CN114786685A - Mitochondria-based drug delivery system and application thereof - Google Patents
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Abstract
A mitochondrial-based drug delivery system, the drug delivery system comprising: mitochondria, and a drug loaded to said mitochondria, wherein said drug is loaded to said mitochondria by means other than free diffusion. The drug delivery system can effectively deliver proteins, nucleic acids or other drugs into cells, can be used for treating monogenic genetic diseases, polygenic genetic diseases or other diseases, and can also be used for healthy individuals to achieve the purposes of delaying aging, enhancing body functions or preventing diseases and the like.
Description
The present invention relates to a mitochondria-based drug delivery system, a method for the preparation of said drug delivery system, the use of said drug delivery system for delivering a drug, and the use of said drug delivery system for the manufacture of a medicament and/or formulation for the treatment and/or prevention of a disease, cosmetology, weight loss, fatigue resistance, growth and development promotion, enhancement of body function or senescence delay.
The absence or defect of a particular biological macromolecule in a human cell can cause a variety of significant diseases, such as cancer, hemophilia, albinism, and the like. The common treatment method is to introduce exogenous biomolecular drugs into target cells to replace, compensate, block or correct defective molecules in patient cells, thereby achieving the purpose of preventing and treating diseases.
However, due to the strong polarity, large molecular weight, and easy degradation, biomacromolecule drugs such as proteins and nucleic acids must be loaded on a certain carrier to successfully enter target cells. The natural excellent transmembrane transport capacity of viruses makes them common drug delivery vehicles. Although viral vectors achieve primary therapeutic effects in the clinic, their limitations, such as potential carcinogenicity, cytotoxicity, strong immunogenicity, limited loading capacity and difficult quality control, are not negligible. At the same time, the high preparation cost of viral vectors further limits their widespread use. For example, lipoprotein lipase deficient patients can cost up to a million euros on the gene therapy drug Glybera. This extremely high medical cost far exceeds the economic viability of the average patient and eventually the drug can only overshadow the market.
Another common biological macromolecular vector is a non-viral vector. Such carriers currently include mainly cationic polymers, liposomes or other nanoparticles. However, non-viral vectors still face many challenges, mainly in terms of low drug loading, low transfection efficiency, high cytotoxicity, etc. These deficiencies also limit their widespread clinical use. Therefore, there is still a need in the field of drug delivery to develop non-viral biomacromolecule vectors with high transfection efficiency and low toxicity. Disclosure of Invention
Mitochondria are important organelles responsible for energy synthesis, cellular differentiation, information transfer, and apoptosis in eukaryotic cells. Mitochondria have evolved from 15 million years ago ancient bacteria and have maintained some characteristics of bacteria so far, and have "infectivity" similar to intracellular bacteria, for example, mitochondria are isolated from cells and co-cultured with other cells, and mitochondria can rapidly and efficiently enter these cells (Clark, M.A. and J.W.Shay (1982). "Mitochondrial transformation of mammalian cells." Nature 295(5850): 605-607.). Inspired by this, the inventors of the present invention have creatively recognized that mitochondria can be used as a biomacromolecule carrier to achieve safe and effective intracellular delivery of a target drug.
Until now, there have been only studies to load small molecule anticancer drugs or quantum dots into Mitochondria to achieve their intracellular delivery by free diffusion or electroporation, respectively (Li, w. -q., Wang, z., Hao, s., Sun, l., Nisic, m., Cheng, g., … Zheng, s. -Y. (2018) mitochonddria-based aircraft engineering in vivo imaging of carbon quantum dots and delivery of anticancer drug, nanoscale,10(8), 3744-. However, after entering the cell, these drugs need to pass through the inner and outer membranes of the mitochondria in addition to be released, which undoubtedly reduces their delivery efficiency. Meanwhile, the drugs that can enter the inside of mitochondria through diffusion are very limited, and electroporation transfection is generally considered to be capable of destroying cell membranes, which results in high cell lethality. Therefore, it is an object of the present invention to provide a novel, widely applicable and safe mitochondrial loading approach to achieve 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 comprising:
(1) mitochondria, and
(2) a drug loaded on the mitochondria in the presence of a drug,
wherein the drug is loaded to the mitochondria by means other than free diffusion.
Wherein, the source of mitochondria can be autologous, allogeneic or xenogeneic and the combination thereof. The drug may be a proteinaceous drug such as a polypeptide drug, an antibody drug, a cytokine, an enzyme, a proteinaceous vaccine, a polypeptide hormone, a protein translated from a normal gene, a protein derivative, a nucleic acid drug such as DNA, RNA, or a nucleic acid analog, or any other substance that is unable or difficult to penetrate a cell membrane, and combinations thereof. Preferably, the drug is a proteinaceous drug or a nucleic acid drug and combinations thereof. The drug may be loaded reversibly or irreversibly on the outer mitochondrial membrane, inner mitochondrial membrane, interstitial space or mitochondrial matrix and combinations thereof, by covalent or non-covalent bonding.
The drug delivery system according to the present invention enables the delivery of drugs into cells, which may include the inner surface of cell membranes, cytoplasm, nucleus and combinations thereof. The drug can exist in a state of being bound to or separated from mitochondria after being delivered into a cell.
In the drug delivery system according to the present invention, the drug loaded on mitochondria may be a protein-based drug. Wherein the proteinaceous agent can be bound to the mitochondria by any method, including but not limited to: (1) the proteinaceous drugs form fusion proteins with mitochondrial localization sequences, e.g. mitochondrial outer membrane localization sequences such as fis1.mts, mitochondrial matrix localization sequences such as cox8.mts, through which they bind to mitochondria; (2) the proteinaceous agent is fused to an amino acid sequence that is capable of specifically binding to mitochondria, such as an antibody, antibody-binding antigen variable region, nanobody, or other non-antibody but capable of binding to mitochondria, to form a fusion protein, thereby binding to mitochondria; (3) binding to mitochondria occurs by means of chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der waals forces, hydrogen bonds, aromatic ring stacking interactions, hydrophobic forces, and halogen bonds, and combinations thereof.
The protein drug to be loaded on mitochondria may be a single protein or a combination of a plurality of proteins. The mitochondrial-loaded proteinaceous drugs may be intact proteins such as GFP, or individual portions of a protein divided into several fractions such as GFP1-11 and GFP12 from Split-GFP. The mitochondrial-supported proteinaceous drugs may or may not be labeled with a fluorescent protein or other protein Tag (e.g., His-Tag).
In the drug delivery system according to the present invention, the drug loaded on the mitochondria may be a nucleic acid-based drug. Nucleic acid-based drugs can bind to mitochondria through nucleic acid binding proteins or nucleic acid binding domains of nucleic acid binding proteins, and can also bind to mitochondria by means of 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. For example, DNA is negatively charged, and if protein X is positively charged, anchoring X-MOM (mitochonddrial outer membrane localization sequence) to the outer membrane of mitochondria, DNA can bind to the protein X and become loaded on the outer membrane of mitochondria. The nucleic acid drug to be loaded on mitochondria may be one gene or a plurality of genes.
The nucleic acid drug can be DNA, RNA, analogs of DNA and RNA, and combinations thereof. The DNA may be circular or linear, single-or double-stranded, including but not limited to plasmids, PCR products, genomic DNA, and the like; RNA includes, but is not limited to, mRNA, miRNA, shRNA, siRNA, or an aptamer. DNA or RNA analogs refer to substances such as morpholino oligos and the like that can be transcribed, replicated, or bound to DNA or RNA by base-like complementarity.
In a further aspect, the present invention also provides the use of a drug delivery system as described above for delivering a drug, such as a proteinaceous drug, a nucleic acid drug or other macromolecular drug.
In a further aspect, the present invention also provides the use of the above drug delivery system for the preparation of a formulation and/or composition for the treatment and/or prevention of diseases, cosmetology, weight loss, fatigue resistance, growth and development promotion, enhancement of body functions or senescence delay. Diseases that the formulation or composition prepared using the drug delivery system of the present invention can be used for treatment and/or prevention include nervous system diseases, immune system diseases, respiratory system diseases, circulatory system diseases, urinary system diseases, reproductive system diseases, motor system diseases, endocrine system diseases, digestive system diseases, monogenic genetic diseases, polygenic genetic diseases, infectious diseases, tumors, diabetes, neurodegenerative diseases or cardiovascular and cerebrovascular diseases, and combinations thereof.
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows that a protein of interest NeoR is supported on the outer membrane of mitochondria through a mitochondrial outer membrane targeting sequence and is introduced into cells in example 1 according to the present invention. Wherein (A) CellMask Deep-red marks cell membranes. (B) The red fluorescent protein mScarlet-i shows the protein of interest NeoR. (C) The cell membrane signal overlaps with the target protein signal, indicating that the target protein NeoR is delivered into the cell.
FIG. 2 shows that the protein of interest NeoR is introduced into cells by optogenetic protein pairs loaded on the outer membrane of mitochondria in example 2 according to the present invention. Wherein (A) CellMask Deep-red marks cell membranes. (B) The red fluorescent protein mScalet-i shows the protein of interest NeoR. (C) The cell membrane signal overlaps with the target protein signal, indicating that the target protein NeoR is delivered into the cell.
FIG. 3 shows simultaneous mitochondrial delivery of two proteins of interest into cells according to example 3 of the present invention. Wherein (A) CellMask Deep-red marks cell membranes. (B) The red fluorescent protein mScarlet-i shows protein 1 of interest (Catalase). (C) Green fluorescent protein GFP shows protein 2 of interest (IL 10). (D) The cell membrane signal overlaps with the target protein signal, indicating that the target proteins Catalase and IL10 were delivered into the cell.
FIG. 4 shows introduction of a protein of interest into cells by an antibody binding to a mitochondrial outer membrane protein in example 4 according to the present invention. Wherein (A) CellMask Deep-red staining was performed to label the cell membrane. (B) GFP labels and mimics mitochondrial outer membrane proteins. (C) And the target protein NeoR is connected to the nano antibody for resisting GFP. (D) After the overlap, it was shown that the target protein NeoR had been introduced into the cells.
FIG. 5 shows that plasmid DNA was introduced into cells using mitochondria as a vector and successfully expressed in example 5 according to the present invention. Wherein (A) is a bright field showing cell contours. (B) GFP is shown labeling the nucleus (bright signal in the middle) and mitochondria (weaker signal in the periphery). (C) After the overlap, it was shown that some cells successfully expressed GFP, which marks the mitochondria and nuclei, indicating that the plasmid DNA had been successfully introduced into the cells and expressed.
FIG. 6 shows that mRNA was introduced into cells using mitochondria as a vector and successfully expressed in example 6 according to the present invention. Wherein (A) CellMask Deep-red staining was performed to label the cell membrane. (B) Mitochondrial red fluorescent protein was labeled, indicating that Mito-mScarlet-i had been introduced into the cell and translated. (C) Overlapping (a) with (B) shows that the translated protein is located intracellularly, indicating successful delivery and expression of mRNA into the cell.
The invention will now be described in more detail with reference to the following examples. These examples are provided only for illustrating 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 may be loaded on the outer membrane of mitochondria by fusion with a fis1.MTS protein, wherein the MTS protein is a targeting polypeptide (MTS) of the mitochondrial outer membrane protein FIS1. The drug is fused with the FIS1.MTS protein, and the targeting part of the FIS1 is contained, so that the drug can be loaded on the outer membrane of mitochondria.
In one embodiment of the invention, the drug may be fused to a fluorescent protein and a fish 1.mts protein, and then loaded on the surface of mitochondria. Among them, the fluorescent protein serves to label the drug, merely to follow and monitor the drug delivery, and is not a necessary means for achieving the 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 mReclet-I, and the like.
In one embodiment of the invention, the drug is loaded onto the outer membrane of the mitochondria by optogenetic means, for example by pMag and nMAG, Cry2-CIB or any other pair of optogenetic proteins. Specifically, in one embodiment of the invention, for example, the drug forms a fusion protein with pMag, nMag forms a fusion protein with a mitochondrial outer membrane localization sequence, such as fis1.mts protein, and the pair of pMag and nMag proteins pair under the influence of blue light bind in pair, such that the drug is loaded on the outer membrane of the mitochondria.
In some embodiments of the invention, in addition to using optogenetic proteins, the drug delivery system according to the invention may also use protein pairs that bind under the action of chemical substances, such as rapamycin regulated FKBP12 and FRB or any other reversibly or non-reversibly bound protein pair. In some embodiments of the invention, a pair of protein pairs may be used between the drug and the mitochondrial outer membrane localization sequence, multiple pairs of protein pairs may be used, one type of protein pair may be used, and multiple types of protein pairs may be used.
In one embodiment of the invention, the drug may be fused to a fluorescent protein and the above-described protein pair, and then loaded on the outer membrane of mitochondria. Among them, the fluorescent protein serves as a marker of the drug, only for tracking and monitoring the drug delivery, and is not necessary for achieving the 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 the like.
In one embodiment of the present invention, the drug delivery system according to the present invention is prepared by a process comprising the steps of:
(1) design of an X-Y-FIS1.MTS protein, wherein the protein comprises three parts: x is any target drug; y is a fluorescent protein for labeling a target drug; the MTS is a targeting polypeptide of mitochondrion outer membrane protein FIS 1;
(2) constructing plasmid of X-Y-FIS1.MTS protein by using pcDNA3.1 vector and transfecting HEK293T cell; and
(3) after successful expression, the X-Y-FIS1.MTS marked mitochondria in the cell are extracted and then co-cultured with the unmarked cell.
In one embodiment of the present invention, the drug delivery system according to the present invention is prepared by a process comprising the steps of:
(1) respectively designing X-Y1-pMag protein and nMAGHigh1-Y2-FIS1.MTS protein;
(2) constructing plasmids of the two proteins by using pcDNA3.1 vector and transfecting HEK293T cells to simultaneously express the two proteins;
(3) after 24 hours the cells were irradiated with blue light for 10 minutes to allow binding of the pair of pMag and nMagHigh1 proteins; and
(4) intracellular Y1 and Y2 labeled mitochondria were extracted and then co-cultured with unlabeled cells.
Wherein, X is any target medicine, Y1 and Y2 are fluorescent proteins for marking, pMag and nMaghigh1 are a pair of optogenetic protein pairs, FIS1.MTS is the target polypeptide of mitochondrion outer membrane protein FIS1.
Further, the fluorescent protein may be GFP, EGFP, mScarlet-i, YFP, CFP, or other fluorescent protein.
The plasmid vector may be pcDNA3.1, pEGFP, pCMVp-NEO-BAN, pSV2pUC19DNA, pUC18DNA, PQE30 or any other commonly used cloning vector.
The transfected cells may be HEK293T, CHO-K1, COS-7, HeLa, NIH-3T3, PC-3, A549, MCF-7, HuH-7, U-2OS, HepG2 or any other cells which may be transfected.
The transfection reagent may be Lipofectamine, X-tremeGENE 9, EscortTM III、Escort TM IV、NeuroPorter TMDOTAP methosulfate, Calcium phospate, or any other transfection reagent.
The transfection method may be liposome transfection, calcium phosphate transfection, electroporation transfection or any other transfection method.
The time for co-culturing the mitochondria and the cells can be 0.5 to 120 hours, preferably 6 to 24 hours.
< example 1>
Protein NeoR-mScplet-i-FIS 1.MTS was designed. Wherein NeoR is anti-Neomycin (Neomycin) protein and is a target drug; mScarlet-i is a red fluorescent protein used for marking a target drug; mts is a targeting sequence for the mitochondrial outer membrane protein FIS1, whereby the NeoR-mScarlet-i-FIS1 mts protein can be targeted to the outer membrane of mitochondria. NeoR-mScalet-i-FIS 1.MTS plasmid (see sequence Listing, SEQ ID NO:1) was constructed using pcDNA3.1 vector and transfected into HEK293T cells. After 24 hours of successful expression, the NeoR-mScarlet-i-fis1. mts-labeled mitochondria, i.e. mitochondria loaded with NeoR-mScarlet-i-fis1.mts fusion protein, in HEK293T cells were extracted and then co-cultured with unlabeled 293A cells for 6 hours. Cell membranes are stained by using a cell membrane dye CellMask Deep-red, and the 293A cells are observed to be mSclet-i positive by using a laser confocal microscope, so that the target drug NeoR is successfully delivered into the 293A cells. The results of the detection are shown in FIG. 1.
< example 2>
The proteins NeoR-mScalet-i-pMag and nMagHigh1-GFP-FIS1.MTS were designed. Wherein NeoR is the target drug, and mScarlet-i is a red fluorescent protein used for marking the target drug. The nMagHigh1-GFP-fis1.mts fusion protein can bind to the outer membrane of mitochondria via the mitochondrial outer membrane targeting sequence fis1. mts. pMag and nMAGH 1 are a pair of optogenetic proteins which bind to each other under blue light irradiation. Under the action of blue light, the fusion protein NeoR-mScarlet-i-pMag and the fusion protein nMAGHigh1-GFP-FIS1.MTS are combined with each other, so that the target drug NeoR is loaded on the outer membrane of mitochondria. Specifically, a NeoR-mScarlet-i-pMag plasmid (see a sequence table, SEQ ID NO:2) and an nMAGhigh1-GFP-FIS1.MTS plasmid (see a sequence table, SEQ ID NO:3) are constructed by using a pcDNA3.1 vector, the pcDNA3.1-NeoR-mScarlet-i-pMag and pcDNA3.1-nMAG high1-GFP-FIS1.MTS are co-transfected into 293T cells, and after 24 hours, the cells are irradiated with intense light for 10 minutes to combine the NeoR-mScarlet-i-pMag fusion protein with the nMAG high1-GFP-FIS1.MTS fusion protein, so that the target protein NeoR is loaded on the outer membrane of mitochondria. Then extracting mitochondria loaded with NeoR protein (mScarlet-i label), diluting the extracted mitochondria with DMEM complete culture medium, adding the diluted mitochondria into cultured 293A cells without labels, incubating the cells at 37 ℃ for 6-8 hours, staining cell membranes by using cell membrane dye CellMask Deep-red, and detecting by laser confocal microscope imaging, wherein partial mScarlet-i label target protein NeoR enters the 293A cells. The results of the detection are shown in FIG. 2.
< example 3>
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) were constructed. Wherein NeoR is a target drug, mScplet-i is red fluorescent protein and is used for marking the target drug, and NB. GFP serves to label mitochondria and mimic mitochondrial outer membrane proteins, and fis1.mts is the targeting sequence for the mitochondrial outer membrane protein FIS1. 293T cells of 2 10cm culture dishes were transfected with each plasmid, and 24 hours later, cells expressing NeoR-mScardlet-i-NB.GFP were harvested, swelled with distilled water for 5 minutes, added with 1/9 volumes of 10 XPBS, disrupted with a glass homogenizer, centrifuged at 10000g for 10 minutes, and the supernatant containing NeoR-mScardlet-i-NB.GFP was taken for use. Cells expressing GFP-FIS1.MTS were harvested, mitochondria were extracted and resuspended in a supernatant containing NeoR-mSCarlet-i-NB. GFP, incubated with unlabeled 293A cells for 10 minutes at room temperature, incubated for 6-8 hours in a 37 ℃ medium, stained for cell membrane with the cell membrane dye CellMask Deep-red, and examined by confocal laser microscopy imaging, to find that a portion of the GFP-labeled mitochondria had successfully transferred the mSCarlet-i-labeled NeoR into the cells. The results of the detection are shown in FIG. 3.
< example 4>
Protein COX8. MTS-hCATALase-mSCARET-i, IL10-mNeonGreen-FIS1.MTS was designed. In the protein COX8. MTS-hCATALase-mSALLE-i, hCATALase is human Catalase and is a target drug, mSALLE-i is a red fluorescent protein and is used for marking the target drug, COX8.MTS is a targeting sequence of mitochondrial matrix protein COX8, and the protein COX8. MTS-hCATALase-mSALLE-i can be positioned to a matrix of mitochondria through the targeting sequence COX8. MTS. In the protein IL10-mNeon Green-FIS1.MTS, IL10 is a target drug, mNeon Green is a high-brightness green fluorescent protein which is used for marking the target drug, and the protein IL10-mNeon Green-FIS1.MTS can be combined to the outer membrane of mitochondria through a targeting sequence FIS1. MTS. Specifically, the pcDNA3.1 vector was used to construct COX8 MTS-hCATALase-mSCarlet-i plasmid (see sequence listing, SEQ ID NO:6) and IL10-mNeon Green-FIS1 MTS plasmid (see sequence listing, SEQ ID NO:7), 293T cells were co-transfected with both plasmids, hCATALase (mSCALE-I tag) and/or IL10(mNeon Green tag) loaded mitochondria were extracted after 24 hours, and the extracted mitochondria were diluted in DMEM complete medium, added to cultured 293A cells without tags, and after incubation for 6-8 hours at 37 ℃ in medium, membrane stain Maslk Deep-red, and detection was performed using confocal laser microscopy to find that part of the mNeon Green tagged hCATALase and mSCarlet-i tagged IL10 had entered the cell membrane. The results of the detection are shown in FIG. 4.
< example 5>
Plasmid pcDNA3.1-hTFAM1-CRT-mScarlet-i-FIS1.MTS (see sequence Listing, SEQ ID NO:8) was constructed in which hTFAM1 is the DNA binding domain of human TFAM1 and CRT is the DNA binding domain of TopII alpha, both of which bind DNA. mScalet-i is a red fluorescent protein used to label mitochondria carrying the fusion protein; mts is a signal peptide that targets the outer mitochondrial membrane. Transfecting a mouse 3T3 cell with pcDNA3.1-hTFAM 1-CRT-mScalet-i-FIS 1.MTS, expressing a fusion protein after 24 hours, and taking 107Mitochondria were extracted from each cell, resuspended in 100. mu.L DMEM complete medium, and 200ng plasmid pcDNA3.1-Mito-EGFP-2A-n.EGFP (see sequence listing, SEQ ID NO:9) was added and incubated at room temperature for 1 hour, where 2A allowed translation of one mRNA into two proteins, Mito-EGFP labeling mitochondria, and nEGFP labeling nuclei. Mitochondria were then co-cultured with unlabeled 293A cells and after 24 hours fluorescence microscopy images a visible portion of the cells expressing GFP labeled mitochondria and nuclei, indicating that the plasmid had been successfully transferred into the cells and expressed. The results of the detection are shown in FIG. 5.
< example 6>
Construction of plasmid pcDNA3.1-UP1-FIS1.MTS (see sequence Listing, SEQ ID NO:10), wherein UP1 is the non-specific RNA binding domain of human hnRNP A1 protein and FIS1.MTS is a signal peptide that targets the outer mitochondrial membrane. 293T cell was transfected with pcDNA3.1-UP1-FIS1.MTS, 10 hours later7Mitochondria were extracted from cells, resuspended in 100 μ l LDMEM complete medium, 200ng mRNA of Mito-mScarlet-I (obtained by in vitro transcription of plasmid pcdna3.1-Mito-mScarlet-I (see sequence listing, SEQ ID NO: 11)), incubated at room temperature for 0.5 hour, then incubated with unlabeled 293A cells, 24 hours later the cell membranes were stained with the cell membrane dye CellMask Deep-red, laser confocal microscopy imaged a red signal indicating that the labeled mitochondria were expressed within the visible portion of the cell, indicating that mRNA has been successfully transferred to and translated into the cell. The results of the detection are shown in FIG. 6.
Claims (10)
- A mitochondrial-based drug delivery system, the drug delivery system comprising:(1) mitochondria, and(2) a drug loaded on the mitochondria so as to be capable of inducing a drug-induced response,wherein the drug is loaded to the mitochondria by means other than free diffusion.
- The drug delivery system of claim 1, wherein the source of mitochondria is autologous, allogeneic or xenogeneic, and combinations thereof.
- The drug delivery system of claim 1, wherein the drug comprises a proteinaceous drug such as a polypeptide drug, an antibody drug, a cytokine, an enzyme, a proteinaceous vaccine, a polypeptide hormone, a protein translated from a normal gene, a protein derivative, a nucleic acid drug such as DNA, RNA, or the like, and combinations thereof.
- The drug delivery system of claim 1, wherein the drug is loaded reversibly or irreversibly on the outer mitochondrial membrane, inner mitochondrial membrane, interstitial space or mitochondrial matrix and combinations thereof by covalent or non-covalent bonding.
- The drug delivery system of claim 1, wherein the drug delivery system effects delivery of a drug into a cell, wherein the cell comprises an inner surface of a cell membrane, a cytoplasm, a nucleus, and combinations thereof.
- The drug delivery system of claim 1, wherein the drug is present in a state of being bound to or separated from the mitochondria after being delivered into a cell.
- The drug delivery system of claim 1, wherein the drug is a proteinaceous drug, wherein the proteinaceous drug is loaded onto the mitochondria in a manner comprising:(1) fused to a mitochondrial localization sequence, e.g., a mitochondrial outer membrane localization sequence such as fis1.mts or a mitochondrial matrix localization sequence such as cox8.mts, bound to mitochondria as a fusion protein;(2) fused to an amino acid sequence that specifically binds to mitochondria, such as an antibody, a variable region of an antibody-binding antigen, or a nanobody, or other non-antibody but capable of binding to mitochondria, thereby binding to mitochondria;(3) binding to mitochondria occurs by means of chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der waals forces, hydrogen bonds, aromatic ring stacking interactions, hydrophobic forces, and halogen bonds, and combinations thereof.
- The drug delivery system of claim 1, wherein the drug is a nucleic acid drug, wherein the nucleic acid drug is loaded on the mitochondria in a manner comprising:(1) binding to mitochondria via a nucleic acid binding protein or nucleic acid binding domain thereof, or(2) Binding to mitochondria is by means of chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der waals forces, hydrogen bonds, aromatic ring stacking interactions, hydrophobic forces, and halogen bonds, and combinations thereof.
- Use of a drug delivery system according to any one of claims 1 to 8 for the preparation of a formulation for the treatment of a disease, prevention of a disease, cosmetic, weight loss, anti-fatigue, promotion of growth and development, enhancement of body function or delay of aging.
- The use of claim 9, wherein the disease comprises a neurological disease, an immune system disease, a respiratory system disease, a circulatory system disease, a urinary system disease, a reproductive system disease, a motor system disease, an endocrine system disease, a digestive system disease, a monogenic genetic disease, a polygenic genetic disease, an infectious disease, a tumor, diabetes, a neurodegenerative disease, or a cardiovascular disease and combinations thereof.
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| US20100111911A1 (en) * | 2007-02-16 | 2010-05-06 | John Guy | Mitochondrial nucleic acid delivery systems |
| EP2992892A1 (en) * | 2014-09-05 | 2016-03-09 | Universität zu Köln | Fusion protein for use in the treatment of mitochondrial diseases |
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