CN117487737A - Fe-containing material 3 O 4 Enucleated cells of nanoparticles, preparation thereof and application thereof in anti-aging - Google Patents

Abstract

The invention relates to a Fe-containing alloy ₃ O ₄ Preparation of enucleated cells of nanoparticles and application of enucleated cells in anti-aging, and Fe-containing nanoparticles ₃ O ₄ The size of the enucleated cells of the nano particles is 10 mu m-15 mu m, and the enucleated cells comprise Fe ₃ O ₄ Nanoparticle, mitochondria, do not contain nuclei. The enucleated cells have large diameters and can wrap and transmit more mitochondria. The enucleated cells and target cells are incubated in VSV-G concentrated solution, vesicular stomatitis virus proteins are inserted on cell membranes, and the distance between the membranes is changed under the action of magnetic forceAnd the cells are fused under the action of citric acid buffer solution by changing the membrane tension. The invention improves the cell fusion efficiency through magnetic force and higher fusion temperature, and the transplanted mitochondria maintain normal reticular structure and membrane potential, improves the biological activity of the mitochondria, can effectively enter target cells, can solve the problems of mitochondrial mutation and dysfunction, is used for transferring biological macromolecules and organelles, and can also be used for resisting aging.

Description

An enucleated cell containing Fe3O4 nanoparticles and its preparation and use in anti-aging application

Technical field

The invention belongs to synthetic biology technology, and specifically relates to an enucleated cell containing Fe ₃ O ₄ nanoparticles and its preparation and application in anti-aging.

Background technique

Mitochondria are semi-autonomous organelles that provide most of the energy required for cellular biochemical reactions. Mitochondria are also involved in numerous basic activities of the cell. Mitochondrial DNA (mtDNA) is prone to mutations. These mutations may be Mendelian inherited, maternally inherited, sporadic, or break out at a certain stage of development, leading to mitochondrial dysfunction, which plays an important role in aging, metabolic diseases, and neurodegeneration. Diseases, neuromuscular diseases, and cancers all play an important role. Therefore, mitochondria are now increasingly becoming a research hotspot and a target of concern for biomedical companies.

Mitochondrial transplantation occurs naturally in the body and can also be transferred in cell cultures outside the body through structures called tunnel nanotubes. Additionally, cells can release vesicles containing mitochondria and transfer them to surrounding cells. The above-mentioned mitochondrial transplantation has been reported to result in reduction of free radicals (ROS) and apoptosis. However, the efficiency of mitochondria transplanted by these pathways is underground, limiting their biomedical value.

There are currently many studies reporting purified mitochondrial transplantation, and mitochondrial transplantation is said to be effective against mitochondrial co-dysfunction in various research models and even clinical studies. McCully and colleagues reported that mitochondrial transplantation reduced myocardial infarct size, reduced cardiomyocyte loss, and improved postischemic myocardial function. Zhang et al reported that mitochondrial transplantation can reduce cerebral infarct volume and reverse neurological deficits in a stroke rat model. However, the biological mechanism underlying the above studies has not been elucidated, leading to the application of purified mitochondria being questioned.

Enucleated cells are well-known biological carriers that can be used to deliver normal mitochondria or patient mitochondria containing mutated DNA to cells lacking mitochondria (Rho0 cells). In the above situation, mitochondria are surrounded by a plasma membrane, which protects them from severe environmental damage and remains intact. Plasma membrane vesicles (PMVs), a type of small enucleated cells obtained by mechanically squeezing cells, can be used to transplant functional mitochondria. Rho0 cells and acetaminophen-injured HepG2 liver cells were well protected after mitochondrial transplantation, and the cells were protected from death and gained proliferative activity. However, this approach relies heavily on cell membrane fusion, which is notoriously difficult and often results in cell death.

Membrane fusion is a fundamental process in biological cells, involved in membrane biogenesis, intracellular transport, hormone secretion, and viral infection. Membrane fusion undergoes a series of well-defined intermediate steps, the so-called semifusion model. These steps are governed by energy barriers, and various types of fusogenic reagents, such as proteins, peptides, lipids, and ions, have been reported to overcome the energy barriers of the fusion process. Energy barriers can also be released through mechanical or chemical techniques, such as PEG induction and electrical stimulation, but these methods are inefficient and lead to severe cytotoxicity.

Contents of the invention

The purpose of the present invention is to provide enucleated cells containing Fe ₃ O ₄ nanoparticles and their preparation and application in anti-aging, and to promote cell fusion mediated by the fusion-capable vesicular stomatitis virus glycoprotein VSV-G under the action of magnetism. And the transplantation of mitochondria can achieve efficient delivery of functional mitochondria to solve the problems existing in the existing technology.

A method for preparing enucleated cells containing Fe ₃ O ₄ nanoparticles, including the following steps:

(1) Plate BMSC cells in a well plate; preferably a 24-well plate, the cell density is about 50-70% (about 1x10 ³ -1x10 ⁶ cells), and the culture medium volume is 500 μL;

(2) Add 0.5-2 mg/mL Fe ₃ O ₄ nanoparticles, place the well plate on a magnet and incubate for 12-24 hours, discard the supernatant, and wash;

(3) Place the tube upside down in a centrifuge tube and add centrifuge solution; place the tube in a preheated centrifuge and centrifuge for 40-60 minutes at 30-35°C and 5000-10000 rpm;

(4) Add cell culture medium and place in a cell culture incubator to recover for 30-60 minutes to obtain enucleated cells containing Fe ₃ O ₄ nanoparticles.

Further, the centrifuge solution contains 10% sucrose, 1 mg/mL cytochalasin B, 100 μM calcium chloride, 500 mM NAC antioxidant and 10 mg/mL colchicine.

Enucleated cells containing Fe ₃ O ₄ nanoparticles prepared by the above preparation method.

An enucleated cell containing Fe ₃ O ₄ nanoparticles, with a size of 10µm~15µm; including Fe ₃ O ₄ nanoparticles and mitochondria, but does not contain a cell nucleus. Fe ₃ O ₄ nanoparticles are loaded into bone marrow mesenchymal stem cells BMSCs; the enucleated cells contain stem macromolecules of stem cells; the surface of the enucleated cells integrates the fusogenic protein VSV-G.

Nanoparticles can wrap proteins and some small molecule drugs to easily enter cells. However, the larger the size of the nanoparticles, the efficiency of delivery will decrease, and the nanoparticles cannot wrap larger substances, such as mitochondria with a diameter of microns. The size of the enucleated cells containing Fe ₃ O ₄ nanoparticles of the present invention is about 10µm-15µm. Due to the magnetic force and higher fusion temperature, the fusion efficiency between the enucleated cells and the target cells is greatly improved, thereby significantly Promotes mitochondrial transfer efficiency.

As a new type of nanomaterial, iron oxide nanoparticles have good targeting, biocompatibility, biodegradability and biosafety; they show excellent physical and chemical properties such as biodistribution, metabolic rate and thermal stability. stability. The present invention effectively promotes membrane fusion efficiency by changing the membrane tension between cells and increasing intracellular calcium ion transients and cytoskeleton formation under the action of an external magnetic field.

BMSC bone marrow mesenchymal stem cells were selected, and the cells contained Fe ₃ O ₄ nanoparticles, and were enucleated by high-speed centrifugation. The enucleated cells obtained contained a large number of mitochondria. Mitochondria have a mesh-like structure and the membrane potential is normal.

Enucleated cells containing Fe ₃ O ₄ nanoparticles fuse with target cells under the influence of magnetism and high temperature, delivering a large number of functional mitochondria to cells with defective mitochondria. The transplanted mitochondria maintain normal network structure and membrane potential, function in the target cells and restore the normal functions of the cells.

An enucleated cell containing Fe ₃ O ₄ nanoparticles and integrating VSV-G.

A method for fusing enucleated cells containing Fe ₃ O ₄ nanoparticles with mitochondria-deficient cells, including the following steps:

(1) Digest the enucleated cells containing Fe ₃ O ₄ nanoparticles with digestive juice and collect by centrifugation;

(2) The collected enucleated cells containing Fe ₃ O ₄ nanoparticles are resuspended in VSV-G concentrated solution and added to the plated mitochondria-deficient cells;

(3) Place the well plate on a magnet and incubate it in a cell culture incubator for 2-4 hours;

(4) Aspirate the supernatant and add citrate buffer at 37-55°C for 1 minute;

(5) Aspirate the supernatant, add DMEM cell culture medium containing 10% serum and place it in a cell culture incubator for incubation.

The enucleated cells containing Fe ₃ O ₄ nanoparticles of the present invention are incubated with the target cells in a concentrated solution containing VSV-G. The cell membrane is attached with the vesicular stomatitis virus glycoprotein VSV-G, and is induced at 45°C under the influence of magnetism. Cell fusion can effectively promote mitochondrial transplantation.

^AccutaseTM digestive juice is preferred in the present invention. AccutaseTM digestive juice contains proteolytic enzyme and collagenase activities, which can gently and effectively digest cells without destroying cell surface antigens. It can achieve the separation of adherent cells within a few minutes and increase cell yield and survival. rate and enhance cell adhesion efficiency.

VSV-G concentrate is obtained by collecting the VSV-G conditioned culture medium and then concentrating it by ultracentrifugation. VSV-G conditioned culture medium was collected from Ad293 cells 48 hours after transfection with VSV-G expression plasmid.

Further, the preparation of VSV-G conditioned culture medium includes the following:

(1) Add 0.8 μg of vesicular stomatitis virus protein expression particles into a centrifuge tube, add 50 μL of serum-free culture medium and mix well;

(2) Add 3 μL Polyjet transfection reagent and 50 μL serum-free culture medium into another centrifuge tube, shake gently; then add to the solution obtained in step (1); obtain the transfection mixture, and let stand at room temperature for 15 minutes;

(3) After 15 minutes, aspirate the culture medium and add 1 mL of fresh serum-free culture medium. Immediately add the transfection mixture to the Ad293 cells and shake gently;

(4) Replace with new culture medium after 12 hours.

Further, the preparation method of VSV-G concentrate is as follows:

(1) Transiently transfect Ad293 cells with 0.8 μg vesicular stomatitis virus protein expressing plasmid;

(2) After 48 hours, the supernatant will be collected and the cells will be digested with trypsin and collected;

(3) The collected cells are resuspended in cell culture medium, freeze and thaw repeatedly 5 times, and then centrifuged to collect the supernatant;

(4) Collect the supernatants of (2) and (3) together, and collect the supernatant after centrifugation at 300g, 10min, 2000g, 10min and 10000rpm for 30min;

(5) Place (4) into a 6mL ultracentrifuge tube, ultracentrifuge at 47000 rpm, 4°C for 2 hours, and collect 1 mL of precipitate from each tube, which is the concentrated VSV-G protein conditioned culture medium.

Further, the mitochondria-deficient cells include Rho0 cells and senescent cells.

Further, the number of enucleated cells containing Fe ₃ O ₄ nanoparticles is: 1x10 ³ -1x10 ⁶ , the concentration of the VSV-G concentrated solution is 0.5-5mg/mL, and the incubation volume is 10-100L.

Further, the citric acid concentration is 0.8-1mM, and the pH is 4.5-6.0. Add the citric acid solution to the cell culture medium and account for 1/10 of the total volume, which is the citric acid buffer. The magnetic force of the magnet is 1500Gs-5000Gs.

The application of the above-mentioned enucleated cells containing Fe ₃ O ₄ nanoparticles in delivering biological macromolecules and organelles is non-disease diagnosis and treatment.

Enucleated cells containing Fe ₃ O ₄ nanoparticles can be used for mitochondrial transplantation into target cells.

The target cell may be the mitochondria-deficient cell Rho0. Rho0 cells were obtained from C2C12 cells after continuous treatment with dideoxycytosine (10 μM) and ethidium bromide (5 μM) for 7 days.

Target cells can also be senescent cells. Senescent cells can be induced from C2C12 cells. Induction conditions were: incubate in 2% FBS serum culture medium containing D-gal (40 g/L) for one week, and then incubate in culture medium containing R6G (5 μg/mL) for 1 day.

Application of the above-mentioned enucleated cells containing Fe ₃ O ₄ nanoparticles in anti-aging.

The invention introduces a magnetic force to bring enucleated bone marrow mesenchymal stem cells (BMSCs) into close contact with target cells. pH-induced membrane fusion is performed at 45ºC, with higher temperatures favoring breaking the liquid crystalline state of the cell membrane. Fusion results in enucleated BMSCs with intact mitochondria being transplanted into target cells such as Rho0 cells and senescent cells. Similarly, mitochondria were successfully transplanted into gastrocnemius muscle cells.

Compared with the existing technology, the present invention greatly improves cell fusion efficiency through magnetic force and higher fusion temperature. Fusion leads to mitochondrial transplantation, and the transplanted mitochondria maintain normal network structure and membrane potential, thereby improving the biological activity of mitochondria. The enucleated cells containing Fe ₃ O ₄ nanoparticles of the present invention have a larger diameter and can wrap and deliver more mitochondria. Enucleated cells and target cells are co-incubated in VSV-G concentrated solution. The vesicular stomatitis virus protein is inserted into the cell membrane and pulled into the distance between the membranes under the action of magnetic force, changing the membrane tension. Under the action of 45ºC citrate buffer Next, fusion occurs between cells. Mitochondria in enucleated cells can effectively enter target cells, which can solve the problem of mitochondrial mutations and dysfunction and can be used for anti-aging.

Description of drawings

Figure 1 is a schematic diagram of the preparation and fusion of enucleated cells containing Fe ₃ O ₄ nanoparticles of the present invention;

Figure 2 is a schematic diagram after loading cells with Fe ₃ O ₄ nanoparticles at different concentrations; and loading 293 cells with nanoparticles at different concentrations and incubating them under magnetic conditions for 24 hours, taking pictures and performing CCK8 detection;

Figure 3 (A) is a diagram of cell fusion under different concentrations of melt protein. The 1x representative concentration is: 0.5 mg/mL. One group of 293 cells is loaded with nanoparticles and marked in green, and the target cell 293 is marked in red; on the far right One column of pictures is a patchwork of cells, and the other three columns, from left to right, are cell DiI fluorescence, bright field, Calcein and cell nuclei; (B) is a statistical diagram of the diameter of cells after fusion; (C) is a statistical diagram of cell fusion rate;

Figure 4 (A) shows the effect of different concentrations of Fe ₃ O ₄ nanoparticles on cell fusion; a group of 293 cells are loaded with different concentrations of nanoparticles and marked in green, and the target cells 293 are marked in red; the rightmost column shows the cells Mosaic diagram, the other three columns from left to right are cell DiI fluorescence, bright field, Calcein and cell nucleus; (B) is the statistical diagram of cell diameter after fusion; (C) is the statistical diagram of cell fusion rate;

Figure 5 (A) shows pictures of cell fusion under different magnetic forces; one group of 293 cells is loaded with 2 mg/mL nanoparticles and marked in green, and the target cells 293 are marked in red; at a 1x concentration of VSV-G, the concentration is 0.5 mg. /mL; the rightmost column is a patchwork of cells, and the other three columns from left to right are cell DiI fluorescence, bright field, Calcein and nucleus; (B) is a statistical diagram of the diameter of cells after fusion; (C) is a statistical graph of cell fusion rate;

Figure 6 (A) shows the cell fusion efficiency under different temperatures of citrate buffer and incubation temperature; (B) shows the fused cells stained with Calcein and PI to mark live and dead cells; (C) shows the statistics under different conditions The cell fusion efficiency is made into a histogram;

Figure 7 shows the mitochondria-deficient cell C2C12 Rho0 (mitochondria-deficient) constructed after adding ethidium bromide (EB), dideoxycytosine (Zalcitabine, ddC), and uridine, from left to right: Mitochondria staining reagents, brightfield and mosaic images;

Figure 8 shows the mitochondria-deficient cells C2C12 Rho0 (mitochondria-deficient) and BMSC cells containing nanoparticles after enucleation after removing ethidium bromide (EB), zalcitabine (ddC), and uridine. (mitochondria have red fluorescence) fusion, and fluorescence staining identification of mitochondrial morphology was performed 30 minutes after fusion. From left to right are CFSE, mitochondrial staining reagent, bright field, cell nucleus and stitched image;

Figure 9 shows the fusion of mitochondria-deficient C2C12 Rho0 cells with enucleated nanoparticle-containing BMSC cells after removing ethidium bromide (EB), zalcitabine (ddC), and uridine. (A ) is the observation of cell morphology two to three days after fusion, (B) is cell number statistics, (C) is PCR analysis, (D) is quantitative PCR analysis of mitochondrial DNA.

Figure 10 is an analysis diagram after 30 minutes of fusion of senescent cells C2C12 and senescent cells with enucleated BMSC cells;

Figure 11 is a picture of cell senescence β-galactosidase staining of normal C2C12 cells, senescent C2C12 cells, and senescent cells fused with enucleated BMSC cells for 3 days.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below with reference to the accompanying drawings.

Example 1

1. A method for preparing enucleated cells containing Fe ₃ O ₄ nanoparticles, including the following steps:

(1) Plate BMSC cells in 24 wells, the cell density is about 70% (number of cells 2x10 ⁵ ), and the culture medium volume is 500 μL;

(2) Add Fe ₃ O ₄ nanoparticles (0.5-2 mg/mL) to (1), and place the well plate on a magnet and incubate for 12-24 hours;

(3) Place upside down in a 50mL centrifuge tube; the centrifuge tube contains 10mL centrifuge solution, which contains 10% sucrose, 1mg/mL cytochalasin B, 100μM calcium chloride, 500mM NAC antioxidant and 10mg/mL colchicine;

(4) Place the centrifuge tube in a preheated centrifuge, centrifuge at 30-35°C, 5000-10000 rpm for 40-60 minutes;

(5) Add cell culture medium and put it into a cell culture incubator to recover for 30-60 minutes to obtain enucleated cells containing Fe ₃ O ₄ nanoparticles.

2. The fusion method of enucleated cells containing Fe ₃ O ₄ nanoparticles and Rho0 cells or senescent cells includes the following steps:

(1) Digest the enucleated cells containing Fe ₃ O ₄ nanoparticles with Accutase ^TM digestion solution and collect by centrifugation;

(2) The collected cells are resuspended in VSV-G concentrated solution (concentration 0.5mg/ml) and added to the Rho0 cells or senescent cells plated the day before (the cell density is about 20%, that is, the approximate number is 5x10 ⁴ );

(3) Place the well plate on a magnet and incubate it in a cell culture incubator for 2-4 hours;

(4) Aspirate the supernatant and add 45°C citric acid buffer for 1 minute;

(5) Aspirate and discard the supernatant, add DMEM cell culture medium containing 10% serum and place it in a cell culture incubator for incubation. These are fused cells.

Among them, the preparation of VSV-G conditioned culture medium includes the following steps:

(1) Add 0.8 μg of vesicular stomatitis virus protein expression particles into a centrifuge tube, add 50 μL of serum-free culture medium and mix well;

(2) Add 3 μL Polyjet transfection reagent and 50 μL serum-free culture medium into another centrifuge tube, shake gently; then add to the solution obtained in step (1); obtain the transfection mixture, and let stand at room temperature for 15 minutes;

(3) After 15 minutes, aspirate the culture medium and add 1 mL of fresh serum-free culture medium. Immediately add the transfection mixture to the Ad293 cells and shake gently;

(4) Replace with new culture medium after 12 hours.

The preparation method of VSV-G concentrated solution includes the following steps:

(1) Transiently transfect Ad293 cells with 0.8 μg vesicular stomatitis virus protein expressing plasmid;

(2) After 48 hours, the supernatant will be collected and the cells will be digested with Accutase ^TM digestion solution and then collected;

(3) The collected cells are resuspended in cell culture medium, freeze and thaw repeatedly 5 times, and then centrifuged to collect the supernatant;

(4) Collect the supernatants of (2) and (3) together, and collect the supernatant after centrifugation at 300g, 10min, 2000g, 10min and 10000rpm for 30min;

(5) Place (4) into a 6mL ultracentrifuge tube, ultracentrifuge at 47000 rpm, 4°C for 2 hours, and collect 1 mL of precipitate from each tube, which is the concentrated VSV-G protein conditioned culture medium. The concentration is: 0.5-5mg/ mL.

Example 2

Monitoring of cells loaded with nanoparticles

Ad293 cells with a confluence of about 50% (number of about 1x10 ⁵ ) were loaded with Fe ₃ O ₄ nanoparticles at different concentrations, placed on magnets and incubated in cell culture for 24 hours. Afterwards, the cells were stained for nuclei and digested. Observe and take pictures under a fluorescence microscope. The results in Figure 2 show that as the concentration of Fe ₃ O ₄ nanoparticles continues to increase, the loading amount of Fe ₃ O ₄ nanoparticles in the cells also gradually increases; three-dimensional photography of the cells shows that the Fe ₃ O ₄ nanoparticles are confirmed to be located inside the cells. In addition, the above cells were tested for CCK8, and the results showed that loading the cells with Fe ₃ O ₄ nanoparticles did not affect cell viability and proliferation.

Example 3

A fusion between _Ad293 cells and normal Ad293 cells containing _Fe3O4 nanoparticles

Ad293 cells are plated in a 24-well plate with a cell confluence of about 50% (the number is about 1x10 ⁵ ). According to the experimental conditions, 0.01-2mg/mL nanoparticles of different concentrations are added and placed on the magnet for 12-24 hours, and then incubated. The cells were labeled with Calcein staining, digested and collected by centrifugation. According to the experimental design, different VSV-G (concentration range 0.5-5 mg/mL) conditioned culture medium was used to resuspend and add DiI-labeled target cells 293 (cell density was about 30%, The number is about 5x10 ⁴ ), placed on magnets with different magnetic strengths of 1500Gs-5000Gs and incubated in the incubator for 30 minutes. After 30 minutes, acidify using 0.8-1mM citrate buffer at different temperatures of 37-55°C according to the experimental design. 1 min, pH 4.5-6.0, then add cell culture medium and incubate for 30 min, and the cells will be stained for nuclear staining.

Cells labeled with the two different dyes mentioned above were subjected to the action of external magnetism and VSV-G, and finally the membrane fusion was induced by lowering the pH value to 5.5. The fused cells had two different colors of fluorescence. By counting the cell diameter size and cell area for analysis, cells below 20 μm are unfused cells, and cells with a diameter above 20 μm are fused cells.

From Figure 3 to Figure 6, it can be seen that after cells are loaded with nanoparticles and incubated with target cells in VSV-G conditioned medium, the fusion efficiency increases, and the higher the concentration of VSV-G, the higher the fusion efficiency; when the citric acid buffer is increased When the temperature of the liquid and the incubation temperature were adjusted, the cell fusion efficiency increased to a certain extent; according to the experimental results, it can be seen that after the cells were loaded with 2 mg/ml nanoparticles, the conditioned culture medium of 1x VSV-G (concentration: 0.5 mg/mL) Incubate at 45°C for 30 minutes, acidify with 45°C citric acid buffer for 1 minute, and then incubate in an incubator to achieve the best fusion effect.

Select the optimal experimental conditions to prepare enucleated nanocells. The size of the prepared enucleated nanocells is 10µm-15µm, which contains a large number of mitochondria. Under the external magnetic force and VSV-G, membrane fusion is induced by lowering the pH value to 5.5, releasing Transfer the contents of the enucleated cells to the target cells. Deliver the target biological macromolecules and organelles into cells with defects in biological macromolecules and organelles.

Example 4

Verification of delivering mitochondria from BMSC cells to Rho0 cells

Rho0 cells were obtained by treating C2C12 cells with ethidium bromide (EB), dideoxycytosine (Zalcitabine, ddC), and uridine. Normal cells and Rho0 cells were stained with 1 μM MitoTracker (mitochondria green staining reagent) and observed under a confocal microscope; in addition, in order to detect the cell membrane potential, 500 nMTMRE (mitochondria membrane potential staining reagent) was used for staining, as shown in Figure 7 It shows that the mitochondrial morphology of the Rho0 cells obtained was significantly damaged after analysis under a confocal microscope.

The BMSC cells loaded with nanoparticles were enucleated and fused with Rho0 cells; briefly, after the cells were enucleated, they were resuspended in VSV-G and the CSFE-labeled Rho0 cells were placed on a magnet and incubated in an incubator for 2 hours, and then used at 45°C. Acidify with citrate buffer for 1 minute, then add cell culture medium and incubate in the incubator for 30 minutes. After 30 minutes, perform mitochondrial fluorescence staining and nuclear staining on the fused cells and Rho0 cells. It can be seen from Figure 8 that Rho0 cells contain a large number of normal-shaped mitochondria. The above results prove that the BMSC cells of the present invention can effectively deliver mitochondria to Rho0 cells.

Example 5

Functional verification of BMSC cells delivering mitochondria to Rho0 cells.

The BMSC cells loaded with nanoparticles were enucleated and then fused with Rho0 cells; the fused cells obtained by the fusion method in Example 4 above were observed and detected under a microscope three days later. In addition, cell counting methods, real-time PCR technology and gel electrophoresis were also used for characterization. It can be seen from Figure 9 that the cell morphology of Rho0 cells obviously changed to normal after 3 days of fusion, while the cell morphology of the cells that did not undergo fusion was not full, stringing and death occurred with or without the addition of uracil. In Figure 9 (B), it can be seen that on the second day of cell fusion, the number of fused cells was significantly higher than that of the other two groups, and after the third day, the number of cells in the fusion group was about two times more than that of the other groups. The above results further prove that after cell fusion The cells resumed growth; the quantitative PCR test in the result (C) in Figure 9 shows that, using nuclear β-2M as the standard, the mitochondrial DNA copy number of Rho0 cells increased significantly after transplantation; further analysis of the PCR electrophoresis strips revealed that the cells after fusion The content of inner mitochondria increased significantly. The above results prove that enucleated BMSCs can effectively deliver mitochondria to Rho0 cells and exert biological functions, allowing them to restore the growth and proliferation of Rho0 cells in normal cell culture medium without uracil addition.

Example 6

Monitoring and verification of mitochondrial delivery from BMSC cells to senescent C2C12 cells.

C2C12 cells were treated with 2% FBS and D-gal for seven days and then treated with R6G for one day to obtain senescent C2C12 cells. R6G is a membrane-permeable cationic fluorescent dye. After entering the cell, it can freely pass through the outer mitochondrial membrane and localize on the inner mitochondrial membrane. In addition, it is an effective inhibitor of oxidative phosphorylation, inhibiting ATP synthase and blocking hydrogen ion channels, thereby inhibiting ATP synthesis. Endogenous mitochondria and mtDNA can be eliminated after cells are treated with R6G. The senescent cells and the enucleated BMSC cells containing nanoparticles were fused by the fusion method of Example 3; it can be seen in Figure 10 that after the cells were treated with R6G, the endogenous mitochondria basically disappeared; while the senescent cells and the enucleated BMSC cells After fusion, mitochondria with normal morphological structure appeared in senescent cells; the above performance once again proved that enucleated BMSC cells can effectively deliver mitochondria to senescent cells under the conditions of magnetism, VSVG and high temperature. Three days after cell fusion, cell senescence β-galactosidase staining was performed with normal cells and senescent cells respectively. As can be seen from Figure 11, the staining degree of cells became lighter after fusion, proving that after enucleated cells effectively delivered mitochondria to senescent cells, , mitochondria can replicate and perform biological functions in aging cells.

Claims (10)

1. A method for preparing enucleated cells containing Fe ₃ O ₄ nanoparticles, which is characterized by comprising the following steps: (1) Plate BMSC cells in a well plate; (2) Add 0.5-2 mg/mL Fe ₃ O ₄ nanoparticles, and place the well plate on a magnet and incubate for 12-24 hours; aspirate the supernatant and wash; (3) Place the tube upside down in a centrifuge tube and add centrifuge solution; place the tube in a preheated centrifuge and centrifuge for 40-60 minutes at 30-35°C and 5000-10000 rpm; (4) Add cell culture medium and place in a cell culture incubator to recover for 30-60 minutes to obtain enucleated cells containing Fe ₃ O ₄ nanoparticles. 2. The preparation method according to claim 1, characterized in that the centrifuge contains 10% sucrose, 1 mg/mL cytochalasin B, 100 μM calcium chloride, 500 mM NAC antioxidant and 10 mg/mL colchicine. 3. Enucleated cells containing Fe ₃ O ₄ nanoparticles prepared according to the method of claim 1 or 2. 4. An enucleated cell containing Fe ₃ O ₄ nanoparticles and integrating VSV-G. 5. A method for fusion of enucleated cells containing Fe ₃ O ₄ nanoparticles and mitochondria-deficient cells, characterized by comprising the following steps: (1) Digest the enucleated cells containing Fe ₃ O ₄ nanoparticles with digestive juice and collect by centrifugation; (2) The collected enucleated cells containing Fe ₃ O ₄ nanoparticles are resuspended in VSV-G concentrated solution and added to the plated mitochondria-deficient cells; (3) Place the well plate on a magnet and incubate it in a cell culture incubator for 2-4 hours; (4) Aspirate the supernatant and add citrate buffer at 37-55°C for 1 minute; (5) Aspirate and discard the supernatant, add DMEM cell culture medium containing 10% serum and place it in a cell culture incubator for incubation. 6. The method of claim 5, wherein the mitochondria-deficient cells include RhoO cells and senescent cells. 7. The method according to claim 5, characterized in that the number of enucleated cells containing Fe ₃ O ₄ nanoparticles is: 1x10 ³ -1x10 ⁶ /well, and the VSV-G concentrate is The concentration is 0.5-5mg/mL. 8. The method according to claim 5, characterized in that the citric acid concentration is 0.8-1mM, the pH is 4.5-6.0, and the magnetic force of the magnet is 1500Gs-5000Gs. 9. The application of enucleated cells containing Fe ₃ O ₄ nanoparticles according to claim 1 in delivering biological macromolecules and organelles, and the application is not for the diagnosis and treatment of diseases. 10. Application of enucleated cells containing Fe ₃ O ₄ nanoparticles in anti-aging according to claim 1.