WO2021004477A1 - Mitochondria-based drug delivery system and use thereof - Google Patents

Abstract

A mitochondria-based drug delivery system. The drug delivery system comprises mitochondria and a drug loaded on the mitochondria, wherein the drug is loaded on the mitochondria by means of non-free diffusion. The drug delivery system can effectively deliver protein, nucleic acid or other drugs into cells, can be used for treating monogenic diseases, polygenic diseases or other diseases, and can also be used in healthy individuals to achieve the purposes such as delaying senility, enhancing body functions, or preventing diseases.

Description

A drug delivery system based on mitochondria and its use Technical field

The present invention relates to a mitochondrial-based drug delivery system, a preparation method of the drug delivery system, the use of the drug delivery system for delivering drugs, and the preparation of the drug delivery system for treating and/or preventing diseases, Use in medicines and/or preparations for cosmetology, weight loss, anti-fatigue, growth promotion, enhancement of body function or anti-aging.

Background technique

The absence or defect of specific biological macromolecules in human cells can cause a variety of major diseases, such as cancer, hemophilia, and albinism. A common treatment method is to introduce exogenous biomolecule drugs into target cells to replace, compensate, block or correct defective molecules in the patient's cells, so as to achieve the purpose of preventing and treating diseases.

However, biological macromolecular drugs such as proteins and nucleic acids must be loaded on a certain carrier in order to successfully enter target cells due to their strong polarity, large molecular weight, and easy degradation. The natural excellent transmembrane transport ability of viruses makes them a common drug delivery vehicle. Although viral vectors have achieved initial clinical efficacy, their limitations should not be ignored, such as potential carcinogenicity, cytotoxicity, strong immunogenicity, limited loading capacity, and difficult quality control. At the same time, the high preparation cost of viral vectors further restricts their widespread use. For example, patients with lipoprotein lipase deficiency can spend millions of euros on the gene therapy drug Glybera. This extremely high medical cost far exceeds the financial affordability of ordinary patients, and in the end the drug can only be delisted from the market.

Another common biological macromolecule vector is a non-viral vector. At present, such carriers mainly include cationic polymers, liposomes or other nanoparticles. However, non-viral vectors still face many challenges, mainly in the aspects of low drug load, low transfection efficiency, and high cytotoxicity. These shortcomings also limit its wide clinical application. Therefore, in the field of drug delivery, there is still a need to develop non-viral biological macromolecule vectors with high transfection efficiency and low toxicity. Summary of the invention

Mitochondria are important organelles responsible for energy synthesis, cell differentiation, information transmission and apoptosis of eukaryotic cells. Mitochondria evolved from ancient bacteria 1.5 billion years ago. They still retain some of the characteristics of bacteria and have the "infectious ability" similar to intracellular bacteria. For example, mitochondria are separated from cells and co-cultured with other cells. It can enter these cells quickly and efficiently (Clark, MA and JWShay (1982). "Mitochondrial transformation of mammalian cells." Nature 295(5850):605-607.). Inspired by this, the inventor of the present invention creatively realized that mitochondria can be used as biomacromolecule carriers to achieve safe and effective intracellular delivery of target drugs.

So far, there are only studies on loading small molecule anticancer drugs or quantum dots into mitochondria by free diffusion or electroporation to achieve their intracellular delivery (Li, W.-Q., Wang, Z., Hao, S .,Sun,L.,Nisic,M.,Cheng,G.,…Zheng,S.-Y.(2018).Mitochondria-based aircraft carrier enhances in vivo imaging of carbon quantum dots and delivery of anticancer drug.Nanoscale, 10(8), 3744–3752). However, after entering the cell, these drugs need to pass through the two layers of mitochondrial membranes in order to be released, which undoubtedly reduces their delivery efficiency. At the same time, the drugs that can diffuse into the mitochondria are very limited, and the electroporation transfection method is generally considered to be able to destroy the cell membrane and lead to high cell lethality. Therefore, the purpose of the present invention is to provide a new, widely applicable and safe mitochondrial loading method, so as to realize the efficient transmembrane delivery of various drugs, especially biological macromolecules such as proteins and nucleic acids.

In one aspect, the present invention provides a mitochondrial-based drug delivery system, the drug delivery system comprising:

(1) Mitochondria, and

(2) Drugs loaded on the mitochondria,

The drug is loaded on the mitochondria through non-free diffusion.

Among them, the source of mitochondria can be autologous, allogeneic or heterologous and combinations thereof. Drugs can be protein drugs such as peptide drugs, antibody drugs, cytokines, enzymes, protein vaccines, peptide hormones, proteins translated from normal genes, protein derivatives, nucleic acid drugs such as DNA, RNA or nucleic acid analogs , Or any other substances that cannot or are difficult to penetrate the cell membrane, and combinations thereof. Preferably, the drugs are protein drugs or nucleic acid drugs and combinations thereof. The drug can be reversibly or irreversibly loaded on the outer mitochondrial membrane, inner membrane, interstitial membrane or mitochondrial matrix and combinations thereof in the form of covalent bonds or non-covalent bonds.

The drug delivery system according to the present invention can realize the delivery of drugs into cells, where the cells may include the inner surface of the cell membrane, the cytoplasm, the nucleus, and combinations thereof. After the drug is delivered into the cell, it can exist in a state of being bound to or separated from mitochondria.

In the drug delivery system according to the present invention, the drug loaded on the mitochondria may be a protein drug. Protein drugs can be bound to mitochondria by any method, including but not limited to: (1) Protein drugs and mitochondrial localization sequences, such as mitochondrial outer membrane localization sequences such as FIS1.MTS, and mitochondrial matrix localization sequences such as COX8.MTS to form fusion proteins , Bind to mitochondria through the positioning sequence; (2) Protein drugs and other proteins that can specifically bind to mitochondria, such as antibodies, antibody-binding variable regions of antigens, nanobodies, or other non-antibodies that can bind to mitochondria The amino acid sequence is fused to form a fusion protein to bind to mitochondria; (3) Through chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der Waals forces, hydrogen bonds, aromatic ring stacking, hydrophobic forces and halogen bonds, and Combines to mitochondria.

The protein drug loaded on the mitochondria can be a single protein or a combination of multiple proteins. The protein drug loaded on the mitochondria can be a complete protein, such as GFP, or each part of a protein divided into several parts, such as GFP1-11 and GFP12 of Split-GFP. The protein drugs loaded on the mitochondria can be labeled with fluorescent protein or other protein tags (such as His-Tag, etc.) or not.

In the drug delivery system according to the present invention, the drug loaded on the mitochondria may be a nucleic acid drug. Nucleic acid drugs can bind to mitochondria through nucleic acid binding proteins or nucleic acid binding domains of nucleic acid binding proteins, or through chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der Waals forces, hydrogen bonds, aromatic ring stacking, and hydrophobic forces. And the halogen bond, and its combination, bind to the mitochondria. For example, DNA is negatively charged. If protein X is positively charged, X-MOM (Mitochondrial outer membrane, mitochondrial outer membrane positioning sequence) is anchored to the outer membrane of mitochondria, then DNA can bind to the protein X, and then load the outer membrane of the mitochondria.膜上。 The membrane. Nucleic acid drugs loaded on mitochondria can be one gene or multiple genes.

Nucleic acid drugs can be DNA, RNA, DNA and RNA analogs, and combinations thereof. DNA can be circular or linear, single-stranded or double-stranded, including but not limited to plasmids, PCR products, genomic DNA, etc.; RNA includes but not limited to mRNA, miRNA, shRNA, siRNA or aptomer. DNA or RNA analogs refer to substances that can be transcribed, replicated, or bind to DNA or RNA through similar base complementation, such as morpholino and oligos.

In another aspect, the present invention also provides the use of the above-mentioned drug delivery system for delivering drugs, such as protein drugs, nucleic acid drugs or other macromolecular drugs.

In yet another aspect, the present invention also provides the above-mentioned drug delivery system in the preparation of preparations and/or compositions for treating and/or preventing diseases, beauty, weight loss, anti-fatigue, promoting growth and development, enhancing body function or delaying aging use. The preparations or compositions prepared by the drug delivery system of the present invention can be used to treat and/or prevent diseases including nervous system diseases, immune system diseases, respiratory system diseases, circulatory system diseases, urinary system diseases, reproductive system diseases, and sports system diseases , Endocrine system disease, digestive system disease, single gene genetic disease, polygenic genetic disease, infectious disease, tumor, diabetes, neurodegenerative disease or cardiovascular and cerebrovascular disease and combinations thereof.

Description of the drawings

Through the following detailed description in conjunction with the accompanying drawings, the above content and other objectives, features and other advantages of the present invention will be understood more clearly, among which:

Fig. 1 shows that the target protein NeoR in Example 1 according to the present invention is loaded on the outer membrane of mitochondria through the mitochondrial outer membrane targeting sequence, and then is introduced into the cell. Among them, (A) CellMask Deep-red marks the cell membrane. (B) The red fluorescent protein mScarlet-i shows the target protein NeoR. (C) The cell membrane signal overlaps with the target protein signal, showing that the target protein NeoR is delivered into the cell.

Fig. 2 shows that the target protein NeoR in Example 2 of the present invention is loaded on the outer membrane of mitochondria through an optogenetic protein pair and then introduced into the cell. Among them, (A) CellMask Deep-red marks the cell membrane. (B) The red fluorescent protein mScarlet-i shows the target protein NeoR. (C) The cell membrane signal overlaps with the target protein signal, showing that the target protein NeoR is delivered into the cell.

Figure 3 shows that in Example 3 according to the present invention, mitochondria simultaneously deliver two target proteins into the cell. Among them, (A) CellMask Deep-red marks the cell membrane. (B) The red fluorescent protein mScarlet-i shows the target protein 1 (Catalase). (C) Green fluorescent protein GFP shows target protein 2 (IL10). (D) The cell membrane signal overlaps with the target protein signal, showing that the target protein Catalase and IL10 are delivered into the cell.

Fig. 4 shows the introduction of a target protein into a cell by an antibody bound to the outer mitochondrial membrane protein in Example 4 according to the present invention. Among them, (A) CellMask Deep-red staining, marking the cell membrane. (B) GFP labels the outer mitochondrial membrane and mimics the outer mitochondrial membrane protein. (C) The target protein NeoR linked to the anti-GFP Nanobody. (D) After overlapping, it shows that the target protein NeoR has been introduced into the cell.

Fig. 5 shows that the plasmid DNA is introduced into the cell using mitochondria as a vector and successfully expressed in Example 5 according to the present invention. Among them, (A) bright field, showing cell outline. (B) shows GFP marking the nucleus (bright signal in the middle) and mitochondria (weak signal around). (C) After overlap, it shows that some cells successfully express GFP that marks mitochondria and nucleus, indicating that the plasmid DNA has been successfully introduced into the cell and expressed.

Fig. 6 shows that in Example 6 of the present invention, mitochondria is used as a vector to introduce mRNA into a cell and successfully express it. Among them, (A) CellMask Deep-red staining, marking the cell membrane. (B) The red fluorescent protein labeled with mitochondria indicates that Mito-mScarlet-i has been introduced into the cell and translated. (C) Overlap (A) and (B), showing that the translated protein is located in the cell, indicating that the mRNA is successfully delivered into the cell and expressed.

Detailed ways

The present invention will now be explained in more detail with reference to the following examples. These examples are provided only to illustrate the present invention and should not be construed as limiting the scope and spirit of the present invention.

In one embodiment of the present invention, the drug can be fused with FIS1.MTS protein and then loaded on the outer membrane of mitochondria, where FIS1.MTS protein is a targeting polypeptide (MTS, mitochondrial targeting sequence) of the outer mitochondrial membrane protein FIS1 . After the drug is fused with FIS1.MTS protein, it contains the targeting part of FIS1, so it can be loaded on the outer membrane of mitochondria.

In one embodiment of the present invention, the drug can be fused with the fluorescent protein and FIS1.MTS protein, and then loaded on the surface of the mitochondria. Among them, the role of fluorescent protein is to mark drugs, only for tracking and monitoring drug delivery, not a necessary means to achieve drug delivery. Fluorescent proteins include but are not limited to green fluorescent protein GFP, red fluorescent protein RFP, enhanced green fluorescent protein EGFR, red fluorescent protein mScarlet-I and so on.

In one embodiment of the present invention, the drug is loaded onto the outer membrane of the mitochondria by optogenetic methods, such as pMag and nMag, Cry2-CIB or any other optogenetic protein pair. Specifically, in one embodiment of the present invention, for example, the drug forms a fusion protein with pMag, nMag forms a fusion protein with the mitochondrial outer membrane positioning sequence such as FIS1.MTS protein, and the pair of pMag and nMag proteins are paired and bound under the action of blue light, so that The drug is loaded on the outer membrane of the mitochondria.

In some embodiments of the present invention, in addition to using optogenetic proteins, the drug delivery system according to the present invention can also use proteins that bind under the action of chemical substances, such as FKBP12 and FRB regulated by rapamycin, or any other reversibility. Or irreversibly bound protein pair. In some embodiments of the present invention, a pair of protein pairs can be used between the drug and the mitochondrial outer membrane localization sequence, or multiple pairs of proteins can be used, one type of protein pair can be used, or multiple types of protein pairs can be used. Protein pair.

In one embodiment of the present invention, the drug can be fused with the fluorescent protein and the aforementioned protein pair, and then loaded on the outer membrane of the mitochondria. Among them, the fluorescent protein plays the role of labeling drugs, only for tracking and monitoring the delivery of drugs, and is not necessary to achieve drug delivery. Fluorescent proteins include but are not limited to green fluorescent protein GFP, red fluorescent protein RFP, enhanced green fluorescent protein EGFR, red fluorescent protein mScarlet-I and so on.

In one embodiment of the present invention, the drug delivery system according to the present invention is prepared by a method including the following steps:

(1) Design X-Y-FIS1.MTS protein, where the protein includes three parts: X is any target drug; Y is a fluorescent protein used to label the target drug; FIS1.MTS is a targeting polypeptide of the mitochondrial outer membrane protein FIS1;

(2) Construct the plasmid of X-Y-FIS1.MTS protein with pcDNA3.1 vector and transfect HEK 293T cells; and

(3) After successful expression, extract the mitochondria labeled with X-Y-FIS1.MTS in the cells, and then co-culture with unlabeled cells.

In one embodiment of the present invention, the drug delivery system according to the present invention is prepared by a method including the following steps:

(1) Design X-Y1-pMag protein and nMagHigh1-Y2-FIS1.MTS protein respectively;

(2) Constructing plasmids of the two proteins using the pcDNA3.1 vector and transfecting HEK293T cells to express the two proteins simultaneously;

(3) After 24 hours, the cells are irradiated with blue light for 10 minutes to bind the pMag and nMagHigh1 protein pair; and

(4) Extract the Y1 and Y2 labeled mitochondria in the cells, and then co-culture with unlabeled cells.

Among them, X is any target drug, Y1 and Y2 are fluorescent proteins for labeling, pMag and nMagHigh1 are a pair of optogenetic proteins, and FIS1.MTS is the targeting peptide of the outer mitochondrial membrane protein FIS1.

Further, the fluorescent protein can be GFP, EGFP, mScarlet-i, YFP, CFP or other fluorescent proteins.

The plasmid vector can be pcDNA3.1, pEGFP, pCMVp-NEO-BAN, pSV2pUC19DNA, pUC18DNA, PQE30 or any other commonly used cloning vectors.

The transfected cells can be HEK 293T, CHO-K1, COS-7, HeLa, NIH-3T3, PC-3, A549, MCF-7, HuH-7, U-2OS, HepG2 or any other transfectable cells.

The transfection reagent can be Lipofectamine, X-tremeGENE 9, Escort ^™ III, Escort ^™ IV, NeuroPorter ^™ DOTAP methosulfate, Calcium Phosphate or any other transfection reagent.

The transfection method can be liposome transfection method, calcium phosphate transfection method, electroporation transfection method or any other transfection method.

The co-cultivation time of mitochondria and cells can be 0.5-120 hours, preferably 6-24 hours.

<Example 1>

Design protein NeoR-mScarlet-i-FIS1.MTS. Among them, NeoR is the anti-neomycin (Neomycin) protein, which is the target drug; mScarlet-i is the red fluorescent protein, used to label the target drug; FIS1.MTS is the targeting sequence of the mitochondrial outer membrane protein FIS1, so NeoR-mScarlet- i-FIS1.MTS protein can target the outer membrane of mitochondria. The NeoR-mScarlet-i-FIS1.MTS plasmid (see sequence table, SEQ ID NO:1) was constructed using pcDNA3.1 vector and transfected into HEK 293T cells. After 24 hours of successful expression, extract NeoR-mScarlet-i-FIS1.MTS-labeled mitochondria in HEK 293T cells, that is, mitochondria loaded with NeoR-mScarlet-i-FIS1.MTS fusion protein, and then co-culture with unlabeled 293A cells6 hour. Using the cell membrane dye CellMask Deep-red to stain the cell membrane, and then using a laser confocal microscope to observe that some mitochondria in 293A cells are mScarlet-i positive, indicating that the target drug NeoR has been successfully delivered into 293A cells. The test results are shown in Figure 1.

<Example 2>

Design proteins NeoR-mScarlet-i-pMag and nMagHigh1-GFP-FIS1.MTS. Among them, NeoR is the target drug, and mScarlet-i is the red fluorescent protein used to label the target drug. The nMagHigh1-GFP-FIS1.MTS fusion protein can bind to the outer mitochondrial membrane through the mitochondrial outer membrane targeting sequence FIS1.MTS. pMag and nMagHigh1 are a pair of optogenetic proteins that can bind to each other under blue light irradiation. Therefore, under the action of blue light, the fusion protein NeoR-mScarlet-i-pMag and the fusion protein nMagHigh1-GFP-FIS1.MTS bind to each other, so that the target drug NeoR is loaded on the outer membrane of the mitochondria. Specifically, the NeoR-mScarlet-i-pMag plasmid (see Sequence Listing, SEQ ID NO: 2) and the nMagHigh1-GFP-FIS1.MTS plasmid (see Sequence Listing, SEQ ID NO: 3) were constructed using the pcDNA3.1 vector. pcDNA3.1-NeoR-mScarlet-i-pMag and pcDNA3.1-nMagHigh1-GFP-FIS1.MTS were co-transfected into 293T cells. After 24 hours, the cells were irradiated with strong light for 10 minutes to make NeoR-mScarlet-i-pMag fusion protein Combine with nMagHigh1-GFP-FIS1.MTS fusion protein to load the target protein NeoR onto the outer membrane of mitochondria. Then extract the mitochondria loaded with NeoR protein (mScarlet-i label), and dilute the extracted mitochondria with DMEM complete medium, and then add them to the cultured unlabeled 293A cells. After incubating with the medium at 37°C for 6-8 hours, use Cell membrane dye CellMask Deep-red stains the cell membrane, and then uses laser confocal microscope imaging to detect that some of the target protein NeoR labeled with mScarlet-i has entered the 293A cells. The test results are shown in Figure 2.

<Example 3>

Construct plasmids pcDNA3.1-NeoR-mScarlet-i-NB.GFP (see Sequence Listing, SEQ ID NO: 4) and pcDNA3.1-GFP-FIS1.MTS (see Sequence Listing, SEQ ID NO: 5). Among them, NeoR is the target drug, mScarlet-i is a red fluorescent protein used to label the target drug, and NB.GFP is a nanobody that can specifically bind to GFP. The role of GFP is to mark mitochondria and mimic mitochondrial outer membrane protein. FIS1.MTS is the target sequence of mitochondrial outer membrane protein FIS1. Two 293T cells in a 10cm culture dish were transfected with the two plasmids. After 24 hours, the cells expressing NeoR-mScarlet-i-NB.GFP were harvested. The cells were swelled with distilled water for 5 minutes, and then 1/9 volume of 10X PBS was added. The cells were broken by a glass homogenizer, centrifuged at 10,000g for 10 minutes, and the supernatant containing NeoR-mScarlet-i-NB.GFP was taken for later use. The cells expressing GFP-FIS1.MTS were harvested, mitochondria were extracted, and resuspended in the supernatant containing NeoR-mScarlet-i-NB.GFP, placed at room temperature for 10 minutes, and then co-cultured with unlabeled 293A cells, 37℃ medium After 6-8 hours of incubation, the cell membrane was stained with the cell membrane dye CellMask Deep-red, and then imaged with a laser confocal microscope. It was found that some GFP-labeled mitochondria had successfully transferred mScarlet-i labeled NeoR into the cells. The test results are shown in Figure 3.

<Example 4>

Design protein COX8.MTS-hCatalase-mScarlet-i, IL10-mNeonGreen-FIS1.MTS. In the protein COX8.MTS-hCatalase-mScarlet-i, hCatalase is human Catalase, which is the target drug, mScarlet-i is a red fluorescent protein used to label the target drug, and COX8.MTS is the target sequence of the mitochondrial matrix protein COX8. The protein COX8.MTS-hCatalase-mScarlet-i can be located to the mitochondrial matrix through the targeting sequence COX8.MTS. In the protein IL10-mNeonGreen-FIS1.MTS, IL10 is the target drug, mNeonGreen is a high-brightness green fluorescent protein, used to label the target drug, the protein IL10-mNeonGreen-FIS1.MTS can be bound to the target sequence FIS1.MTS The outer membrane of the mitochondria. Specifically, the pcDNA3.1 vector was used to construct the COX8.MTS-hCatalase-mScarlet-i plasmid (see the sequence list, SEQ ID NO: 6) and the IL10-mNeonGreen-FIS1.MTS plasmid (see the sequence list, SEQ ID NO: 7) 293T cells were co-transfected with two plasmids, 24 hours later, mitochondria loaded with hCatalase (mScarlet-i label) and/or IL10 (mNeonGreen label) were extracted, and the extracted mitochondria were diluted with DMEM complete medium and added to the culture In the unlabeled 293A cells, after incubating in 37℃ medium for 6-8 hours, stain the cell membrane with the cell membrane dye CellMask Deep-red, and then use laser confocal microscope imaging to detect some mNeonGreen-labeled hCatalase and mScarlet-i labeled IL10 has entered the cell. The test results are shown in Figure 4.

<Example 5>

Construct the plasmid pcDNA3.1-hTFAM1-CRT-mScarlet-i-FIS1.MTS (see Sequence Listing, SEQ ID NO: 8), where hTFAM1 is the DNA binding domain of human TFAM1, CRT is the DNA binding domain of TopII alpha, both Can bind to DNA. mScarlet-i is a red fluorescent protein used to label mitochondria carrying the fusion protein; FIS1.MTS is a signal peptide targeting the outer mitochondrial membrane. Mouse 3T3 cells transfected with pcDNA3.1-hTFAM1-CRT-mScarlet- i-FIS1.MTS, fusion protein expression after 24 hours, take 10 ⁷ cell extract mitochondria, resuspended in 100μL DMEM complete medium, plus 200ng plasmid pcDNA3.1-Mito-EGFP-2A-n.EGFP (see Sequence Listing, SEQ ID NO: 9), incubated at room temperature for 1 hour, where 2A can translate one mRNA into two proteins, Mito-EGFP marks mitochondria, nEGFP Mark the nucleus. Then the mitochondria were co-cultured with unlabeled 293A cells. After 24 hours, fluorescence microscope imaging showed that some cells expressed GFP labeled with mitochondria and nuclei, indicating that the plasmid had been successfully transferred into the cells and expressed. The test results are shown in Figure 5.

<Example 6>

The plasmid pcDNA3.1-UP1-FIS1.MTS (see Sequence Listing, SEQ ID NO: 10) was constructed, where UP1 is the non-specific RNA binding domain of human hnRNP A1 protein, and FIS1.MTS is a signal peptide targeting the outer mitochondrial membrane. 293T cells were transfected with pcDNA3.1-UP1-FIS1.MTS, mitochondrial ¹⁰⁷ cell extracts taken after 24 hours, resuspended in mitochondria 100μLDMEM complete medium, plus the mRNA 200ng Mito-mScarlet-i (manufactured by plasmid pcDNA3.1-Mito -mScarlet-I (see sequence listing, SEQ ID NO: 11) was transcribed in vitro), incubated at room temperature for 0.5 hours, then co-cultured mitochondria with unlabeled 293A cells, and stained the cell membrane with the cell membrane dye CellMask Deep-red after 24 hours , Laser confocal microscope imaging shows that some cells express red signals that mark mitochondria, indicating that mRNA has been successfully transferred into cells and translated. The test results are shown in Figure 6.

Claims (10)

1. A mitochondrial-based drug delivery system, the drug delivery system comprising:

   (1) Mitochondria, and

   (2) Drugs loaded on the mitochondria,

   The drug is loaded on the mitochondria through non-free diffusion.
2. The drug delivery system according to claim 1, wherein the source of the mitochondria is autologous, allogeneic or xenogeneic, and combinations thereof.
3. The drug delivery system according to claim 1, wherein the drugs include protein drugs such as polypeptide drugs, antibody drugs, cytokines, enzymes, protein vaccines, polypeptide hormones, proteins translated from normal genes, protein-derived Drugs, nucleic acid drugs such as DNA, RNA or their analogs, and combinations thereof.
4. The drug delivery system according to claim 1, wherein the drug is reversibly or irreversibly loaded on the outer mitochondrial membrane, inner membrane, interstitial membrane or mitochondrial matrix and combinations thereof through covalent bonds or non-covalent bonds.
5. The drug delivery system according to claim 1, wherein the drug delivery system realizes the delivery of drugs into cells, wherein the cells include inner surface of cell membrane, cytoplasm, nucleus, and combinations thereof.
6. The drug delivery system according to claim 1, wherein after the drug is delivered into the cell, it exists in a state of being bound to or separated from the mitochondria.
7. The drug delivery system according to claim 1, wherein the drug is a protein drug, wherein the protein drug is loaded on the mitochondria in a manner including:

   (1) Fusion with mitochondrial positioning sequence, such as mitochondrial outer membrane positioning sequence such as FIS1.MTS or mitochondrial matrix positioning sequence such as COX8.MTS, and bind to mitochondria in the form of a fusion protein;

   (2) Fusion with a protein that specifically binds to mitochondria, such as antibodies, variable regions where antibodies bind to antigens, or nanobodies, or other non-antibodies but capable of binding to mitochondria with amino acid sequences that bind to mitochondria;

   (3) Binding to mitochondria through chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der Waals forces, hydrogen bonds, aromatic ring stacking, hydrophobic forces, and halogen bonds, and combinations thereof.
8. The drug delivery system according to claim 1, wherein the drug is a nucleic acid drug, wherein the nucleic acid drug is loaded on the mitochondria in a manner including:

   (1) Binding to mitochondria through a nucleic acid binding protein or its nucleic acid binding domain, or

   (2) Binding to the mitochondria through chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der Waals forces, hydrogen bonds, aromatic ring stacking, hydrophobic forces and halogen bonds, and combinations thereof.
9. The use of the drug delivery system according to any one of claims 1 to 8 in the preparation of a preparation for treating diseases, preventing diseases, cosmetology, weight loss, anti-fatigue, promoting growth and development, enhancing body function or delaying aging.
10. The use according to claim 9, wherein the disease includes nervous system disease, immune system disease, respiratory system disease, circulatory system disease, urinary system disease, reproductive system disease, motor system disease, endocrine system disease, digestive system disease, Single-gene genetic diseases, polygenic genetic diseases, infectious diseases, tumors, diabetes, neurodegenerative diseases or cardiovascular and cerebrovascular diseases and combinations thereof.

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