copolymer wherein 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.

[0096] In a preferred embodiment, the zwitterionic protective polymer is selected from: poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyethyleneimine-g-poly(2-methacryloyloxyethyl phosphorylcholine) (PEI-g-PMPC), co-assembly of cationic (carboxyl functionalized) and anionic (amino functionalized) copolyesters based on 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).

[0097] In some embodiments, the protective layer is connected to the targeting moiety, wherein the protective layer connected to the targeting moiety is optionally electrostatically linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof). In preferred embodiments, the targeting moiety is an antibody or a carbohydrate molecule. In further preferred embodiments, the targeting moiety is contained on the outer surface of the protective layer. The outer surface of the protective layer is the surface in contact with the environment, wherein the inner surface is close to or in contact with the mitochondria and the payload contained therein. In some embodiments, the protective layer is connected to the targeting moiety, wherein the protective layer connected to the targeting moiety is optionally covalently linked to one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof). In preferred embodiments, the targeting moiety is an antibody or a carbohydrate molecule. In further preferred embodiments, the targeting moiety is contained on the outer surface of the protective layer.

[0098] In a further embodiment, a protective layer is attached to the antibody, wherein the protective layer attached to the antibody is optionally electrostatically linked to one or more payloads (eg, nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0099] Furthermore, in some embodiments, a protective layer is attached to a carbohydrate, wherein the protective layer attached to the carbohydrate is optionally electrostatically linked to one or more payloads (eg, nucleic acid molecules, polypeptides, drugs, or combinations thereof).

[0100] The carbohydrate or antibody connected to the protective layer of the present invention is preferably a targeting moiety, i.e., a moiety that targets cells or tissues or molecules contained therein through affinity interactions. The targeting mechanism generally requires that the targeting moiety is located on the surface of the protective layer so that the targeting moiety can be used to interact with the target (e.g., a cell surface receptor). The targeting moiety enhances the association of the entity to which they are connected with the target cell, tissue, specific cell type, or molecules contained therein (e.g., cell surface molecules). The targeting moiety can be a carbohydrate, such as lactose, galactose, N-acetylgalactosamine (NAG), mannose, mannose-6-phosphate (M6P), or a derivative thereof, but is not limited to these examples. The term "antibody" as used herein 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 fragments (scFv)), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, single-domain antibodies (e.g., camel or llama VHH antibodies), multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of immunoglobulin molecules comprising antigen recognition sites of desired 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 their subclasses), and antibodies do not necessarily belong to any particular class. Immunoglobulins can be classified into different classes based on the amino acid sequence of the constant domain of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known. Exemplary targeting antibodies include, but are not limited to, monoclonal antibodies, complete antibodies or antibody fragments. The antibody as a targeting antibody can be any antibody as defined above. Standard methods for connecting a targeting moiety (e.g., carbohydrate or antibody) can be used. For example, the targeting moiety can be covalently linked to the protective layer through an amide bond, a thioester bond, a disulfide bond or a hydrazone bond. The covalent attachment of the targeting moiety can be performed before the protective layer is formed, by covalently attaching the targeting moiety to a polymer or lipid contained in the protective layer, or the targeting moiety can be covalently attached to the protective layer after the protective layer is formed.Methods for connecting targeting moieties are well known to those skilled in the art and are described in many review articles (e.g., Z. Zhao et al., Cell, 2020, 181, p151-167; MJ 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 can be electrostatically connected, for example, to a payload or a protective polymer.

[0101] In another aspect, any of the mitochondria described herein can be incorporated into a composition. Thus, the present invention provides a composition comprising a mitochondria of the present invention, wherein the mitochondria comprises one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0102] The compositions can comprise any of the mitochondria described herein and any additional compounds that help facilitate delivery.

[0103] The mitochondria and compositions of the present invention can be formulated into pharmaceutical compositions comprising an acceptable carrier (e.g., 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 tissues, organs, and / or body fluids of a subject without excessive toxicity, irritation, allergic reaction, or other problems or complications, and with a reasonable benefit / risk ratio, within the scope of sound medical judgment. As used herein, the term "pharmaceutically acceptable carrier" refers to physiologically compatible solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption delaying agents, among others. The composition may include a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt.

[0104] Thus, the present invention relates to a pharmaceutical composition comprising the mitochondria of the present invention as described above and a pharmaceutically acceptable carrier. The pharmaceutical composition may comprise the mitochondria as described herein and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is formulated as a solution. The pharmaceutical composition may comprise the mitochondria as described herein and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is formulated as an aerosol.

[0105] The products, compositions and mitochondria described herein can be used for treatment. The mitochondria used for treatment in this application can be used in an allogeneic or autologous manner. The present invention provides mitochondria, compositions and pharmaceutical compositions for treating diseases that may benefit from the use of healthy mitochondria and a combination of healthy mitochondria and nucleic acid molecules. It is envisaged that the expression of certain target proteins may be increased by, for example, delivering messenger RNA (mRNA) or reduced by delivering small interfering RNA (siRNA). Therefore, the present invention provides treatments for human cardiovascular disease (CVD), such as ischemic heart disease, ischemia-reperfusion injury and atherosclerosis, treatments for aging-related diseases, such as sarcopenia, Parkinson's disease and Hutchinson-Gilford progeria syndrome (HGPS), treatments for kidney disease, such as autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis and Fabry disease, methods and treatments using in vitro / in vivo gene transfection and editing using CRISPR-Cas9, for gene therapy treatments for diseases such as cystic fibrosis and cancer.

[0106] The terms "drug" and "pharmaceutical composition" are used interchangeably herein. Therefore, the definitions and explanations provided herein in relation to "pharmaceutical composition" apply, mutatis mutandis, to the term "drug". Accordingly, the present invention provides mitochondria for use as a medicament, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0107] The present invention provides a composition for use as a medicament, comprising mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0108] The present invention provides a pharmaceutical composition for use as a medicament, comprising mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0109] The mitochondria, compositions and pharmaceutical compositions of the present invention can be used for gene therapy. The present invention provides a delivery platform for a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), which is particularly suitable for in vivo, ex vivo or in vitro gene therapy. It is known to those skilled in the art that in vivo gene therapy or gene editing methods may involve treatment or gene editing methods in a subject, ex vivo gene therapy or gene editing methods may involve, for example, treatment or gene editing methods in an organ artificially maintained outside the subject's body, and in vitro gene therapy or gene editing methods may involve, for example, treatment or gene editing methods in cultured cells or tissues. "Gene therapy" as used herein involves modifying the subject's genes to treat or cure a disease. The terms "gene editing" and "genome editing" can be used interchangeably herein. Therefore, the present invention provides mitochondria for gene therapy, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; d) Attached to small molecules that target mitochondria.

[0110] The present invention provides a composition for gene therapy, comprising mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0111] The present invention provides a pharmaceutical composition for gene therapy, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0112] Thus, the present invention provides mitochondria for in vitro, ex vivo or in vivo genome editing, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or combinations thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or combinations thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0113] The present invention provides a composition for in vitro, ex vivo or in vivo genome editing, comprising a plurality of mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0114] The present invention provides a pharmaceutical composition for in vitro, ex vivo or in vivo genome editing, comprising a plurality of mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0115] The mitochondria, compositions, and pharmaceutical compositions of the present invention can be used to treat a condition or disease in a subject. The term "subject" generally refers to any individual, such as an animal. Within the meaning 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.

[0116] A disease can be any condition or state of an unhealthy individual. A disease can also be a state of discomfort or well-being. According to the present invention, the disease can preferably be a cardiovascular disease, a disease associated with aging, a kidney disease, or a cancer. In a preferred embodiment, the disease is ischemic heart disease, atherosclerosis, muscular dystrophy, Parkinson's disease, or Hutchinson-Gilford progeria syndrome.

[0117] The terms "treatment," "treating," and the like are used herein to generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, i.e., completely or partially preventing a disease or its symptoms and / or preventing the progression of a disease or its symptoms. As used herein, the term "treatment" is understood to refer to any form of therapy.

[0118] Thus, the present invention provides mitochondria for treating cardiovascular disease, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0119] Preferably, the present invention provides mitochondria for treating ischemic heart disease, ischemia-reperfusion injury or atherosclerosis, wherein the mitochondria comprises one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0120] The present invention provides a composition for treating cardiovascular disease, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0121] Preferably, the present invention provides a composition for treating ischemic heart disease, ischemia-reperfusion injury or atherosclerosis, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0122] The present invention provides a pharmaceutical composition for treating cardiovascular disease, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0123] Preferably, the present invention provides a pharmaceutical composition for treating ischemic heart disease, ischemia-reperfusion injury or atherosclerosis, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0124] Therefore, the present invention provides mitochondria for treating aging-related diseases, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0125] Preferably, the present invention provides mitochondria for treating sarcopenia, Parkinson's disease or Hutchinson-Gilford Progeria Syndrome, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0126] The present invention provides a composition for treating aging-related diseases, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0127] Preferably, the present invention provides a composition for treating sarcopenia, Parkinson's disease or Hutchinson-Gilford Progeria Syndrome, comprising a plurality of mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0128] The present invention provides a pharmaceutical composition for treating aging-related diseases, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0129] Preferably, the present invention provides a pharmaceutical composition for treating sarcopenia, Parkinson's disease or Hutchinson-Gilford Progeria Syndrome, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0130] Thus, the present invention provides mitochondria for treating kidney disease, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0131] Preferably, the present invention provides mitochondria for treating autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis or Fabry disease, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0132] The present invention provides a composition for treating kidney disease, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0133] Preferably, the present invention provides a composition for treating autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis, or Fabry disease, comprising a plurality of mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0134] The present invention provides a pharmaceutical composition for treating kidney disease, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0135] Preferably, the present invention provides a pharmaceutical composition for treating autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis or Fabry disease, comprising a plurality of mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0136] Thus, the present invention provides mitochondria for treating cancer, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0137] The present invention provides a composition for treating cancer, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0138] The present invention provides a pharmaceutical composition for treating cancer, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0139] As described above, the mitochondria, compositions or pharmaceutical compositions of the present invention are used to treat various diseases, including cardiovascular disease, ischemia-reperfusion injury, kidney disease, cancer, mitochondrial dysfunction, metabolic disorders, autoimmune diseases, infectious diseases, inflammatory diseases, muscle diseases and aging-related diseases.

[0140] The cardiovascular disease is preferably selected from the group consisting of ischemic heart disease, myocardial ischemia, atherosclerosis, myocardial infarction, acute coronary syndrome heart failure and hypertensive heart disease.

[0141] Ischemia-reperfusion injury can be any disease involving ischemia, preferably, ischemia-reperfusion injury is selected from liver ischemia-reperfusion injury, ischemic injury-compartment syndrome, chronic ischemia, hypertension and any injury involving ischemia, such as myocardial infarction, stroke, organ transplantation, etc.

[0142] The renal disease is preferably selected from the group consisting of autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis and Fabry disease.

[0143] The cancer is preferably selected from acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., glioblastoma), breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer 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), cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors, 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, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0144] The autoimmune disease is preferably selected from the group consisting of 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, Sjögren's syndrome, psoriasis and celiac disease.

[0145] 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.

[0146] Mitochondrial dysfunction diseases are preferably selected from diseases caused by mutations in mtDNA, such as Keams-Sayre syndrome, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, Leber hereditary optic neuropathy, Pearson syndrome, progressive external ophthalmoplegia, mitochondrial myopathy, diabetes and deafness (DAD), Leigh syndrome, "neuropathy, ataxia, retinitis pigmentosa and ptosis" (NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with ragged red fibers (MERRF syndrome), encephalomyopathy, lactic acidosis, Parkinson's disease and stroke-like symptoms (MELAS syndrome), etc. MERRF syndrome, MELAS syndrome, Leber disease, Barth syndrome and diabetes.

[0147] Metabolic disorders are preferably selected from obesity and its related metabolic diseases (e.g., type 2 diabetes). By administering the mitochondria, compositions or pharmaceutical compositions of the present invention to the white adipose tissue of a subject, metabolic disorders can be treated or prevented. White adipose tissue or white fat is one of the two types of adipose tissue found in mammals. It is generally used by the body as energy storage and includes many white fat cells. Another type of adipose tissue is brown adipose tissue. The function of brown adipose tissue is to convert the energy in food into heat. White fat cells generally contain single lipid droplets. In contrast, brown fat cells contain many smaller droplets and a large number of mitochondria. Due to the recognition that the brown adipose tissue of adults is an organ with considerable energy dissipation capacity, targeting brown adipose tissue thermogenesis is now considered as a method for treating or preventing metabolic disorders (e.g., obesity and its related metabolic diseases (e.g., type 2 diabetes)). Brown adipose tissue has been described for use in the treatment of obesity and diabetes, for example, in Cypess, Aaron M., and C.Ronald Kahn. "Brown fat as atherapy for obesity and diabetes." Current opinion in endocrinology, diabetes, and obesity 17.2 (2010): 143, which is incorporated herein by reference in its entirety. Because a major difference between brown adipocyte and the white adipocyte is the mitochondrial quantity in the cell, present disclosure provides the method for treating and preventing metabolic disorders by mitochondrion, the compositions or pharmaceutical composition that comprise mitochondrion being applied to the white adipose tissue of experimenter. Mitochondrion of the present invention is applied to white adipocyte and white adipocyte can be converted into brown adipocyte, thereby white adipose tissue is converted into brown adipose tissue.

[0148] The infectious disease is preferably selected from viral infection (eg HIV, HCV, RSV), bacterial infection, fungal infection and sepsis.

[0149] The muscle disease is preferably selected from Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), anisomia muscular dystrophy (OMD), Emory-Dreyfus 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 disease), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy (DD) and congenital muscular dystrophy (CMD). Muscle diseases may also include diseases or disorders involving or potentially involving voluntary muscle cell death or inflammation, including myositis, polymyositis, dermatomyositis and inclusion body myositis, and myopathies.

[0150] Aging-related diseases are preferably selected from neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, dementia, etc.), sarcopenia, Hutchinson-Gilford progeria syndrome, osteopenia, osteoporosis, arthritis, atherosclerosis, cardiovascular disease, hypertension, cataracts, presbyopia, glaucoma, type 2 diabetes, metabolic syndrome, hair loss, chronic inflammation, immunosenescence and age-related vision loss.

[0151] 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 radiopharmaceuticals that can be used for radiotherapy. Such radiopharmaceuticals for radiotherapy can be delivered to solid tumors by the delivery system of the present invention. The present invention is not particularly limited to any agent for radiotherapy. Iodine 131 is an exemplary agent for radiotherapy of thyroid cancer. Therefore, the present invention provides mitochondria for radiotherapy, comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0152] The present invention provides a composition for use in radiation therapy, comprising a plurality of mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0153] The present invention provides a pharmaceutical composition for radiation therapy, comprising a plurality of mitochondria, wherein the mitochondria comprise one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0154] The present invention provides mitochondria for use in radiotherapy, the mitochondria comprising one or more radiopharmaceuticals attached to the outer membrane of the mitochondria, wherein the one or more radiopharmaceuticals: a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0155] In another aspect, the present invention provides a method for delivering a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to an organ of a subject by administering the delivery platform of the present invention to the subject. The terms "administer," "introduce," and "deliver" are used interchangeably in the context of the present invention. For example, the delivery platform of the present invention (i.e., a mitochondrial payload complex) can be introduced into a subject by a method or route that results in at least a partial localization of the introduced complex at a desired site (e.g., a site believed to produce a desired effect (e.g., treatment or therapy)). The mitochondria, compositions, or pharmaceutical compositions of the present invention can be administered by,For example, but not limited to, enteral (into the intestine), gastrointestinal, epidural (into the dura mater), oral (through the mouth), transdermal, peridural, intracerebral (into the brain), intraventricular (into the ventricles), epidermal (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical (into the eye), intracavernous (into a pathological cavity), intracavernous (into the penis). root), intravaginal administration, intrauterine, extraamniotic administration, transdermal (systemic distribution by diffusion through intact skin), transmucosal (diffusion through mucous membranes), vaginal, insufflation (nasal inhalation), sublingual, sublabial, enema, eye drops (on the conjunctiva), intraaural drops, intraaural (in or through the ear), buccal (towards the cheek), conjunctiva, skin, dental (to one or more teeth), electroosmosis, intracervical, intrasinus, intratracheal, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (inside the cartilage), intracaudal (inside the cauda equina), intracisternal (inside the cisterna magna), intracorneal (inside the cornea), intracoronary (inside the dental crown) intracornal), intracoronary (inside a coronary artery), intracavernous (inside the expandable space of the corpus cavernosum), intradiscal (inside an intervertebral disc), intraductal (inside a glandular duct), intraduodenal (inside the duodenum), intradural (inside or below the dura mater), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (inside the stomach), intragingival (inside the gums), intraileal (inside the distal portion of the small intestine), intralesional (inside a local lesion or introduced directly into a local lesion), intraluminal (inside the lumen), intralymphatic (inside the lymph), intramedullary (inside the bone marrow cavity), intrameningeal (inside the meninges), intramuscular (inside the heart muscle), intraocular (inside the eye), intraovarian (inside the ovary), intrapericardial (inside the pericardium), Intrapleural (inside the pleura), intraprostatic (inside the prostate), intrapulmonary (inside the lung or its bronchi), intrasinus (inside the nose or periorbital sinuses), intraspinal (inside the spine), intrasynovial (inside the synovial cavity of a joint), intratendinous (inside a tendon), intratesticular (inside a testicle), intrathecal (inside the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (inside the chest), intraductal (inside a tube of an organ), intratumoral (inside a tumor), intratympanic (inside the middle ear), intravascular (inside one or more blood vessels), intraventricular (inside the brain ventricles), iontophoresis (passage of an electric current in which soluble salt ions migrate into body tissues), irrigation (soaking or irrigating an open wound or body cavity), laryngeal (directly in the throat), nasogastric (through the nose into the stomach), occlusive dressing techniques (topical route of administration,Then covered with a dressing to occlude the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, transcutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhaled into the respiratory tract through the mouth or nasal cavity for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramuscular (into the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or through the placenta), transtracheal (through the tracheal wall), transtympanic (through or through the tympanic cavity), ureteral (to the ureters), urethral (to the urethra), vaginal, caudal blocks, diagnostic, nerve blocks, biliary instillation, cardiac instillation, photochemotherapy, and spinal.

[0156] Modes of administration include injection, infusion, instillation and / or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal fluid and intrasternal injection and infusion. In some instances, the route is intravenous. The mitochondria, composition or pharmaceutical composition of the present invention are administered in a single dose or in at least two or more consecutive doses. Preferably, the mitochondria, composition or pharmaceutical composition of the present invention are administered intravenously or by inhalation. Preferably, the mitochondria, composition or pharmaceutical composition of the present invention are administered into the bloodstream upstream of the target organ. Preferably, the mitochondria, composition or pharmaceutical composition of the present invention are administered into an organ. Preferably, the mitochondria, composition or pharmaceutical composition of the present invention are administered directly into the target organ, such as an organ in need of treatment. Preferably, the mitochondria, composition or pharmaceutical composition of the present invention are administered directly into 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 are administered by inhalation.

[0157] In certain embodiments, the target organ is the kidney. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the kidney of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the kidney, i.e., into the subject's renal artery. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the kidney.

[0158] In certain embodiments, the target organ is the heart. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the heart of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the heart, i.e., into the subject's coronary arteries. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the heart.

[0159] In certain embodiments, the target organ is the liver. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the liver of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the liver, i.e., into the hepatic artery or portal vein of the subject. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the liver.

[0160] In certain embodiments, the target organ is the pancreas. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the pancreas of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the pancreas, i.e., into the subject's hepatic artery. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the pancreas.

[0161] In certain embodiments, the target organ is the duodenum. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the duodenum of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the duodenum, i.e., into the subject's hepatic artery. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the duodenum.

[0162] In certain embodiments, the target organ is the spleen. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the spleen of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the spleen, i.e., into the subject's splenic artery. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the spleen.

[0163] In certain embodiments, the target organ is the lung. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the lungs of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the lungs, i.e., into the subject's pulmonary artery. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the lungs.

[0164] In certain embodiments, the target organ is the intestine. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the intestine of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the intestine, i.e., into the superior mesenteric artery of the subject. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the intestine.

[0165] In certain embodiments, the target organ is the bladder. In certain embodiments, the mitochondria, compositions, or pharmaceutical compositions of the present invention are delivered to the bladder of a subject. To this end, the mitochondria, compositions, or pharmaceutical compositions of the present invention are preferably administered upstream of the bladder, i.e., into the superior and inferior vesical arteries of the subject. Alternatively, the mitochondria, compositions, or pharmaceutical compositions of the present invention are injected directly into the bladder.

[0166] For delivery of mitochondria, compositions or pharmaceutical compositions, administration may be by injection or infusion. Mitochondria, compositions or pharmaceutical compositions may be administered systemically. The phrases "systemic administration," "systemically administered," "peripherally administered," and "peripherally administered" refer to the administration of mitochondria, compositions or pharmaceutical compositions other than by direct injection into a target site, cell, tissue or organ so that they enter the subject's circulatory system for metabolism and other similar processes. Preferably, the mitochondria, compositions or pharmaceutical compositions of the present invention are delivered to cells by direct incubation with cells in cell culture medium. In a further preferred embodiment, the mitochondria, compositions or pharmaceutical compositions of the present invention are delivered directly to the site in need of treatment by injection. In a further preferred embodiment, the mitochondria, compositions or pharmaceutical compositions of the present invention are delivered systemically by intravenous injection. In a further preferred embodiment, the mitochondria, compositions or pharmaceutical compositions of the present invention are delivered by injection into the bloodstream upstream of the target organ in need of treatment. In a further preferred embodiment, the aerosolized mitochondria, aerosolized compositions or aerosolized pharmaceutical compositions of the present invention are delivered by inhalation.

[0167] The mitochondria, compositions, or pharmaceutical compositions of the present invention can be administered into the bloodstream upstream of a target organ. Thus, the present invention provides a method for delivering a nucleic acid molecule to a target organ, the method comprising administering a pharmaceutical composition into the bloodstream of a subject in need thereof, the pharmaceutical composition comprising: mitochondria, the mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) linked to a small molecule that targets mitochondria; and A pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered into the bloodstream upstream of a target organ.

[0168] The present invention provides a method for delivering a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to a target organ, the method comprising administering a pharmaceutical composition into the bloodstream of a subject suffering from cardiovascular disease, an aging-related disease, a kidney disease, or cancer, the pharmaceutical composition comprising: mitochondria, the mitochondria comprising one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) attached to the outer mitochondrial membrane, wherein the one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) linked to a small molecule that targets mitochondria; and A pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered into the bloodstream upstream of a target organ.

[0169] The present invention provides a method for delivering a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to a target organ, the method comprising the step of administering a pharmaceutical composition into the bloodstream of a subject suffering from ischemic heart disease, atherosclerosis, sarcopenia, Parkinson's disease, Hutchinson-Gilford Progeria Syndrome, or cancer, the pharmaceutical composition comprising: mitochondria comprising one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) attached to the outer mitochondrial membrane, wherein the one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) linked to a small molecule that targets mitochondria; and A pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered into the bloodstream upstream of a target organ.

[0170] The mitochondria, compositions, or pharmaceutical compositions of the present invention can be administered by inhalation. Accordingly, the present invention provides a method for delivering a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the lung, the method comprising the step of delivering a pharmaceutical composition to a subject in need thereof, the pharmaceutical composition comprising: mitochondria comprising one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) linked to a small molecule that targets mitochondria; and A pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered by inhalation.

[0171] The present invention provides a method for delivering a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the lung, the method comprising administering a pharmaceutical composition to a subject suffering from cardiovascular disease, an aging-related disease, a kidney disease, or cancer, the pharmaceutical composition comprising: mitochondria, the mitochondria comprising one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) attached to the outer mitochondrial membrane, wherein the one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) linked to a small molecule that targets mitochondria; and A pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered by inhalation.

[0172] The present invention provides a method for delivering a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the lung, the method comprising the step of administering a pharmaceutical composition to a subject suffering from ischemic heart disease, atherosclerosis, sarcopenia, Parkinson's disease, Hutchinson-Gilford Progeria Syndrome, or cancer, the pharmaceutical composition comprising: mitochondria, the mitochondria comprising one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) attached to the outer membrane of the mitochondria, wherein the one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof): a) electrostatic attachment of positively charged species to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) linked to a small molecule that targets mitochondria; and A pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered by inhalation.

[0173] In certain embodiments, the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) or the compositions or pharmaceutical compositions containing the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are delivered to the kidney of the experimenter. In certain embodiments, by being injected into the renal artery or by being directly injected into the kidney to realize delivery to the kidney. In certain embodiments, by being injected into the renal artery or by being directly injected into the kidney to realize delivery to the kidney, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are electrostatically attached to the outer membrane of the mitochondria by positively charged material. In certain embodiments, by being injected into the renal artery or by being directly injected into the kidney to realize delivery to the kidney, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are covalently attached to the outer membrane of the mitochondria. In certain embodiments, by being injected into the renal artery or by being directly injected into the kidney to realize delivery to the kidney, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are connected to the antibody of the antigen contained in the specific binding mitochondrial outer membrane. In certain embodiments, delivery to the kidney is achieved by injection into the renal artery or by injection directly into the kidney, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are linked to small molecules that target mitochondria.

[0174] In certain embodiments, the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) or the compositions or pharmaceutical compositions containing the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are delivered to the heart of the subject. In certain embodiments, delivery to the heart is achieved by injection into the coronary artery or by direct injection into the heart. In certain embodiments, delivery to the heart is achieved by injection into the coronary artery or by direct injection into the heart, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are electrostatically attached to the outer membrane of the mitochondria by positively charged substances. In certain embodiments, delivery to the heart is achieved by injection into the coronary artery or by direct injection into the heart, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are covalently attached to the outer membrane of the mitochondria. In certain embodiments, delivery to the heart is achieved by injection into the coronary artery or by direct injection into the heart, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are connected to antibodies that specifically bind to the antigen contained in the mitochondrial outer membrane. In certain embodiments, delivery to the heart is achieved by injection into the coronary artery or by injection directly into the heart, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are linked to a small molecule that targets mitochondria.

[0175] In certain embodiments, the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) or the composition or pharmaceutical composition containing the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are delivered to the liver of the subject. In certain embodiments, delivery to the liver is achieved by injection into the hepatic artery or portal vein or by direct injection into the liver. In certain embodiments, delivery to the liver is achieved by injection into the hepatic artery or portal vein or by direct injection into the liver, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are electrostatically attached to the outer membrane of the mitochondria by positively charged substances. In certain embodiments, delivery to the liver is achieved by injection into the hepatic artery or portal vein or by direct injection into the liver, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are covalently attached to the outer membrane of the mitochondria. In certain embodiments, delivery to the liver is achieved by injection into the hepatic artery or portal vein or by direct injection into the liver, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are connected to the antibody that specifically binds the antigen contained in the mitochondrial outer membrane. In certain embodiments, delivery to the liver is achieved by injection into the hepatic artery or portal vein, or by direct injection into the liver, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are linked to small molecules that target mitochondria.

[0176] In certain embodiments, the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) or the composition or pharmaceutical composition containing the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are delivered to the pancreas of the subject. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or by direct injection into the pancreas. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or by direct injection into the pancreas, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are electrostatically attached to the outer membrane of the mitochondria by positively charged substances. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or by direct injection into the pancreas, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are covalently attached to the outer membrane of the mitochondria. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or by direct injection into the pancreas, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are connected to the antibody that specifically binds the antigen contained in the mitochondrial outer membrane. In certain embodiments, delivery to the pancreas is achieved by injection into the hepatic artery or by direct injection into the pancreas, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are linked to a small molecule that targets mitochondria.

[0177] In certain embodiments, the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) or the compositions or pharmaceutical compositions containing the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are delivered to the duodenum of the experimenter. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or by direct injection into the duodenum. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or by direct injection into the duodenum, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are electrostatically attached to the outer membrane of the mitochondria by positively charged material. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or by direct injection into the duodenum, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are covalently attached to the outer membrane of the mitochondria. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or by direct injection into the duodenum, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are connected to the antibody for the antigen contained in the specific binding mitochondrial outer membrane. In certain embodiments, delivery to the duodenum is achieved by injection into the hepatic artery or by direct injection into the duodenum, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) are linked to a small molecule that targets mitochondria.

[0178] In certain embodiments, the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) or the compositions or pharmaceutical compositions containing the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are delivered to the spleen of the subject. In certain embodiments, the delivery to the spleen is achieved by injection into the splenic artery or by direct injection into the spleen. In certain embodiments, the delivery to the spleen is achieved by injection into the splenic artery or by direct injection into the spleen, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are electrostatically attached to the outer membrane of the mitochondria by positively charged substances. In certain embodiments, the delivery to the spleen is achieved by injection into the splenic artery or by direct injection into the spleen, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are covalently attached to the outer membrane of the mitochondria. In certain embodiments, the delivery to the spleen is achieved by injection into the splenic artery or by direct injection into the spleen, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are connected to antibodies that specifically bind the antigen contained in the outer membrane of the mitochondria. In certain embodiments, delivery to the spleen is achieved by injection into the splenic artery or by direct injection into the spleen, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are linked to a small molecule that targets mitochondria.

[0179] In certain embodiments, the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) or the compositions or pharmaceutical compositions containing the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are delivered to the lung of the subject. In certain embodiments, by being injected into the pulmonary artery or by being directly injected into the lung to realize delivery to the lung. In certain embodiments, by being injected into the pulmonary artery or by being directly injected into the lung to realize delivery to the lung, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are electrostatically attached to the outer membrane of mitochondria by positively charged material. In certain embodiments, by being injected into the pulmonary artery or by being directly injected into the lung to realize delivery to the lung, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are covalently attached to the outer membrane of mitochondria. In certain embodiments, by being injected into the pulmonary artery or by being directly injected into the lung to realize delivery to the lung, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or its combination) are connected to the antibody for the antigen included in the specific binding mitochondrial outer membrane. In certain embodiments, delivery to the lungs is achieved by injection into the pulmonary artery or by direct injection into the lungs, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are linked to small molecules that target mitochondria.

[0180] In certain embodiments, the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) or the compositions or pharmaceutical compositions containing the mitochondria comprising one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are delivered to the intestinal tract of the subject. In certain embodiments, delivery to the intestinal tract is achieved by injection into the superior mesenteric artery or by direct injection into the intestinal tract. In certain embodiments, delivery to the intestinal tract is achieved by injection into the superior mesenteric artery or by direct injection into the intestinal tract, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are electrostatically attached to the outer membrane of the mitochondria by positively charged substances. In certain embodiments, delivery to the intestinal tract is achieved by injection into the superior mesenteric artery or by direct injection into the intestinal tract, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are covalently attached to the outer membrane of the mitochondria. In certain embodiments, delivery to the intestinal tract is achieved by injection into the superior mesenteric artery or by direct injection into the intestinal tract, and one or more payloads (such as nucleic acid molecules, polypeptides, drugs or their combinations) are connected to the antibodies for the antigens contained in the specific binding mitochondrial outer membrane. In certain embodiments, delivery to the intestine is achieved by injection into the superior mesenteric artery or by direct injection into the intestine, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are linked to small molecules that target mitochondria.

[0181] In certain embodiments, mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) or compositions or pharmaceutical compositions containing mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are delivered to the bladder of the subject. In certain embodiments, delivery to the bladder is achieved by injection into the superior and inferior vesical arteries or by direct injection into the bladder. In certain embodiments, delivery to the bladder is achieved by injection into the superior and inferior vesical arteries or by direct injection into the bladder, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are electrostatically attached to the outer membrane of the mitochondria by positively charged substances. In certain embodiments, delivery to the bladder is achieved by injection into the superior and inferior vesical arteries or by direct injection into the bladder, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are covalently attached 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 by direct injection into the bladder, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are connected to antibodies that specifically bind to the antigens 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 by direct injection into the bladder, and one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) are linked to a small molecule that targets mitochondria.

[0182] In one aspect, the present invention provides methods for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to mitochondria to produce mitochondria of the present invention. In the sense of the present invention, "contacting" refers to placing a first substance and a second substance in close physical proximity so that the two can react. For example, mitochondria can be contacted with a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance in a solution (e.g., a buffer).

[0183] In the sense of the present invention, any payload (e.g., a nucleic acid, a polypeptide, or a drug) can be attached to the mitochondria. The payload can be attached to the mitochondria by contacting the mitochondria with the payload (e.g., a nucleic acid, a drug, or a polypeptide). In the sense of the present invention, the mitochondria and the payload can be contacted in the presence of a positively charged substance (e.g., a polycationic substance). The step of contacting the mitochondria with the nucleic acid, drug, or polypeptide and / or the positively charged substance can be performed under any feasible reaction conditions for successful complex formation, i.e., successful attachment of the nucleic acid, drug, or polypeptide.

[0184] The mitochondria can be contacted with a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a buffer (e.g., a binding buffer). The mitochondria can be contacted with a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and a positively charged substance (e.g., a polycationic substance) in a buffer (e.g., a binding buffer). The mitochondria can be contacted with a polypeptide in a buffer (e.g., a binding buffer). The mitochondria can be contacted with a polypeptide and a positively charged substance (e.g., a polycationic substance) in a buffer (e.g., a binding buffer).

[0185] In some embodiments, the concentration of mitochondria is 0.1 to 5 mg / mL. In some embodiments, the concentration of mitochondria in the binding 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 binding 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, 3.8, 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 a preferred embodiment, the concentration of mitochondria in the binding buffer is 2 mg / mL. In a preferred embodiment, the concentration of mitochondria in the binding buffer is 4 mg / mL.

[0186] In some embodiments, the concentration of mitochondria is 0.5 to 30 billion / mL. In some embodiments, the concentration of mitochondria in the binding buffer is 0.5 to 30 billion / mL. In some embodiments, the concentration of mitochondria in the binding buffer is 0.5 to 30 billion / mL. In some embodiments, the concentration of mitochondria is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 55, 58, 60, 62, 64 , 68, 70, 72, 75, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 125, 129, 132, 134, 136, 138, 140, 150, 160, 170, 180, 200, 220, 240, 260, 280, or 30 billion / mL. In some embodiments, the concentration of mitochondria in the binding buffer is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 55, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 , 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 125, 129, 132, 134, 136, 138, 140, 150, 160, 170, 180, 200, 220, 240, 260, 280, or 30 billion / mL. In a preferred embodiment, the concentration of mitochondria in the binding buffer is 6 billion / mL. In a preferred embodiment, the concentration of mitochondria in the binding buffer is 12 billion / mL. In a preferred embodiment, the concentration of mitochondria in the binding buffer is 15 billion / mL.

[0187] In some embodiments, the mitochondria are contacted with 0.002 to 5000 pmol of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof). In some embodiments, the mitochondria are contacted with 0.002 to 5000 pmol of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a binding buffer. In some embodiments, the mitochondria are contacted with 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, 270 0, 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, 40 50, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000 pmol of payload (e.g., nucleic acid molecule, polypeptide, drug, or combination thereof), optionally in binding buffer. In some embodiments, the mitochondria are contacted with 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 of payload (e.g., nucleic acid molecule, polypeptide, drug or combination thereof), optionally in binding buffer. In preferred embodiments, the mitochondria are contacted with 0.1 to 50 pmol of payload (e.g., nucleic acid molecule, polypeptide, drug or combination thereof). In preferred embodiments, the mitochondria are contacted with 0.1 to 50 pmol of payload (e.g., nucleic acid molecule, polypeptide, drug or combination thereof) in binding buffer.

[0188] In some embodiments, the mitochondria are contacted with 0.002 to 5 μg / μL of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof). In some embodiments, the mitochondria are contacted with 0.002 to 5 μg / μL of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a binding buffer. In some embodiments, the mitochondria are contacted with 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 of the nucleic acid molecule, optionally in binding buffer. 4.5, 4.6, 4.7, 4.8, 4.9, or 5 μg / μL of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), optionally in binding buffer. In a preferred embodiment, the mitochondria are contacted with 0.1 to 2 μg / μL of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof). In a preferred embodiment, the mitochondria are contacted with 0.1 to 2 μg / μL of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a binding buffer.

[0189] In some embodiments, the mitochondria are contacted with 0.004 to 40 mg / mL of the positively charged substance. In some embodiments, the mitochondria are contacted with 0.004 to 40 mg / mL of the positively charged substance in binding buffer. In some embodiments, the mitochondria are contacted with 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 of the positively charged substance. In some embodiments, the mitochondria are contacted with 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 of the positively charged species in binding buffer. In preferred embodiments, the mitochondria are contacted with 0.02 to 1.0 mg / mL of the positively charged species, optionally in binding buffer. In preferred embodiments, the mitochondria are contacted with 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 of the positively charged species, optionally in binding buffer.

[0190] In some embodiments, the mitochondria are contacted with 0.004 to 40 mg / mL of a protective polymer. In some embodiments, the mitochondria are contacted with 0.004 to 40 mg / mL of a protective polymer in binding buffer. In some embodiments, the mitochondria are contacted with 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 of a protective polymer. In some embodiments, the mitochondria are contacted with 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 of a protective polymer in binding buffer. In preferred embodiments, the mitochondria are contacted with 0.02 to 2 mg / mL of a protective polymer, optionally in binding buffer. In preferred embodiments, the mitochondria are 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. 1.5, 1.8, 2.0 mg / mL of protective polymer are optionally contacted in binding buffer.

[0191] In a further preferred embodiment, the mitochondria are contacted with 0.002 to 5000 pmol of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and 0.004 to 40 mg / mL of a positively charged substance, optionally in a binding buffer, wherein the concentration of the mitochondria is 0.1 to 5 mg / mL.

[0192] In a further preferred embodiment, mitochondria are contacted with 0.1 to 50 pmol of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and 0.02 to 1.0 mg / mL of a positively charged substance, optionally in a binding buffer, wherein the concentration of mitochondria is 1 mg / mL.

[0193] In a further preferred embodiment, the mitochondria are contacted with 0.1 to 50 pmol of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and 0.02 to 1.0 mg / mL of a positively charged substance in a binding buffer, wherein the concentration of the mitochondria is 1 mg / mL. In a further preferred embodiment, 50 μg of mitochondria are contacted with 0.1 to 50 pmol of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and 0.02 to 1.0 mg / mL of a positively charged substance in a binding buffer, wherein the concentration of the mitochondria is 1 mg / mL.

[0194] In some embodiments, 50 μg of mitochondria are contacted with 0.1 to 50 pmol of a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof, or a peptide) and 0.02 to 1.0 mg / mL of a positively charged substance. In some embodiments, 50 μg of mitochondria are contacted with 0.1 to 50 pmol of a plurality of payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof attached to a small molecule targeting mitochondria). In a preferred embodiment, 50 μg of mitochondria are contacted with 0.1 to 50 pmol of a plurality of payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and 0.02 to 1.0 mg / mL of a positively charged substance.

[0195] In some embodiments, 50 μg of mitochondria are contacted with 0.1 to 50 pmol siRNA or mRNA. In some embodiments, 50 μg of mitochondria are contacted with 0.1 to 50 pmol fluorescently labeled ssDNA or ssRNA or plasmid DNA. In some embodiments, 50 μg of mitochondria are contacted with 0.1 to 2 μL 10 mg / mL poly-L-lysine. The concentration of mitochondria is preferably 1 mg of mitochondria per 1 mL of binding buffer, i.e., 1 mg / mL. It will be appreciated by those skilled in the art that the above embodiments can be combined to promote successful binding of a payload (e.g., a nucleic acid or polypeptide) to mitochondria.

[0196] In some embodiments, an amount of 50 μg to 200 μg of mitochondria is contacted with 0.1 to 50 pmol of the nucleic acid molecule and 0.02 to 10 μg, preferably 0.02 to 5 μg, of the positively charged substance.

[0197] In some embodiments, an amount of 50 μg to 200 μg of mitochondria is contacted with 0.1 to 50 pmol of a nucleic acid molecule linked to a mitochondrial-targeted small molecule. -50 μg of mitochondria is equivalent to approximately 150 million mitochondria. 1 mg / mL of mitochondria (based on Qubit protein assay) is equivalent to approximately 3B mitochondria / mL (based on particle counter) The concentration of the protective polymer preparation used to prepare the mitochondria of the present invention is 1 mg / mL. The amount of protective polymer is between 0.1 mg and 10 mg. The concentration of the nanoparticle preparation used to prepare the mitochondria of the present invention is 1 mg / mL. The amount of protective polymer is between 0.1 mg and 10 mg.

[0198] Thus, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0199] At least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) can be contacted with a positively charged substance and a mitochondria simultaneously. At least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) can be contacted with a positively charged substance first to form a positively charged complex, and then the positively charged complex can be contacted with the mitochondria. Alternatively, the mitochondria can be contacted with a positively charged substance first and then with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof). Accordingly, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance, wherein contacting at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) with the positively charged substance and the mitochondria simultaneously; or contacting at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) with a positively charged substance to form a positively charged complex, and then contacting the positively charged complex with mitochondria; or contacting the mitochondria with a positively charged substance and subsequently with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof); and c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0200] In the methods of the present invention, the method may comprise contacting the mitochondria with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance, wherein a) contacting at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) with a positively charged substance and a mitochondria simultaneously; b) wherein at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) is contacted with a positively charged species to form a positively charged complex, and the positively charged complex is then contacted with the mitochondria; or c) contacting the mitochondria with a positively charged substance and subsequently with at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof).

[0201] In preferred embodiments, the methods of the invention involve contacting mitochondria with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged species, wherein the mitochondria are contacted with the positively charged species and subsequently contacted with the at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof).

[0202] The step of contacting the mitochondria with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and a positively charged substance can be performed in a suitable buffer. Thus, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance; c) attaching at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance, Therein, mitochondria are contacted with at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and a positively charged substance in a suitable buffer.

[0203] Preferably, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) in the presence of a positively charged substance; c) attaching at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance, wherein the mitochondria are contacted with one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) and a polycationic substance in a buffer comprising a 4:1 mixture of solution X and solution Y, wherein solution X comprises or consists of 20 mM HEPES, 1 mM EGTA, and 300 mM trehalose (pH 7.2), and solution Y comprises or consists of 0.1 M CHES (pH 10) and 0.2 M sodium dihydrogen phosphate dihydrate.

[0204] The contacting step of the present invention is not particularly limited to any reaction conditions, time or reaction time. Generally, any reaction conditions that help to attach a payload (such as a nucleic acid molecule, a polypeptide, a drug or a combination thereof) to mitochondria by a positively charged substance can be used, thereby promoting the formation of a delivery complex. However, it is preferred that mitochondria be contacted with at least one payload (such as a nucleic acid molecule, a polypeptide, a drug or a combination thereof) and a positively charged substance for more than 5 minutes at room temperature, preferably in the dark. Therefore, the present invention provides a method for 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 the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance, wherein the mitochondria are contacted with the plurality of nucleic acid molecules and the positively charged substance for at least 5 minutes, e.g., at least 10 minutes, 20 minutes, or 30 minutes at room temperature; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0205] The present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance, wherein the mitochondria are contacted with the at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and the positively charged substance in the dark; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0206] The present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance, wherein the mitochondria are contacted with the plurality of payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) and the positively charged substance in the dark at room temperature for at least 5 minutes, e.g., at least 10 minutes, 20 minutes, or 30 minutes; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0207] Preferably, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a positively charged substance, wherein the mitochondria are contacted with the at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) and the positively charged substance in the dark at room temperature for 30 minutes; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0208] As described above, the present invention provides mitochondria comprising one or more payloads, such as nucleic acid molecules, polypeptides, drugs, or combinations thereof, attached to the outer membrane of the mitochondria. The nucleic acid molecules are preferably DNA or RNA. Accordingly, the present invention also provides a method for attaching a DNA molecule to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one DNA molecule in the presence of a positively charged substance; c) attaching at least one DNA molecule to the mitochondria via a positively charged substance.

[0209] The present invention also provides a method for attaching an RNA molecule to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one RNA molecule in the presence of a positively charged substance; c) attaching at least one RNA molecule to the mitochondria via a positively charged substance.

[0210] As described above, the present invention provides a method for attaching nucleic acid molecules to mitochondria via a positively charged substance (preferably a polycationic substance). Therefore, the present invention provides a method for attaching nucleic acid molecules to the outer membrane of mitochondria, the method comprising the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one nucleic acid molecule in the presence of a positively charged substance; c) attaching at least one nucleic acid molecule to the mitochondria via a polycationic substance.

[0211] In the sense of the present invention, polycationic material can be linear or branched polycationic polymer.Linear or branched polycationic polymer can be electrostatically connected to payload, such as nucleic acid molecules, polypeptide, drug or its combination, such as DNA or RNA molecule.The present invention is not particularly limited to any polycationic polymer.In general, any polycationic polymer that helps payload (such as nucleic acid molecules, polypeptide, drug or its combination) to be attached to mitochondria can be used to help form the delivery complex.However, linear or branched polycationic polymer is preferably 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 derivatives, diethylaminoethyl (DEAE)-dextran, poly (N-alkyl-4-vinyl pyridinium), poly (amidoamine), cationic gelatin, cationic cellulose or its combination. Thus, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of a linear or branched polycationic polymer; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via the polycationic substance.

[0212] The present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., 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 (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) contained in a plurality of nucleic acids; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0213] The present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., 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, histidylated polylysine, polyornithine, polyarginine, high mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimers, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), a polyallylamine derivative, diethylaminoethyl (DEAE)-dextran, poly(N-alkyl-4-vinylpyridinium), poly(amidoamine), cationic gelatin, cationic cellulose, or a combination thereof, optionally wherein the polycationic polymer is electrostatically linked to at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof); c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0214] As described above, the negative surface charge distribution of mitochondria can also be used to electrostatically attach one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) to the outer membrane of mitochondria via positively charged nanoparticles or positively charged particles. Payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) can be attached to the surface of positively charged nanoparticles / particles or can be encapsulated therein. The present invention is not particularly limited to any nanoparticle or particle. In general, any positively charged nanoparticle / particle that helps attach a payload (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) to mitochondria, thereby helping to form a delivery complex, can be used. However, the positively charged nanoparticles / particles are preferably lipid nanoparticles / particles, dendrimer nanoparticles / particles, micellar 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 oxide nanoparticles / particles, graphene or graphene oxide nanoparticles. Accordingly, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of positively charged nanoparticles; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a positively charged substance.

[0215] The present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of positively charged nanoparticles; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via the positively charged nanoparticles.

[0216] In the methods of the present invention, the method may include contacting mitochondria with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of positively charged nanoparticles, wherein the method further includes a) attaching at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the surface of a positively charged nanoparticle; or b) encapsulating at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) within the positively charged nanoparticle.

[0217] Thus, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of positively charged nanoparticles, wherein prior to step (b), the further steps of: a') attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the surface of the positively charged nanoparticle; or b') encapsulating at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) within the positively charged nanoparticle; c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via the positively charged nanoparticles.

[0218] The present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in the presence of positively charged nanoparticles, wherein prior to step (b), the further steps of: a') attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the surface of the positively charged nanoparticle; or b') encapsulating at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) within the positively charged nanoparticles, c) attaching at least one payload (e.g., a nucleic acid molecule, a peptide, a drug, or a combination thereof) to the mitochondria via positively charged nanoparticles; 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.

[0219] In another aspect, the present invention provides methods for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria. As described above, the present invention provides payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) that can be covalently attached to the outer membrane of a mitochondria directly or indirectly (e.g., via an intermediate entity). Exemplary intermediate entities include activated esters, such as N-hydroxysuccinimide (NHS) esters. Therefore, the present invention provides methods for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the following steps: a) providing a mitochondrial preparation; b) providing a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) that has been modified to contain an activated ester; and c) attaching the payload (eg, nucleic acid molecule, polypeptide, drug, or combination thereof) provided in step (b) to an amine contained in the polypeptide in the outer mitochondrial membrane.

[0220] Preferably, the present invention provides a method for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) providing a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) that has been modified to comprise an N-hydroxysuccinimide (NHS) ester; and c) attaching the payload (eg, nucleic acid molecule, polypeptide, drug, or combination thereof) provided in step (b) to an amine contained in the polypeptide in the outer mitochondrial membrane.

[0221] In another aspect, the present invention provides a method for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) providing a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) that has been modified to include a chemical group; and c) attaching the payload (eg, nucleic acid molecule, polypeptide, drug, or combination thereof) provided in step (b) to an amine contained in the polypeptide in the mitochondrial outer membrane via a chemical group.

[0222] The present invention provides a method for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) providing a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) that has been modified to comprise a chemical group selected from the group consisting of isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imido ester, carbodiimide, anhydride, and fluorophenyl ester; and c) attaching the nucleic acid molecule provided in step (b) to an amine contained in the polypeptide in the outer mitochondrial membrane via a chemical group.

[0223] As described above, a payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) can be attached to or encapsulated within a nanoparticle, which can then be covalently attached to a mitochondria via a covalent bond (eg, an amide bond).

[0224] The present invention provides a method for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) encapsulating a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a nanoparticle, wherein the surface of the nanoparticle comprises an activated ester; and c) attaching the nanoparticles provided in step (b) to amines contained in the polypeptide in the outer mitochondrial membrane.

[0225] Preferably, the present invention provides a method for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) encapsulating a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a nanoparticle, wherein the surface of the nanoparticle comprises an N-hydroxysuccinimide (NHS) ester; and c) attaching the nanoparticles provided in step (b) to amines contained in the polypeptide in the outer mitochondrial membrane.

[0226] 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. In another aspect, the present invention provides a method for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the following steps: a) providing a mitochondrial preparation; b) encapsulating a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a nanoparticle, wherein the surface of the nanoparticle comprises chemical groups; and c) attaching the nanoparticles provided in step (b) to amines contained in the polypeptide in the outer mitochondrial membrane via chemical groups.

[0227] The present invention provides a method for covalently attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) encapsulating a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) in a nanoparticle, wherein the surface of the nanoparticle comprises a chemical group selected from the group consisting of isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, carbodiimide, anhydride, and fluorophenyl ester; and c) attaching the nanoparticles provided in step (b) to amines contained in the polypeptide in the outer mitochondrial membrane via chemical groups.

[0228] In a further embodiment, the present invention provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, wherein the nucleic acid is linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane. Thus, the present invention provides a method for attaching at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) comprising the antigen in its outer membrane with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) linked to an antibody; and c) attaching at least one payload (e.g., 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 mitochondrial membrane.

[0229] In some embodiments, the present invention provides a method for attaching one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria comprising the antigen in their outer membrane provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) encapsulated in a nanoparticle, wherein the nanoparticle is covalently linked to an antibody; and c) attaching at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer mitochondrial membrane.

[0230] In some embodiments, the present invention provides a method for attaching one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) comprising the antigen in its outer membrane with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), wherein the nucleic acid molecule is electrostatically linked to a modified antibody, wherein the modified antibody has one or more positive charges; and c) attaching at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer mitochondrial membrane.

[0231] In some embodiments, the present invention provides a method for attaching one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) comprising the antigen in its outer membrane with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), wherein the nucleic acid molecule is covalently linked to biotin, wherein the biotin is linked to an avidin-binding antibody; and c) attaching at least one payload (e.g., 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 mitochondrial membrane.

[0232] In some embodiments, the present invention provides a method for attaching one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) comprising the antigen in its outer membrane with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), wherein the nucleic acid molecule is covalently linked to an activated ester, wherein the activated ester is linked to the antibody through an amide bond; and c) attaching at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer mitochondrial membrane.

[0233] In some embodiments, the present invention provides a method for attaching one or more nucleic acid molecules to the outer membrane of a mitochondria, wherein the one or more nucleic acid molecules are single-stranded nucleic acid molecules (ssDNA or ssRNA), the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) and comprising the antigen in its outer membrane with at least one single-stranded nucleic acid molecule, wherein the single-stranded nucleic acid molecule can hybridize to one or more complementary single-stranded nucleic acid molecules attached to or to the antibody; and c) attaching at least one single-stranded nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer mitochondrial membrane.

[0234] In some embodiments, the present invention provides a method for attaching one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) comprising the antigen in its outer membrane with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), wherein the one or more nucleic acid molecules are encapsulated in a nanoparticle, wherein the nanoparticle is electrostatically linked to a modified antibody, wherein the modified antibody has one or more positive charges; and c) attaching at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer mitochondrial membrane.

[0235] In some embodiments, the present invention provides a method for attaching one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) comprising the antigen in its outer membrane with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), wherein the one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) are encapsulated in a nanoparticle, wherein the nanoparticle is electrostatically linked to a modified antibody, wherein the modified antibody has one or more negative charges; and c) attaching at least one payload (e.g., 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 mitochondrial membrane.

[0236] In some embodiments, the present invention provides a method for attaching one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or a combination thereof) to the outer membrane of a mitochondria, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) comprising an antigen in its outer membrane with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), wherein the one or more nucleic acid molecules are encapsulated in nanoparticles, wherein the nanoparticles are covalently linked to biotin, wherein the biotin is linked to an avidin-binding antibody; and c) attaching at least one payload (e.g., 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 mitochondrial membrane. In some embodiments, the present invention provides a method for attaching one or more payloads (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of the mitochondria, the method comprising the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) comprising the antigen in its outer membrane with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof), wherein the one or more nucleic acid molecules are encapsulated in a nanoparticle, wherein the nanoparticle is covalently linked to an activated ester, wherein the activated ester is linked to the antibody through an amide bond; and c) attaching at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer mitochondrial membrane.

[0237] The antigen contained in the mitochondrial outer membrane can be any antigen capable of binding to an antibody linked to a payload, such as a nucleic acid molecule, a polypeptide, a drug, or a combination thereof. In a preferred embodiment, the antigen is OPA1, TOM70, TOMM20, Mitofusin 1, Mitofusin 2, or VDAC1.

[0238] The present invention also provides a method for attaching a payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the outer membrane of a mitochondria via a mitochondrial-targeting small molecule, the method comprising the steps of: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one payload (e.g., a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) linked to a mitochondrial-targeting small molecule; and c) attaching at least one payload (eg, a nucleic acid molecule, a polypeptide, a drug, or a combination thereof) to the mitochondria via a mitochondria-targeted small molecule.

[0239] The mitochondrial targeting small molecule can be any mitochondrial targeting small molecule. Preferably, the mitochondrial targeting small molecule is selected from the group consisting of triphenylphosphine (TPP), dequalinium chloride (DQA), E-4-(1H-indol-3-ylvinyl)-N-methylpyridinium iodide (F16), rhodamine 19, biguanide, and guanidine.

[0240] In the sense of the present invention, nucleic acid molecules do not necessarily refer to identical nucleic acid molecules, i.e., molecules having the same sequence. Although it is understood that in some aspects nucleic acid molecules with identical sequences are delivered, in other aspects of the present invention, at least two or more different nucleic acid molecules can be attached to the outer membrane of the mitochondria.

[0241] In some embodiments, the method of the present invention further comprises connecting and / or encapsulating mitochondria comprising one or more payloads (e.g., nucleic acid molecules, polypeptides, drugs, or combinations thereof) with a protective layer. The mitochondria comprising one or more nucleic acid molecules can be any mitochondria as described above and any protective layer as described above. The method of connecting and / or encapsulating mitochondria in a protective layer preferably comprises contacting the mitochondria with a component forming a protective layer (e.g., a protective polymer or protective lipid layer as described above). In a preferred embodiment, the present invention is a method for attaching nucleic acid molecules to the outer membrane of mitochondria, wherein the method comprises the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one nucleic acid molecule in the presence of a positively charged substance; c) attaching at least one nucleic acid molecule to the mitochondria via a positively charged species; and d) connecting and / or encapsulating the mitochondria provided in steps (a) to (c) with a protective layer.

[0242] In some embodiments, the protective layer is a protective polymer. The protective polymer is as described above.

[0243] In some embodiments of the methods of the present invention, the protective polymer is a linear or branched cationic polymer, optionally wherein the linear or branched cationic polymer is electrostatically attached 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.

[0244] In some embodiments of the methods of the present invention, the protective polymer is a linear or branched cationic block copolymer, optionally wherein the linear or branched cationic block copolymer is electrostatically attached 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-polypropylene. alkyleneimine, 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.

[0245] In some embodiments of the methods of the present invention, the protective polymer is a linear or branched cationic graft (g) copolymer, optionally wherein the linear or branched cationic graft (g) copolymer is electrostatically attached 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-polypropylene alkyleneimine, 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.

[0246] In some embodiments of the methods of the present invention, the protective polymer is a linear or branched PEGylated (PEG) cationic polymer, optionally wherein the linear or branched PEGylated (PEG) cationic polymer is electrostatically attached 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.

[0247] In some embodiments of the methods of the present invention, the protective layer is a lipid formulation, optionally wherein the lipid formulation is a cationic lipid formulation, further optionally wherein the cationic lipid formulation is electrostatically attached 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-dilinoleoyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleoylmethyl-4-dimethylaminobutyrate), DODMA (1,2-dioleoyloxy-3-dimethylaminopropane), DOGS (dioctadecylamidoglycylspermine), DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane chloride), DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane chloride), UGG (unsaturated guanidinoglycoside), DOPE (1,2-dioleoyl-sn-glycerophosphoethanolamine), lipofectamine or a combination thereof. In a further embodiment, the lipid formulation further comprises another lipid, preferably wherein the lipid is cholesterol, substituted or unsubstituted cholesterol, a cholesterol derivative such as a hydroxylated cholesterol derivative (e.g., hydroxycholesterol), 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 (dimethyldioctadecyl ammonium), 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, bis(monooleoylglycerol)phosphate, or a combination thereof.

[0248] In some embodiments of the methods of the present invention, the protective polymer is a zwitterionic protective polymer, optionally wherein the zwitterionic protective polymer is electrostatically attached to one or more nucleic acid molecules. Preferably, the zwitterionic protective polymer is selected from the group consisting of poly(2-methacryloyloxyethylphosphorylcholine) (PMPC), polyethyleneimine-g-poly(2-methacryloyloxyethylphosphorylcholine) (PEI-g-PMPC), co-assembly of cationic (carboxyl functionalized) and anionic (amino functionalized) copolyesters based on poly(ε-caprolactone)-block-poly(butylene fumarate)-block-poly(ε-caprolactone) (PCL-b-PBF-b-PCL), poly(lactic-co-glycolic acid) (PLGA)-PCB block copolymer (PLGA-b-PCB).

[0249] In some embodiments of the methods of the present invention, the protective layer is connected to a targeting moiety, and optionally the protective layer connected to the targeting moiety is electrostatically connected to one or more nucleic acid molecules. The connection and targeting and targeting moiety are as described above. Preferably, the targeting moiety is an antibody or a carbohydrate molecule.

[0250] In some embodiments of the methods of the invention, the protective layer is attached to the antibody, optionally wherein the protective layer is attached to the antibody, wherein the antibody is electrostatically attached to one or more nucleic acid molecules.

[0251] In some embodiments of the methods of the present invention, the protective layer is attached to a carbohydrate, wherein the protective layer attached to the carbohydrate is optionally electrostatically attached to one or more nucleic acid molecules.

[0252] In a preferred embodiment, the invention is a method wherein the mitochondria comprise positively charged species, wherein the positively charged species is a polycationic polymer, and wherein the weight ratio of the polycationic polymer to the protective layer is between about 1:2.

[0253] 101. The method according to claim 100, wherein 50 μg to 200 μg of mitochondria are contacted with 0.1 to 50 pmol of the nucleic acid molecule and 0.2 to 10 μg of the protective layer. -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 a particle counter). The concentration of the protective polymer preparation used to prepare the mitochondria of the present invention is 1 mg / mL. The amount of protective polymer is between 0.1 mg and 10 mg. The concentration of the nanoparticle preparation used to prepare the mitochondria of the present invention is 1 mg / mL. The amount of protective polymer is between 0.1 mg and 10 mg.

[0254] In further embodiments, the methods of the present invention may involve a centrifugation step. In the context of the present invention, the centrifugation step can remove components that make up the mitochondrial delivery vehicle, such as unattached payloads, such as nucleic acid molecules, positively charged substances, or protective layers, to facilitate the formation of the delivery vehicle. As known to those skilled in the art, the centrifugation step can be performed after any step in which it is necessary to remove excess components of the delivery vehicle (e.g., excess payload, excess positively charged substances, excess protective layers).

[0255] Therefore, the method of the present invention may comprise the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with at least one nucleic acid molecule in the presence of a positively charged substance; c) attaching at least one nucleic acid molecule to the mitochondria via a positively charged substance; d) centrifuging the mitochondria provided in step (c); and e) optionally connecting and / or encapsulating the mitochondria provided in step (d) in a protective layer.

[0256] In a further embodiment, the method of the present invention may comprise the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) with a positively charged substance; 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) attaching at least one nucleic acid molecule to the mitochondria via a positively charged substance; f) optionally centrifuging the mitochondria provided in step (d); and g) optionally connecting and / or encapsulating the mitochondria provided in step (d) in a protective layer.

[0257] In a further embodiment, the method of the present invention may comprise the following steps: a) providing a mitochondrial preparation; b) contacting at least one nucleic acid molecule with a positively charged substance to form a positively charged complex; c) contacting the mitochondria of (a) with the positively charged complex of (b); d) attaching at least one nucleic acid molecule to the mitochondria via a positively charged substance; f) optionally centrifuging the mitochondria provided in step (d); and g) optionally connecting and / or encapsulating the mitochondria provided in step (d) in a protective layer.

[0258] In some embodiments, the methods of the present invention may include the following steps: a) providing a mitochondrial preparation; b) providing a nucleic acid molecule that has been modified to comprise an activated ester; and c) attaching the nucleic acid molecule provided in step (b) to an amine contained in a polypeptide in the outer mitochondrial membrane; d) centrifuging the mitochondria provided in step (c); and e) optionally connecting and / or encapsulating the mitochondria provided in step (d) in a protective layer.

[0259] In some embodiments, the methods of the present invention may include the following steps: a) providing a mitochondrial preparation; b) encapsulating the nucleic acid molecule in a nanoparticle, wherein the surface of the nanoparticle comprises a chemical group capable of covalently attaching to a polypeptide in the outer mitochondrial membrane; c) attaching 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); and e) optionally connecting and / or encapsulating the mitochondria provided in step (d) in a protective layer.

[0260] In some embodiments, the methods of the present invention may include the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) and comprising the antigen in its outer membrane with at least one nucleic acid molecule linked to an antibody; c) attaching at least one nucleic acid molecule to the mitochondria via an antibody, wherein the antibody specifically binds to an antigen contained in the outer mitochondrial membrane; d) centrifuging the mitochondria provided in step (c); and e) optionally connecting and / or encapsulating the mitochondria provided in step (d) in a protective layer.

[0261] In some embodiments, the methods of the present invention may include the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria provided in step (a) and comprising the antigen in its outer membrane with at least one nucleic acid molecule linked to a small molecule targeting mitochondria; c) attaching at least one nucleic acid molecule to mitochondria via a mitochondrial-targeting small molecule; d) centrifuging the mitochondria provided in step (c); and e) optionally connecting and / or encapsulating the mitochondria provided in step (d) in a protective layer.

[0262] The present invention also provides a mitochondria comprising one or more polypeptides attached to the outer membrane of the mitochondria. Thus, the products, methods, devices and uses of the present invention can be achieved by attaching polypeptides to the mitochondria instead of or in combination with nucleic acid molecules. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length (which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids), as well as polypeptides with modified peptide backbones. In some embodiments, the polypeptides of the present invention comprise 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 contain non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents may be added to proteins by the cells that produce the proteins and vary with the cell type. Certain proteins are defined herein based on their amino acid backbone structure. The term "peptide" as used herein refers to a polypeptide having 2-100 amino acid monomers.

[0263] The present invention is not particularly limited to any polypeptide. Any target polypeptide can be used as a payload attached to the outer mitochondrial membrane. Therefore, the present invention provides polypeptides attached to the outer mitochondrial membrane that can be used, for example, for treatment and / or gene editing. In general, any target polypeptide can be attached to the outer mitochondrial membrane. In the sense of the present invention, mitochondria can be positively charged or negatively charged. In the sense of the present invention, polypeptides can be positively charged or negatively charged. Positively charged polypeptides can be attached to negatively charged mitochondria or entities. Negatively charged polypeptides can be attached to positively charged mitochondria or entities. As long as the mitochondria and the polypeptide have opposite charges or do not have the same charge at their respective pH (e.g., at physiological pH (about 7.2)) in the environment where the polypeptide contacts the mitochondria or entity, any of the above combinations can result in successful attachment through electrostatic interactions. In a preferred embodiment, the positively charged polypeptide includes lysine, arginine or histidine. In a further embodiment, the negatively charged polypeptide includes aspartic acid or glutamic acid.

[0264] Thus, the present invention provides mitochondria comprising one or more polypeptides attached to the outer membrane of the mitochondria, wherein the one or more polypeptides: a) electrostatically attached to the outer membrane of the mitochondria; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) Attached to small molecules that target mitochondria.

[0265] In some embodiments, the polypeptide is negatively charged. In other embodiments, the polypeptide is positively charged.

[0266] The present invention provides mitochondrial-polypeptide complexes that can be used to deliver polypeptides to cells, tissues, or organs. The present invention also provides methods for attaching polypeptides to mitochondria, wherein the polypeptides can be charged. One or more polypeptides can be electrostatically attached to the outer membrane of a mitochondria. One or more polypeptides can be electrostatically attached to the outer membrane of a mitochondria via a positively charged substance. One or more positively charged polypeptides can be electrostatically attached to the outer membrane of a negatively charged mitochondria. One or more negatively charged polypeptides can be electrostatically attached to the outer membrane of a mitochondria via a positively charged substance.

[0267] Mitochondria can electrostatically interact with polypeptides, thereby forming a complex comprising mitochondria and one or more polypeptides. Thus, electrostatic interactions can be used to attach positively charged entities to negatively charged entities. Mitochondria, in the sense of the present invention, can be positively or negatively charged. Polypeptides, in the sense of the present invention, can be positively or negatively charged. Any of the above situations can lead to successful attachment via electrostatic interactions, as long as the mitochondria and polypeptide are oppositely charged or do not have the same charge. Mitochondria have a negatively charged surface to which positively charged polypeptides can electrostatically attach. In one aspect, the present invention provides mitochondria comprising one or more polypeptides attached to the outer mitochondrial membrane. The polypeptide can be electrostatically attached to the outer membrane. The polypeptide can be a charged polypeptide. The polypeptide can be a positively charged polypeptide.

[0268] Mitochondria have a negatively charged surface that can be functionalized with cationic molecules to convert the surface charge of the mitochondrial outer membrane to a positive value (i.e., partially or fully positive). The positively charged mitochondria can then bind to negatively charged polypeptides. In one aspect, the present invention provides mitochondria comprising one or more polypeptides attached to the outer membrane of the mitochondria, wherein the one or more polypeptides are electrostatically attached to the outer membrane of the mitochondria via a positively charged substance. The present invention provides mitochondria comprising one or more polypeptides attached to the outer membrane of the mitochondria, wherein the one or more polypeptides are electrostatically attached to the outer membrane of the mitochondria via a positively charged substance, and wherein the polypeptide is negatively charged.

[0269] The polypeptides of the present invention are preferably positively or negatively charged. As used herein, "charge" or "charged" refers to the total charge 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 knows how to determine the net charge of a given polypeptide at a given pH, for example, at physiological pH (about 7.2). Within the meaning of the present invention, the net charge of the polypeptides of the present invention is preferably negative or positive when in contact with mitochondria. Thus, one or more polypeptides can be electrostatically attached to the outer membrane of the mitochondria via a positively charged substance. One or more polypeptides can be electrostatically attached to the outer membrane of the mitochondria via a polycationic substance, wherein the polycationic substance is a linear or branched polycationic polymer. The one or more polypeptides can be electrostatically attached to the outer membrane of the mitochondria via a linear or branched polycationic polymer, wherein the polycationic material is polylysine, histidyl-lysine, polyornithine, polyarginine, high mobility group proteins (HMG) 1 and 17, modified chitosan, cationized human serum albumin, polyethyleneimine (PEI), polypropyleneimine (PPI), cationic dendrimers, 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.

[0270] In the sense of the present invention, the negative surface charge characteristic of mitochondria can also be used to electrostatically attach 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 attached to the negative surface of mitochondria.

[0271] 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 polypeptides can be attached to the surface of positively charged nanoparticles or positively charged particles, or be encapsulated by positively charged nanoparticles or positively charged particles. Thus, one or more polypeptides can be electrostatically attached to the outer membrane of mitochondria via positively charged nanoparticles, wherein the one or more polypeptides are attached to the surface of positively charged nanoparticles or are encapsulated in positively charged nanoparticles. One or more polypeptides can be electrostatically attached to the outer membrane of mitochondria via positively charged particles, wherein the one or more polypeptides are attached to the surface of positively charged particles or are encapsulated in positively charged particles.

[0272] In general, the present invention is not limited to any particular nanoparticle or particles for attachment to mitochondria and attachment of polypeptides or encapsulation of polypeptides. Thus, one or more polypeptides can be attached to the surface of or encapsulated within 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.

[0273] In addition, one or more polypeptides can be attached to the surface of lipid particles, dendrimer particles, micellar particles, protein particles, liposomes, nonporous 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 oxide particles, graphene, or graphene oxide particles.

[0274] Those skilled in the art will appreciate that the above-mentioned electrostatic attachment method can be applied to all products, methods, devices or uses described herein.

[0275] The mitochondria of the present invention are particularly useful because they can be stored for long periods of time without decomposing, i.e., remain stable. Thus, the present invention provides mitochondria comprising one or more polypeptides attached to the outer membrane of the mitochondria, wherein the one or more polypeptides: a) electrostatically attached to the outer membrane of the mitochondria, optionally via a positively charged species; or b) covalently attached to the outer membrane of the mitochondria; or c) linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane; or d) linked to small molecules targeting mitochondria, wherein the mitochondria are stored in binding buffer at -80°C. The mitochondria of the present invention can be stored in binding buffer at -80°C for at least 2 months, 1 month, 3 weeks, 2 weeks, 1 week, or at least 5 days without disintegration. Mitochondria comprising one or more polypeptides attached to the outer membrane can be stored in binding buffer to maintain high colloidal stability (e.g., no aggregation or disintegration). Mitochondria comprising one or more polypeptides attached to the outer membrane are stored in binding buffer at low temperature (e.g., -80°C) in the dark for up to four months after complex formation.

[0276] The polypeptides of the present invention can be functionalized with targeting molecules (e.g., small targeting molecules, targeting aptamers, targeting peptides, carbohydrates, sugars, and targeting antibodies), drugs, reporter molecules / nanoparticles (e.g., fluorescent molecules, metal nanoparticles, magnetic nanoparticles, etc.), or contact agents.

[0277] The polypeptides of the present invention can be formulated into nanoparticles, cationic lipid nanoformulations, block copolymers, cationic lipids or cationic polymers.

[0278] In the sense of the present invention, polypeptides can also be covalently attached to the outer membrane of mitochondria. Covalent bonds or covalent connections or covalent interactions are formed by chemical bonds involving shared electron pairs between atoms. Polypeptides can be attached to mitochondria through peptide bonds (e.g., amide bonds). Mitochondria with amino groups of mitochondrial membrane-associated proteins of the present invention can be covalently attached to nanoparticles functionalized with N-hydroxysuccinimide ester (NHS), oligonucleotides modified with NHS, or molecules modified with NHS to form covalently bound ligands and more stable conjugates.

[0279] In one embodiment, one or more polypeptides can be covalently attached to the outer membrane of mitochondria. One or more polypeptides can be covalently attached to the polypeptide in the outer membrane of mitochondria by amide bonds. One or more polypeptides can be covalently attached to the polypeptide in the outer membrane of mitochondria by amide bonds, wherein one or more polypeptides have been modified to form amide bonds with the amine functional group contained in the polypeptide in the outer membrane of mitochondria. Polypeptide can also be attached to mitochondria by covalently attaching the nanoparticle comprising polypeptide to mitochondria. Therefore, one or more polypeptides can be covalently attached to the polypeptide in the outer membrane of mitochondria by amide bonds, wherein one or more polypeptides are encapsulated in nanoparticles (such as lipid nanoparticles), and wherein nanoparticles comprise the functional group that allows nanoparticles to be covalently attached to the second polypeptide in the outer membrane of mitochondria. One or more polypeptides can be covalently attached to N-hydroxysuccinimide ester. One or more polypeptides can be covalently attached to N-hydroxysuccinimide ester, wherein N-hydroxysuccinimide ester is conducive to polypeptide attachment to the amine contained in the second polypeptide in the outer membrane of mitochondria. One or more polypeptides are encapsulated in nanoparticles comprising N-hydroxysuccinimide ester, wherein the N-hydroxysuccinimide ester facilitates attachment of the polypeptides to amines contained in a second polypeptide in the outer membrane of the mitochondria via the nanoparticles comprising the N-hydroxysuccinimide ester.

[0280] In the context of the present invention, polypeptides can also be linked to antibodies that specifically bind to antigens contained in the outer mitochondrial membrane. Such antibodies containing polypeptides bind to mitochondria, thereby facilitating the formation of a delivery platform. The present invention is not limited to any particular antigen or antibody; in general, the present invention can be practiced using antibodies that specifically bind to any antigen contained in the outer mitochondrial membrane, thereby facilitating the formation of a mitochondrial-polypeptide complex.

[0281] In general, 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, and VDAC1.

[0282] Preferably, the one or more polypeptides can be linked to any antibody that specifically binds to an antigen contained in the outer mitochondrial membrane. Thus, the one or more polypeptides can be linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane, wherein the preferred antigen is any one of OPA1, TOM70, TOMM20, Mitofusin 1, Mitofusin 2, or VDAC1.

[0283] The polypeptide can be covalently linked to the antibody to form a polypeptide-antibody complex that can bind to an antigen of the mitochondria. Thus, the polypeptide can be covalently linked to the antibody to form a polypeptide-antibody complex that can bind to an antigen contained in the outer mitochondrial membrane.

[0284] The polypeptide can be electrostatically linked to a modified antibody, such as an antibody comprising a positive or negative charge, to form a polypeptide-antibody complex that can bind to an antigen contained in the mitochondrial outer membrane. Thus, the polypeptide can be electrostatically linked to a modified antibody, such as an antibody comprising a positive or negative charge, to form a polypeptide-antibody complex that can bind to an antigen contained in the mitochondrial outer membrane.

[0285] The antibody that specifically binds the antigen comprised in the outer membrane of mitochondria can be used for attachment of the nanoparticle that comprises polypeptide, such as lipid nanoparticle, thereby promotes polypeptide to be attached to mitochondria.Therefore, one or more polypeptides can be connected to the antibody that specifically binds the antigen comprised in the outer membrane of mitochondria, wherein one or more polypeptides are encapsulated in nanoparticle (such as lipid nanoparticle), and wherein nanoparticle is covalently connected to antibody. One or more polypeptides can be connected to the antibody that specifically binds the antigen comprised in the outer membrane of mitochondria, wherein nanoparticle is electrostatically connected to antibody, wherein antibody is the antibody of modification, and wherein the antibody of modification has one or more positive charges. In addition, one or more polypeptides can be connected to the antibody that specifically binds the antigen comprised in the outer membrane of mitochondria, wherein nanoparticle is electrostatically connected to antibody, wherein antibody is the antibody of modification, and wherein the antibody of modification has one or more negative charges.

[0286] The nanoparticles comprising nucleic acid molecules are not particularly limited and can be any nanoparticles as described above. In some embodiments, the nanoparticles are substantially neutral (i.e., net neutral) nanoparticles. In some embodiments, the nanoparticles are charged nanoparticles, such as positively charged or negatively charged nanoparticles. In some embodiments, the nanoparticles are charged nanoparticles, such as nanoparticles with a positive "zeta potential" or positive "surface charge" or nanoparticles with a negative "zeta potential" or negative "surface charge."

[0287] The polypeptide can also be electrostatically linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane. In an environment where the antibody and polypeptide are in contact, the opposite charges of the antibody and polypeptide promote binding. Thus, a positively charged polypeptide can be electrostatically linked to a negatively charged antibody, and vice versa. Thus, one or more polypeptides can be linked to an antibody that specifically binds to an antigen contained in the outer mitochondrial membrane, wherein the one or more polypeptides are electrostatically linked to a modified antibody, wherein the modified antibody has one or more positive or negative charges.

[0288] The polypeptide can also be connected to an entity, which is then connected to an antibody. Such an entity can be biotin, which is connected to an avidin-binding antibody. Thus, one or more polypeptides can be connected to an antibody that specifically binds to the antigen contained in the outer mitochondrial membrane, wherein the one or more polypeptides are covalently bound to biotin, wherein the biotin is connected to the antibody, wherein the antibody is an avidin-binding antibody. In addition, the polypeptide can also be connected to an entity, which is then attached to or encapsulated in a lipid nanoparticle, and the entity is connected to the antibody. Thus, one or more polypeptides can be connected to an antibody that specifically binds to the antigen contained in the outer mitochondrial membrane, wherein the one or more polypeptides are attached to or encapsulated in a nanoparticle, wherein the nanoparticle is covalently bound to biotin, wherein the biotin is connected to the antibody, wherein the antibody is an avidin-binding antibody.

[0289] Polypeptide can also be connected to entity, and then entity is connected to antibody.Such entity can be activated ester, and it is connected to antibody.Therefore, one or more polypeptides can be connected to the antibody of the antigen comprised in specific binding mitochondrial outer membrane, wherein one or more polypeptides are covalently connected to activated ester, and wherein activated ester is connected to antibody by amide bond.In addition, polypeptide can also be connected to entity, and then when this entity is attached to or is encapsulated in nanoparticle (such as lipid nanoparticle), this entity is connected to antibody.Therefore, one or more polypeptides can be connected to the antibody of the antigen comprised in specific binding mitochondrial outer membrane, wherein nanoparticle is covalently connected to activated ester, and wherein activated ester is connected to antibody by amide bond.

[0290] In the sense of the present invention, a "modified antibody" also means that the antibody is modified to have one or more positive or negative charges, for example to attach a negatively or positively charged polypeptide.

[0291] Polypeptide can also be attached to or be encapsulated in positively charged nanoparticle (for example polycationic lipid nanoparticle).The positively charged nanoparticle comprising polypeptide can be covalently linked to the antibody of the antigen comprised in the specific binding mitochondrial outer membrane.The positively charged nanoparticle comprising polypeptide can comprise a phospholipid with a reactive group, and this reactive group can be covalently linked to the antibody of the antigen comprised in the specific binding mitochondrial outer membrane.Polypeptide can also be attached to or be encapsulated in negatively charged nanoparticle (for example polyanionic lipid nanoparticle).The negatively charged nanoparticle comprising polypeptide can be covalently linked to the antibody of the antigen comprised in the specific b...

Claims

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

2. The mitochondria of claim 1, wherein the payload is one or more of: i) nucleic acid molecules; ii) polypeptide; iii) medicines; or iv) a combination of one or more of (i) to (iii).

3. The mitochondria of claim 1 or 2, wherein the payload is charged.

4. The mitochondria of any one of claims 1 to 3, wherein the payload has a net charge that is the same as the net charge of the mitochondria.

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

6. The mitochondria according to claim 5, wherein the positively charged substance is a polycationic substance.

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

8. The mitochondria of claim 7, 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 dendrimers, 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.

9. The mitochondria of claim 5, wherein the positively charged substance is a positively charged nanoparticle.

10. The mitochondria of claim 5, wherein the positively charged species is a positively charged particle.

11. The mitochondria of claim 9, wherein the one or more nucleic acid molecules are attached to the surface of the positively charged nanoparticle or encapsulated in the positively charged nanoparticle.

12. The mitochondria of claim 10, wherein the one or more nucleic acid molecules are attached to the surface of the positively charged particles or are encapsulated in the positively charged particles.

13. The mitochondria according to any one of claims 9 to 12, wherein the positively charged nanoparticles and / or particles are lipid nanoparticles / particles, dendrimer nanoparticles / particles, micellar nanoparticles / particles, protein nanoparticles / particles, liposomes, non-porous silica nanoparticles / particles, mesoporous silica nanoparticles / particles, silicon nanoparticles / particles, gold nanoparticles / particles, gold nanowires, silver nanoparticles / particles, platinum nanoparticles / particles, palladium nanoparticles / particles, titanium dioxide nanoparticles / particles, carbon nanotubes, 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 oxide nanoparticles / particles, graphene or graphene oxide nanoparticles / particles.

14. The mitochondria of any one of claims 1 to 3, wherein the payload has a net charge different from the net charge of the mitochondria.

15. The mitochondria of claim 14, wherein the payload and the mitochondria are attached via a zwitterionic species.

16. The mitochondria of claim 14, wherein the payload is uncharged, and wherein the payload is attached to a positively charged species.

17. The mitochondria according to claim 16, wherein the positively charged species is as defined in any one of claims 6 to 13.

18. The mitochondria of claim 2, wherein the one or more nucleic acid molecules are electrostatically attached to an antibody, optionally wherein the antibody is a modified antibody, optionally wherein the modified antibody has one or more positive charges.

19. The mitochondria of claim 2, wherein the one or more nucleic acid molecules are encapsulated in a nanoparticle, wherein the nanoparticle is electrostatically linked to an antibody, optionally wherein the antibody is a modified antibody, optionally wherein the modified antibody has one or more positive charges.

20. The mitochondria according to claim 18 or 19, wherein the antibody specifically binds to an antigen contained in the outer membrane of the mitochondria, wherein the antigen is OPA1, TOM70, TOMM20, Mitofusin 1, Mitofusin 2 or VDAC1.

21. The mitochondria according to any one of claims 1 to 20, wherein the mitochondria are attached to and / or encased in a protective layer.

22. The mitochondria of claim 21, wherein the protective layer is a protective polymer.

23. The mitochondria of claim 22, wherein the protective polymer is a linear or branched cationic polymer, optionally wherein the linear or branched cationic polymer is electrostatically linked to the one or more payloads.

24. The mitochondria of claim 22, wherein the protective polymer is a linear or branched cationic block copolymer, optionally wherein the linear or branched cationic block copolymer is electrostatically linked to the one or more payloads.

25. The mitochondria of claim 22, wherein the protective polymer is a cationic graft (g) copolymer, optionally wherein the cationic graft (g) copolymer is electrostatically attached to the one or more payloads.

26. The mitochondria of claim 22, wherein the protective polymer is a linear or branched PEGylated (PEG) cationic polymer, optionally wherein the linear or branched PEGylated (PEG) cationic polymer is electrostatically linked to the one or more payloads.

27. The mitochondria of claim 21, wherein the protective layer is a lipid formulation, optionally wherein the lipid formulation is a cationic lipid formulation, further optionally wherein the cationic lipid formulation is electrostatically linked to the one or more payloads.

28. The mitochondria of any one of claims 21 to 27, wherein the protective layer is linked to a targeting moiety.

29. The mitochondria of any one of claims 21 to 28, wherein the protective layer is attached to an antibody, optionally wherein the protective layer attached to the antibody is electrostatically attached to the one or more payloads.

30. The mitochondria of any one of claims 21 to 28, wherein the protective layer is attached to a carbohydrate, optionally wherein the protective layer attached to the carbohydrate is electrostatically attached to the one or more payloads.

31. The mitochondria of claim 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)ethylmethacrylate), RGD-modified poly(2-(dimethylamino)ethylmethacrylate), poly(amidoamine), RGD-modified poly(amidoamine), or a combination thereof.

32. The mitochondria of claim 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-poly The invention relates to 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.

33. The mitochondria of claim 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-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.

34. The mitochondria of claim 26, wherein the PEGylated 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 polypropylene imine, RGD-modified PEGylated polypropylene imine, 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.

35. The mitochondria of claim 27, wherein the lipid preparation comprises DC-cholesterol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), DLinDMA (1,2-dilinoleoyloxy-3-dimethylaminopropane), DLinMC3DMA (dilinoleoylmethyl-4-dimethylaminobutyrate), DODMA (1,2-dioleoyloxy-3-dimethylaminopropane), DOGS (dioctadecyl) acylglycerol hydrochloride), The invention can be used in combination with the present invention to prepare the present invention, wherein the present invention comprises: a) 1,2-dioleoyl-3-trimethylammonium-propane chloride; b) 1,2-dioleoyl-sn-glycerophosphoethanolamine; c) 1,2-dioleoyl-sn-glycerophosphoethanolamine; d ...

36. Mitochondria according to claim 35, wherein the lipid preparation further comprises another lipid, preferably wherein the lipid is cholesterol, substituted or unsubstituted cholesterol, a cholesterol derivative such as a hydroxylated cholesterol derivative (e.g., hydroxycholesterol), a PEG-lipid, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DODAP (1,2-dioleoyl-3-trimethylammoniumpropane), DDA (dimethyldioctadecylammonium), 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dimyristoyl-sn-glycero-3-phosphate, bis(monooleoylglycerol)phosphate, or a combination thereof.

37. The mitochondria of claim 22, wherein the mitochondria are attached to and / or encapsulated in a zwitterionic protective polymer, optionally wherein the zwitterionic protective polymer is electrostatically attached to the one or more payloads.

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

39. A composition comprising a plurality of mitochondria according to any one of claims 1 to 38.

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

41. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition is formulated as a solution.

42. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition is formulated as an aerosol.

43. The mitochondria according to any one of claims 1 to 38, the composition according to claim 39 or the pharmaceutical composition according to any one of claims 40 to 42 for use as a medicament.

44. The mitochondria according to any one of claims 1 to 38, the composition according to claim 39 or the pharmaceutical composition according to any one of claims 40 to 42 for use in gene therapy.

45. The mitochondria according to any one of claims 1 to 38, the composition according to claim 39 or the pharmaceutical composition according to any one of claims 40 to 42 for use in the treatment of cardiovascular diseases, in particular for the treatment of ischemic heart disease, ischemia-reperfusion injury or atherosclerosis.

46. ​​The mitochondria according to any one of claims 1 to 38, the composition according to claim 39 or the pharmaceutical composition according to any one of claims 40 to 42 for use in the treatment of ageing-related diseases, in particular for the treatment of sarcopenia, Parkinson's disease or Hutchinson-Gilford progeria syndrome.

47. Mitochondria according to any one of claims 1 to 38, a composition according to claim 39 or a pharmaceutical composition according to any one of claims 40 to 42 for use in the treatment of a kidney disease, in particular for the treatment of autosomal dominant polycystic kidney disease, Alport syndrome, nephronophthisis or Fabry disease.

48. The mitochondria of any one of claims 1 to 38, the composition of claim 39 or the pharmaceutical composition of any one of claims 40 to 42 for use in treating cancer.

49. The mitochondria of any one of claims 1 to 38, the composition of claim 39 or the pharmaceutical composition of any one of claims 40 to 42 for use in in vitro, ex vivo or in vivo genome editing.

50. The mitochondria according to any one of claims 1 to 38, the composition according to claim 39 or the pharmaceutical composition according to any one of claims 40 to 43 for use in radiotherapy.

51. A method for delivering a payload to a target organ, the method comprising the step of administering a pharmaceutical composition according to any one of claims 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.

52. A method for delivering a payload to the lung, the method comprising the step of administering the pharmaceutical composition of claim 42 to a subject in need thereof, wherein the pharmaceutical composition is administered by inhalation.

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

54. The method of claim 53, wherein a) contacting the at least one payload simultaneously with the positively charged species and the mitochondria; b) contacting the at least one payload with the positively charged species to form a positively charged complex, and then contacting the positively charged complex with the mitochondria; or c) contacting said mitochondria with said positively charged species and subsequently with said at least one payload.

55. The method of claim 52 or 54, wherein the mitochondria are contacted with the at least one payload and the positively charged species in a suitable buffer.

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

57. The method of any one of claims 53 to 56, wherein the mitochondria are contacted with the at least one payload and the positively charged species for at least 5 minutes, e.g., at least 10 minutes, 20, 30, 40, 50, 60 or 120 minutes at room temperature.

58. The method of any one of claims 53 to 57, wherein the mitochondria are contacted with the at least one payload and the positively charged species in the dark.

59. The method of any one of claims 53 to 58, wherein the payload is a nucleic acid molecule which is DNA or RNA.

60. The method of any one of claims 53 to 59, wherein the positively charged species is a polycationic species, wherein the polycationic species is a linear or branched polycationic polymer, optionally wherein the linear or branched polycationic polymer is electrostatically attached to the at least one payload.

61. The method of claim 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 dendrimers, 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.

62. The method of any one of claims 53 to 59, wherein the positively charged species is a positively charged nanoparticle.

63. The method according to claim 62, wherein the method comprises the further steps of: a) attaching the at least one payload to the surface of the positively charged nanoparticle; or b) encapsulating the at least one payload within the positively charged nanoparticle.

64. The method of claim 62 or 63, wherein the positively charged nanoparticles 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.

65. A method for preparing mitochondria containing a payload, wherein the method comprises the following steps: a) providing a mitochondrial preparation; b) contacting the mitochondria with i) contacting with a positively charged species if both the payload and the mitochondria have a net negative charge; ii) contacting the payload, optionally further contacting with a zwitterionic substance, if the payload has a net charge different from the net charge of the mitochondria; or iii) if the payload is uncharged, contacting with a payload attached to a positively charged species; c) obtaining the mitochondria according to any one of claims 1 to 20.

66. The method according to claim 65, further comprising the step of contacting the mitochondria with a component forming a protective layer after step c), and the step of obtaining the mitochondria according to any one of claims 22 to 38.

67. The method according to any one of claims 53 to 66, wherein an amount of 50 to 200 μg of the mitochondria is contacted with 0.1 to 50 pmol of the payload and 0.02 to 10 μg, preferably 0.02 to 5 μg of the positively charged substance.

68. The method of any one of claims 53 to 66, wherein the mitochondria comprise positively charged species, wherein the positively charged species is a polycationic polymer according to any one of the preceding claims, and wherein the ratio of the polycationic polymer to the protective layer is about 1:

2.

69. The method of any preceding method claim, wherein 50 μg to 200 μg of mitochondria are contacted with 0.1 to 50 pmol of payload and 0.2 to 10 μg of the protective layer.

70. A method for delivering a payload to the kidney, the method comprising the step of administering the pharmaceutical composition according to claims 40 to 50 into the renal artery of a subject in need thereof.

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

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

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

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

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

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

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

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

79. A method for delivering a payload to a target organ, the method comprising the step of administering a pharmaceutical composition according to claims 40 to 50 into the body of a subject in need thereof, wherein the pharmaceutical composition is administered by direct injection to the kidney or bladder or intestine or pancreas or duodenum or liver or lung or spleen.

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