CN115068502A - Mitochondrial delivery system and preparation thereof - Google Patents

Abstract

The invention provides a mitochondrial delivery system which is a cell suspension, a vesicle suspension or a combination thereof. The delivery system provides a means of supplementing mitochondria, providing active mitochondria within the recipient's cell, tissue, organ, system via a cell or cell vesicle.

Description

Mitochondrial delivery system and preparation thereof

Technical Field

The present invention is in the field of biomedicine, and in particular relates to a cell and/or vesicle suspension for delivery of mitochondria, and mitochondrial preparations comprising the cell and/or vesicle suspension.

Background

Mitochondria are organelles evolved from archaebacteria billions of years ago, are the site of aerobic respiration within cells, and provide most of the energy for eukaryotic cells. Since all life activities are energy dependent, mitochondria have a vital role for life. Aerobic respiration is accompanied by the production of high concentrations of reactive oxygen species which tend to react with and damage biological macromolecules such as proteins, lipids, nucleic acids, and thus mitochondria are prone to dysfunction. Mitochondrial dysfunction is currently known to be associated with almost all diseases except trauma, including diabetes, neurodegenerative diseases, tumors, autoimmune diseases, and the like. In addition, mitochondrial dysfunction is also considered to be a key cause of aging. With age, the number of mitochondria in the cell decreases and mitochondrial DNA mutations also increase. In a mouse, if the mitochondrial DNA mutation rate is increased, the mouse lifespan is shortened, and if the mitochondrial DNA mutation rate is decreased, the mouse lifespan is extended.

Since mitochondrial dysfunction is responsible for many diseases and aging, correcting mitochondrial dysfunction can prevent and treat diseases and delay aging. Currently, there are two main approaches to correct mitochondrial dysfunction: and (5) repairing and supplementing. Repair is the removal of damaged or mutated mitochondria with specific drugs or methods. The supplementation is by exogenous supplementation of good mitochondria to increase the energy supply of the cell. Although the repair method is low in cost, the effect is limited, and the problem cannot be fundamentally solved. Although the supplementing method theoretically takes effect more quickly and has better effect, a high-efficiency and easy-to-use mitochondrial supplementing method is lacking at present.

Mitochondria isolated from cells have been found to have "infectivity" similar to viruses as early as 1982, and can enter cells from the outside and survive. There have also been some studies reporting that diseases can be treated by injecting isolated mitochondria into ischemic myocardium. However, injection of isolated mitochondria has several disadvantages. First, the extraction efficiency of mitochondria is low, and a certain proportion of mitochondria are lost or active mitochondria are inactivated during the extraction process. In addition, extracellular mitochondria cannot enter the cell one hundred percent, and our experience is that adding extracted mitochondria to cultured cells has about 1/3 cells that can enter, the remaining 2/3 cannot. It has been reported that the efficiency of mitochondrial entry into cells can be increased by coating mitochondria with liposomes, but liposome coating increases the operational difficulty and greatly increases the cost. Finally and most importantly, isolated mitochondria can lose activity quickly. At room temperature, mitochondria lose more than eighty percent of their activity for two to three hours. If cryopreserved, at least ninety percent of the activity is lost after thawing. Even when the inactivated mitochondria can enter the cell, they do not improve the function of the mitochondria. The application scenario of the separated mitochondria as a biological agent is greatly restricted due to the fact that the mitochondria cannot be stored for a long time. Therefore, in order to correct mitochondrial dysfunction or promote mitochondrial function, a highly efficient and easy-to-use method for mitochondrial administration is needed.

Disclosure of Invention

It is therefore an object of the present invention to develop a new method for delivering mitochondria that is safe and convenient. In particular, in one aspect of the invention, the invention provides a mitochondrial delivery system for supplementing active mitochondria, the delivery system being a cell suspension, a vesicle suspension, or a combination thereof, the volume ratio of cells in the cell suspension, the volume ratio of vesicles in the vesicle suspension, or the volume ratio of cells plus vesicles in the composition being from 0.001% to 99.9%. The cells in the cell suspension are single cells, multiple cells, or a combination thereof containing mitochondria. The vesicles in the vesicle suspension are vesicles which are wrapped by the membrane structure of cells, have diameters of more than 100nm, contain mitochondria and have no activity of intact cells, and have or not cell nuclei.

Further, the source of the cell suspension or vesicle suspension is tissue or blood.

Further, the source of the cell suspension or vesicle suspension is cells cultured in vitro.

Further, the cell suspension or vesicle suspension is derived from autologous, allogeneic or xenogeneic sources and combinations thereof.

Further, the source of the cell suspension or vesicle suspension is germ cells and/or somatic cells.

Further, the source of the cell suspension or vesicle suspension is stem cells, non-stem cells, and combinations thereof.

Further, the source of the cell suspension or vesicle suspension is a tumor cell, a non-tumor cell, or a combination thereof.

Further, the source of the cell suspension or vesicle suspension comprises neural cells, glial cells, epidermal cells, osteocytes, chondrocytes, cardiomyocytes, hepatocytes, splenocytes, pancreatic cells, renal cells, adrenal cells, adipocytes, muscle cells, leukocytes, platelets, or a combination thereof.

Further, the source of the cell suspension or vesicle suspension comprises neural tissue, skin, bone, cartilage, heart, liver, spleen, kidney, pancreas, lung, blood vessel, testis, ovary, oral tissue, esophagus, stomach, small intestine, large intestine, fat, muscle, blood, or a combination thereof.

Further, the cell suspension or vesicle suspension is freshly prepared or cryopreserved.

Further, the administration modes of the cell suspension or vesicle suspension include intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, aerosol inhalation, sublingual buccal administration, nasal administration and oral administration.

In another aspect, the invention provides a formulation for delivery of mitochondria comprising a mitochondrial delivery system according to the invention, i.e. comprising a cell suspension or a vesicle suspension according to the invention and combinations thereof. Further, the mitochondrial preparation further comprises physiological saline, albumin, cell culture medium, buffer, blood, plasma, serum, tissue fluid, cryoprotectant antibiotic, serum, growth factor, or a combination thereof.

In one embodiment, the formulation is a cell suspension formulation. Further, the cell suspension consists of single cells, multiple cells, or a combination thereof. Preferably, the cell suspension is a cell suspension prepared from liver, spleen, kidney, muscle, brain tissue, pancreas, fat, leukocytes, platelets, cells cultured in vitro, or a combination thereof.

In one embodiment, the formulation is a vesicle suspension formulation. Preferably, the vesicle suspension is a vesicle suspension prepared from brain tissue. In one embodiment, the vesicle suspension is a cell vesicle suspension without intact cell activity.

In yet another aspect, the present invention provides use of the mitochondrial preparation according to the present invention for the preparation of a medicament and a health product for preventing and treating diseases, promoting physical functions, beautifying, or anti-aging. The innovation points to be protected of the invention are as follows: administration of cells or cell vesicles is used to deliver mitochondria.

The preparation for delivering mitochondria of the present invention has the following advantages:

1. the source of the cells is wide, and a large number of cells can be easily obtained without any modification.

2. The cells can be obtained from a patient body, and the immune rejection reaction can hardly be generated after the cells are injected into the patient body through in vitro culture.

3. The isolated mitochondria are difficult to replicate in vivo except for their DNA, the duration of their effects of transplantation is short, and cells can constantly synthesize new mitochondria and supply them to recipients, and the duration of their effects is long.

4. Mitochondria in cells or vesicles maintain activity for longer periods of time at normal temperature or under frozen conditions relative to isolated mitochondria.

5. Stem cells need not be used. Stem cells are limited in their source, limited in the number of passages that can divide, and require more stringent culture conditions, so the number of cells that can be returned to the body is lower. If the stem cells are not needed, the number of cells obtained is increased, the culture is simpler, and the number of cells which can be input is greatly increased.

Drawings

Figure 1 shows the activity of mitochondria isolated in example 1 compared to mitochondria in cell/vesicle suspensions after thawing in cryopreserved.

FIG. 2 shows the successful transfer of mNeonGreen-labeled mitochondria into different tissues of recipient mice in 293T cells in example 2.

FIG. 3 shows a graph of the successful transfer of GFP-labeled mitochondria into the heart and brain of recipient mice from cell suspensions and vesicle suspensions of mice in example 3.

Detailed description of the preferred embodiments

Preparation of mouse tissue cell suspension

The mouse is killed by cervical dislocation, and tissues and organs such as liver, spleen, kidney, fat, pancreas, thigh muscle, blood and the like are taken, blood is anticoagulated by heparin, and white blood cells are separated by a Solarbio mouse white blood cell separation kit. After the rest of the tissues were minced, 5-10 times by weight of PBS (phosphate buffered saline, pH 7.2-7.4) was added, and the different tissues were dispersed as much as possible into single cells under different homogenization conditions while maintaining the integrity of the cells. Homogenizing adipose tissues at 1200rpm for 2 minutes, filtering by 30 meshes, 60 meshes and 150 meshes in sequence to remove large tissues and cell lumps, filtering by 600 meshes to remove broken cells and free mitochondria, and intercepting by 600 meshes to obtain the adipose cells. After homogenizing the muscles of the liver, spleen, kidney, pancreas and thigh, removing large tissues or overlarge cell masses by low-speed centrifugation and discarding precipitates, and obtaining separated cells by a method of removing broken cells and free mitochondria by medium-speed centrifugation and taking the precipitates. Wherein, liver, spleen and pancreas are homogenized for 1.5min at 1300 rpm, 50g is centrifuged for 3min, supernatant is taken, then 220g is centrifuged for 4min, supernatant is discarded, PBS is resuspended, and then 220g is centrifuged for 4min, and supernatant is discarded, thus obtaining single cell and oligo cell sediment; homogenizing muscle tissue at 7000rpm for 1min, centrifuging at 300g for 3min, collecting supernatant, centrifuging at 220g for 4min, discarding supernatant, resuspending PBS, centrifuging at 220g for 4min, and discarding supernatant to obtain single cell and oligo cell precipitate; homogenizing at 1100 rpm for 2min, centrifuging at 50g for 2min, collecting supernatant, centrifuging at 220g for 4min, discarding supernatant, resuspending in PBS, centrifuging at 220g for 4min, and discarding supernatant to obtain single cell and oligo cell precipitate. The dispersion of the cell suspension and the activity of the cells were examined by staining with MitoTracker Red. Finally, according to the weight of the precipitate, converting the precipitate into volume according to the density of 1g/ml, and resuspending the precipitate with the required solution to prepare the cell suspension with the required concentration.

Preparation of brain tissue vesicle suspension

Killing a mouse by a cervical dislocation method, taking brain tissue, adding PBS (5-10 times of the weight of the brain tissue), homogenizing for 2min by an electric homogenizer at 1100 rpm, centrifuging for 2min at 200g, taking supernatant, centrifuging for 5min at 1500g, discarding the supernatant, resuspending by PBS, repeating the centrifugation for 5min at 1500g, discarding the supernatant, and precipitating to obtain the brain tissue vesicle. Finally, according to the weight of the precipitate, the volume is converted according to the density of 1g/ml, and the required solution is used for resuspension to prepare the vesicle suspension with the required concentration.

Preparation of in vitro cultured cell suspension

293T cells (human embryonic kidney cells) and GC-2spd cells (mouse spermatocyte) are cultured according to a conventional method, firstly, pancreatin is used for enzymolysis, then, the cells are repeatedly blown and beaten into single cell suspension, after the cells are counted, 300g is centrifuged for 5min, supernatant is discarded, and precipitates are resuspended by required solution to prepare the cell suspension with required concentration.

Example 1

After the GC-2spd cells and 293T cells which are cultured conventionally are digested by pancreatin, half of the cells are extracted by a Biyunnan cell mitochondria isolation kit (C3601), and half of the cells are directly prepared into cell suspension by a DMEM complete culture medium (high-sugar DMEM.10% FBS.1xP/S) according to the method. Adding 1M sucrose solution into both the mitochondria suspension and the cell suspension until the final concentration of sucrose is 6%, and putting the mixture into a programmed cooling box for freezing and storing at-80 ℃. Preparing brain tissue vesicles into suspension by using a DMEM complete culture medium, adding 1M sucrose solution until the final concentration of sucrose is 6%, and placing the suspension into a programmed cooling box for freezing and storing at-80 ℃. After 10 days the cells were rapidly thawed in a 37 ℃ water bath and mitochondrial activity was detected by MitoTracker Red staining, CellMask deep staining to mark cell and mitochondrial membranes.

Fig. 1 shows the results of comparing the activity of mitochondria thawed after cryopreservation. (A) Mitochondria extracted from mouse GC-2spd cells were cryopreserved for 10 days and stained after thawing. A1 is CellMask deep Red showing mitochondrial membranes, a2 is MitoTracker Red staining showing mitochondrial activity, A3 is the combination of a1 and a 2. A2 was seen to have almost no staining, indicating that mitochondria purified from GC-2spd cells were almost inactive after cryopreservation and thawing. (B) Mouse GC-2spd cells were cryopreserved for 10 days and stained after recovery. B1 is CellMask deep Red showing cell membranes, mitochondrial membranes and other intracellular membrane structures, B2 is MitoTracker Red staining showing mitochondrial activity, B3 is a combination of B1 and B2. B2 was visibly stained, indicating that the mitochondrial activity remained good after freezing and thawing GC-2spd cells. (C) Mitochondria extracted from 293T cells were cryopreserved for 10 days and stained after thawing. C1 is CellMask deep Red showing mitochondrial membranes, C2 is MitoTracker Red staining showing mitochondrial activity, C3 is the combination of C1 and C2. Little staining was seen with C2, indicating that 293T cell purified mitochondria were virtually inactive after cryopreservation and thawing. (D) 293T cells were cryopreserved for 10 days and stained after recovery. D1 is CellMask deep Red showing cell membranes, mitochondrial membranes and other intracellular membrane structures, D2 is MitoTracker Red staining showing mitochondrial activity, D3 is a combination of D1 and D2. D2 was visibly stained, indicating that the mitochondrial activity of 293T cells remained good after cryopreservation and thawing. (E) The brain tissue vesicles are frozen for 10 days, and are stained after recovery. E1 is CellMask deep Red showing cell membranes, mitochondrial membranes and other intracellular membrane structures, E2 is MitoTracker Red staining showing mitochondrial activity, E3 is a combination of E1 and E2. E2 was clearly stained, indicating that the mitochondrial activity remained good after cryopreservation and thawing of the brain tissue vesicle suspension.

Example 2

293T cell transfection plasmid pcDNA3.1-mNeon Green-FIS1.MTS, 2 days later, collecting transfected cells, a part of which is directly prepared into 1000 ten thousand/ml cell suspension by normal saline, then injecting the cell suspension into a mouse by a tail vein with a dose of 0.05ml/10g, a part of which is prepared into 1000 ten thousand/ml cells by a DMEM complete culture medium, adding 1M sucrose until the final concentration is 6 percent, freezing the cell suspension at-80 ℃ for 10 days, and injecting the cell suspension into the mouse by a tail vein with a dose of 0.05ml/10g after recovery. Mice were sacrificed by cervical dislocation 3 hours after cell injection, and hearts, livers, kidneys, muscles, brains were sliced and stained with MitoTracker Red, and observed by Leica SP8 Confocal imaging, showing scattered mNeon green-labeled mitochondria in different tissues of recipient mice, with the results for hearts and brains as shown in fig. 2. (A) The heart of a recipient mouse receiving fresh 293T cells, A1 stained with MitoTracker Red, A2 marked with donor mitochondria, A3 marked with combination of A1 and A2 shows that mitochondria marked with mNeon Green appear in the myocardium of the recipient mouse, which indicates that the mitochondria in the 293T cells are transferred into the myocardium of the recipient mouse. (B) The result of the staining of the liver of the recipient mouse receiving fresh 293T cells, wherein B1 is MitoTracker Red staining, B2 is mNeon Green labeling donor mitochondria, B3 is the combination of B1 and B2, the appearance of mNeon Green labeled mitochondria in the recipient liver cells can be seen, and the fact that the mitochondria in the 293T cells are transferred into the liver cells of the recipient mouse is indicated. (C) The result of staining of the brain of the recipient mouse receiving fresh 293T cells, wherein C1 is MitoTracker Red staining, C2 is mNeon Green labeling donor mitochondria, C3 is the combination of C1 and C2, the appearance of mNeon Green labeled mitochondria in the recipient brain tissue can be seen, and the fact that the mitochondria in the 293T cells are transferred into the recipient brain is indicated. (D) 293T cells are injected into mice after being frozen for one week, the heart of a receptor mouse is stained, D1 is MitoTracker Red staining, D2 is mNeon Green labeling donor mitochondria, D3 is the combination of D1 and D2, and the appearance of mNeon Green labeled mitochondria in the brain tissue of the receptor can be seen, which indicates that the mitochondria in the 293T cells are transferred into the heart muscle of the receptor.

Example 3

Several 6-8 weeks old C57 mice expressing Mito-GFP (mitochondrial matrix labeled with GFP) were sacrificed by cervical dislocation, cells were obtained by the above method, prepared into 20% cell suspension with physiological saline, and then injected subcutaneously into Kunming mice at the neck at a dose of 0.1ml/10g, and the cell suspension of each tissue organ was injected into 1-2 Kunming mice; brain tissue was collected, and 1% of the brain tissue vesicle suspension was prepared with PBS according to the above method, and then injected into 1-2 Kunming mice at a dose of 0.05ml/10g via tail vein. Mice were sacrificed by cervical dislocation 12 hours after subcutaneous injection or 3 hours after tail vein injection, and hearts and brains were sliced and stained with MitoTracker Red, and observed by Leica SP8 Confocal imaging, with scattered GFP-labeled mitochondria visible in both hearts and brains of recipient mice injected with cell suspensions of heart, liver, spleen, kidney, fat, pancreas, muscle, white blood cells, some of which are shown in fig. 3. (A) The heart of the recipient mouse injected with the hepatocyte suspension, A1 stained with MitoTracker Red, A2 labeled with donor mitochondria, A3 labeled with GFP and combined A1 and A2, shows that the mitochondria labeled with GFP appear in the myocardium of the recipient mouse, and indicates that the mitochondria in the hepatocyte have been transferred into the myocardium of the recipient mouse. (B) The heart of a receptor mouse which receives the injection of the brain tissue vesicle suspension, B1 is MitoTracker Red staining, B2 is GFP for marking donor mitochondria, B3 is the combination of B1 and B2, and the phenomenon that the mitochondria marked by the GFP appear in the receptor myocardial cells is shown, which indicates that the mitochondria in the brain vesicle are transferred into the myocardial cells of the receptor mouse. (C) The brain tissue of a recipient mouse injected by the fat cell suspension, C1 is MitoTracker Red staining, C2 is GFP for marking donor mitochondria, C3 is the combination of C1 and C2, and the phenomenon that the mitochondria marked by the GFP appear in the brain tissue of the recipient can be seen, which indicates that the mitochondria in the fat cell have been transferred into the brain of the recipient. (D) The staining result of the recipient mouse brain tissue injected by the pancreatic cell suspension is received, D1 is MitoTracker Red staining, D2 is GFP for marking donor mitochondria, D3 is the combination of D1 and D2, and the appearance of the GFP-marked mitochondria in the recipient brain tissue is shown, which indicates that the mitochondria in the pancreatic cell are transferred into the recipient brain tissue.

The above embodiments are only preferred embodiments of the present invention, and should not be construed as limiting the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should fall within the protection scope of the present invention. The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be considered as falling within the protection scope of the present invention.

Claims (20)

1. A mitochondrial delivery system, wherein the delivery system is a cell suspension, a vesicle suspension, or a combination thereof.

2. The delivery system of claim 1, wherein the volume ratio of cells in the cell suspension, vesicles in the vesicle suspension, or cells plus vesicles in the combined suspension is 0.001% -99.9%.

3. The delivery system of claim 1 or 2, wherein the cells in the cell suspension are single cells containing mitochondria, multicellular, or a combination thereof.

4. The delivery system of claim 1 or 2, wherein the vesicles in the vesicle suspension are vesicles that are encapsulated by the membrane structure of the cell, are above 100nm in diameter, contain mitochondria, and have no intact cellular activity, with or without nuclei.

5. The delivery system of claim 1 or 2, wherein the source of the cell suspension or vesicle suspension is tissue or blood.

6. The delivery system of claim 1 or 2, the source of the cell suspension or vesicle suspension being cells cultured in vitro.

7. The delivery system of claim 1 or 2, wherein the source of the cell suspension or vesicle suspension comprises neural cells, glial cells, epidermal cells, osteocytes, chondrocytes, cardiomyocytes, hepatocytes, splenocytes, pancreatic cells, renal cells, adrenal cells, adipocytes, muscle cells, leukocytes, platelets, or a combination thereof.

8. The delivery system of claim 1 or 2, the source of the cell suspension or vesicle suspension being liver cells, kidney cells, pancreatic cells, adipocytes, muscle cells, nerve cells, spleen cells, leukocytes, or a combination thereof.

9. The delivery system of claim 1 or 2, wherein the source of the cell suspension or vesicle suspension is autologous, allogeneic or xenogeneic, and combinations thereof.

10. The delivery system of claim 1 or 2, wherein the cell suspension or vesicle suspension source is germ cells, somatic cells, or a combination thereof.

11. The delivery system of claim 1 or 2, wherein the source of the cell suspension or vesicle suspension is a tumor cell, a non-tumor cell, or a combination thereof.

12. The delivery system of claim 1 or 2, wherein the source of the cell suspension or vesicle suspension is non-stem cells.

13. The delivery system of claim 1 or 2, wherein the source of the cell suspension is cells other than mesenchymal liver cells and platelets.

14. The delivery system of claim 1 or 2, wherein the source of the cell suspension or vesicle suspension is a cell line.

15. The delivery system of claim 1 or 2, wherein the cell suspension or vesicle suspension is freshly prepared, cryopreserved, or a combination thereof.

16. The delivery system of claim 1 or 2, wherein the cell or vesicle suspension is administered by a route selected from the group consisting of intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, aerosol inhalation, sublingual buccal, nasal administration, gastric lavage and oral administration.

17. A mitochondrial preparation characterized in that it comprises a delivery system according to any one of claims 1 to 16.

18. The mitochondrial preparation of claim 17, wherein the mitochondrial preparation further comprises physiological saline, albumin, cell culture media, buffers, blood, plasma, serum, interstitial fluid, cryoprotectants, and combinations thereof.

19. Use of the delivery system according to any one of claims 1 to 16 or the mitochondrial preparation according to claim 17 or 18 for the preparation of a medicament or nutraceutical for the prevention and treatment of diseases, for promoting physical function, for cosmetic or anti-aging.

20. The use of claim 19, wherein the disease comprises a mitochondrial dysfunction-related disease.

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