Mitochondrial micro-nano hydrogel microspheres, and preparation method and application thereof
By preparing lipid membrane-encapsulated micro/nano-scale hydrogel microspheres, the problems of bioactivity damage and limitations of existing mitochondrial delivery strategies have been solved, achieving efficient and stable mitochondrial delivery and tissue repair, which is suitable for the treatment of a variety of diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-04
- Publication Date
- 2026-03-24
AI Technical Summary
Existing mitochondrial delivery strategies have limitations such as damage to bioactivity, difficulty in preservation, and local use. Furthermore, exosome delivery faces problems such as low yield and small number of mitochondria encapsulated, making it difficult to achieve efficient and stable mitochondrial function repair.
Micro- and nano-sized hydrogel microspheres with uniform and controllable particle size were prepared by extrusion method using lipid membrane-encapsulated micro- and nano-sized hydrogel microspheres. Active mitochondria were then encapsulated in the hydrogel microspheres using liposome extrusion technology to achieve efficient delivery.
It enables mass production of active mitochondrial delivery, reduces mitochondrial damage, can be absorbed by cells, promotes tissue repair, and is suitable for the treatment of a variety of diseases.
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Figure CN116725976B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of biotechnology, and particularly relates to a mitochondria-loaded micro-nano hydrogel microsphere and a preparation method and application thereof. BACKGROUND
[0002] Mitochondria are considered as the 'energy factory' of cells and are the main organelles for producing adenosine triphosphate (ATP). In cells that rely heavily on oxidative phosphorylation to meet energy demand, such as cardiomyocytes, mitochondria can account for 35% of the cell volume. At the same time, mitochondria are also involved in a variety of cellular processes and the synthesis of a variety of biomolecules, forming a comprehensive network essential for maintaining cellular homeostasis. They regulate cell function by controlling energy production, intermediary metabolism, calcium signaling, and apoptosis. Therefore, dysfunction of mitochondria will lead to collapse of intracellular homeostasis and eventually lead to diseases. A variety of diseases have been found to be related to mitochondrial dysfunction, including cancer, cardiovascular diseases, neurological diseases, metabolic diseases, and aging, etc. Therefore, there is great potential in treating the above diseases by repairing mitochondrial function, and it has attracted widespread attention.
[0003] Mitochondrial delivery is considered as an effective mitochondrial function repair scheme, that is, fresh and healthy mitochondria are delivered to cells to improve the original mitochondrial damage. Existing delivery strategies include direct delivery of free mitochondria and loading mitochondria using block hydrogels, but these strategies also have certain limitations: the bioactivity of directly delivered free mitochondria is easily damaged and difficult to preserve; and although block hydrogels can protect mitochondria to some extent, they can only be used locally and are not suitable for many diseases. Some researchers, such as Professor Shi Liyun of Nanjing University of Chinese Medicine, have proposed that Ad-MSC-derived exosomes can transfer mitochondria of donor cells to macrophages, and the transferred mitochondria of donor cells can be internalized by mitochondria of macrophages and maintain the biological activity of mitochondria (AdMSC-derived exosomes alleviate acute lung injury via transferring mitochondrial component to improve homeostasis of alveolar macrophages; Theranostics. 2022-3-21), but exosome delivery of mitochondria faces problems such as low yield and few mitochondria wrapped. Therefore, it is necessary to seek an efficient and stable mitochondrial delivery strategy for repairing mitochondrial function to treat the above diseases. SUMMARY
[0004] The first object of the present application is to provide a micro-nano hydrogel microsphere loaded with active mitochondria, which can be used for the delivery of mitochondria.
[0005] In some embodiments of the present application, the micro-nano hydrogel microsphere loaded with mitochondria is a micro-nano hydrogel microsphere containing active mitochondria wrapped by a lipid membrane.
[0006] In some embodiments of the present application, the active mitochondria are freshly extracted cell mitochondria.
[0007] Based on the micro-nano hydrogel microsphere loaded with mitochondria prepared in the present application, on the one hand, it can be mass-produced and the particle size is controllable; on the other hand, since the content components have good biocompatibility, can encapsulate organelles and can be phagocytosed by recipient cells, it can be well absorbed by cells and better promote tissue repair.
[0008] In some embodiments of the present application, the micro-nano hydrogel microsphere is a micro-nano hydrogel microsphere wrapped by a lipid membrane.
[0009] In some embodiments of the present application, the micro-nano hydrogel microsphere is a hydrogel microsphere wrapped by a lecithin / cholesterol mixed lipid membrane.
[0010] In some embodiments of the present application, the micro-nano hydrogel microsphere is a GelMA hydrogel microsphere with different concentrations.
[0011] In some embodiments of the present application, the particle size of the micro-nano hydrogel microsphere is 0.05-12 μm, preferably 0.2-10 μm, and more preferably 0.4-3 μm.
[0012] The second object of the present application is to provide a preparation method of the above-mentioned micro-nano hydrogel microsphere loaded with mitochondria, which comprises the following steps:
[0013] The mitochondria in cells are separated and mixed with a hydrogel precursor to obtain a gel microsphere content system, and after adding a lipid membrane, a specific filter structure is used for repeated extrusion, followed by cross-linking to obtain a micro-nano hydrogel microsphere loaded with mitochondria.
[0014] In some embodiments of the present application, the cells are eukaryotic cells containing mitochondria.
[0015] In the present application, the source of cells is not particularly limited, including but not limited to human cells and non-human mammalian cells, examples of mammals include mice, rats, rabbits, pigs, dogs, cows, primates (except humans), etc.
[0016] In some embodiments of the present application, the cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, iPS (induced pluripotent stem cells), tissue cells, etc. according to functions or stages.
[0017] In the present application, the requirement for the cells is fresh living cells, so as to ensure that the cells can obtain mitochondria with biological activity after the method of the present application.
[0018] In some embodiments of the present application, a GelMA hydrogel precursor is used.
[0019] Of course, those skilled in the art can also select other precursors that can be used to form hydrogels for extrusion operation according to actual use requirements, including but not limited to gelatin methacrylate (GelMA), hyaluronic acid methacrylic acid (HAMA), polyvinyl alcohol (PVA), polyacrylamide (PAAM), sodium alginate (SA), chitosan and polyethylene glycol acrylate (PEGDA), preferably GelMA and HAMA.
[0020] In some embodiments of the present application, an artificial lipid membrane formed by mixing lecithin and cholesterol is used.
[0021] Of course, those skilled in the art can also select other natural lipid membranes, artificial lipid membranes and various functionally modified lipid membranes that can be used to form microsphere templates for extrusion operation according to actual use requirements, including but not limited to phospholipid membranes, cholesterol membranes, phospholipid / cholesterol mixed lipid membranes and artificial cationic lipid membranes, mammalian cell membranes, cell microvesicle membranes and other lipid membranes.
[0022] In some embodiments of the present application, the extrusion device with the specific filter pore structure is a liposome extruder with a filter membrane.
[0023] Of course, those skilled in the art can also select other devices with specific filter pore structures for extrusion operation according to actual use requirements, including but not limited to liposome extruders with filter membranes.
[0024] In some embodiments of the present application, the size of the specific filter pore structure is 0.05 μm to 12 μm, preferably 0.2 μm to 10 μm, and more preferably 0.4 μm to 3 μm.
[0025] In some embodiments of the present application, the number of extrusions is greater than or equal to 3.
[0026] In some embodiments of the present application, the cross-linking method is ultraviolet light cross-linking. The specific operation mode of ultraviolet light cross-linking is to directly irradiate the uniform mixed system added with a photo-crosslinking agent, which is one of lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate salt and 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, under ultraviolet light. The temperature of ultraviolet light cross-linking is set to room temperature, and the time is 5-10 minutes.
[0027] In some embodiments of the present application, the cross-linking method is chemical cross-linking. The specific operation mode of chemical cross-linking is to stir the uniform mixed system added with a chemical cross-linking agent, which is one of calcium ions and ammonium persulfate, in a mechanical stirring instrument. The temperature of chemical cross-linking is set to room temperature, and the time is 10 minutes.
[0028] Of course, other methods that can be used for cross-linking can also be selected by those skilled in the art according to actual use requirements, including but not limited to ultraviolet light cross-linking and chemical cross-linking.
[0029] In some embodiments of the present application, the specific operation steps are as follows:
[0030] (1) Separating mitochondria of cells, mixing the mitochondria with a hydrogel precursor to obtain a gel microsphere content system, and then adding a lipid membrane solution to obtain a mixed system;
[0031] (2) Using a syringe to suck the mixed system, connecting the syringe with a liposome extruder clamped with a filter membrane with a pore size of 0.05-12 μm, and then connecting an empty syringe to the other end of the liposome extruder for receiving the material passing through the filter membrane;
[0032] (3) Pushing and pulling the two syringes back and forth so that the mixed system repeatedly passes through the filter membrane in the middle of the extruder for more than 3 times, diluting the system, and then performing ultraviolet light cross-linking to obtain the micro-nano-sized hydrogel microspheres containing mitochondria.
[0033] A third object of the present application is to provide the application of the micro-nano-sized hydrogel microspheres for delivering mitochondria according to the second aspect of the present application in (1)-(3) as follows:
[0034] (1) Preparing a drug for delivering mitochondria to cells;
[0035] (2) Preparing a drug for repairing mitochondrial damage of cells or tissues;
[0036] (3) Preparing a drug for preventing and treating diseases related to mitochondrial dysfunction, such as cancer, cardiovascular system diseases, nervous system diseases, metabolic diseases and aging, etc.
[0037] The present application combines simple liposome extrusion technology and mitochondria / hydrogel complex technology to prepare a kind of micro-nano hydrogel microspheres with high efficiency of loading active mitochondria, which can be used to deliver active mitochondria to cells efficiently. The prior art basically uses chemical reaction to prepare gel microspheres, which is complicated and involves many parameters. Compared with the prior art, the present application overcomes the problem of difficult preparation of micro-nano hydrogel by very simple operation, and the raw materials are abundant and easy to obtain. The amount of mitochondria contained is more abundant than natural vesicles.
[0038] The present application has the following advantages and beneficial effects compared with the prior art:
[0039] (1) The preparation method of the present application reduces the damage of mitochondria caused by the delivery process, so that the mitochondria can be captured by cells while maintaining biological activity.
[0040] (2) The present application is based on the extrusion method to prepare micro-nano hydrogel microspheres. Through this method, micro-nano hydrogel microspheres with controllable and uniform particle size distribution can be obtained. Through simple push-pull operation, mitochondria can be wrapped in hydrogel microspheres by adsorption caused by the difference in electrical properties between materials. Based on the needs of subsequent experiments and applications, the size of the prepared micro-nano hydrogel microspheres can be flexibly adjusted by changing the pore size of the specific filter structure, which has excellent use flexibility.
[0041] (3) The raw materials of the present application are easy to obtain and the operation is simple. Through experiments, it has been verified that different types of key cells can phagocytose the present application to achieve efficient delivery and fusion of mitochondria, so as to play a role in the repair of cell or tissue function loss caused by mitochondrial damage. BRIEF DESCRIPTION OF DRAWINGS
[0042] Figure 1 Fluorescent staining images of hydrogel microspheres with different particle sizes and different gel precursor concentrations prepared by the method in the embodiments of the present application.
[0043] Figure 2 Fluorescent staining images of active mitochondria in hydrogel microspheres prepared by the method in the embodiments of the present application.
[0044] Figure 3 Flow cytometry characterization chart of the content of active mitochondria in the hydrogel microspheres prepared by the method in the embodiments of the present application.
[0045] Figure 4 Fluorescent staining images and flow cytometry characterization chart of the endocytosis efficiency of the hydrogel microspheres prepared by the method in the embodiments of the present application.
[0046] Figure 5 Effect of the hydrogel microspheres prepared by the method in the embodiments of the present application on cell proliferation.
[0047] Figure 6 Figure 1 shows the fluorescence staining of mitochondria fusion of the hydrogel microspheres prepared by the method of the present application. DETAILED DESCRIPTION
[0048] In order to make the inventive purpose, technical scheme and technical effects of the present application clearer, the present application will be further described in detail below in combination with specific embodiments. Unless otherwise defined, all technical and scientific terms used in the present application have the same meanings as commonly understood by those skilled in the art to which the present application belongs. It should be understood that the specific embodiments described in the specification are only for the purpose of explaining the present application, and are not intended to limit the present application.
[0049] The experimental methods not specified in the following embodiments of the present application are generally carried out under conventional conditions, or under conditions recommended by the manufacturers. Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained commercially.
[0050] In the following embodiments, the cells used are rat adipose-derived mesenchymal stem cells. It should be understood, of course, that this cell is only used as an exemplary demonstration, and is not a specific limitation on the type of cell used in the present application. The cells that can be used in the present application include, but are not limited to, rat adipose-derived mesenchymal stem cells.
[0051] In the following embodiments, the artificial lipid membrane used is a lecithin / cholesterol mixed lipid membrane, the natural lipid membrane is a red blood cell membrane, and the hydrogel precursor is a photo-crosslinked GelMA or a chemically crosslinked sodium alginate hydrogel. It should be understood, of course, that these materials are only used as exemplary demonstrations, and are not specific limitations on the artificial lipid membrane and hydrogel precursor used in the present application. The materials that can be used in the present application include, but are not limited to, the above-mentioned materials.
[0052] Example 1
[0053] A method for preparing mitochondria-loaded micro-nano hydrogel microspheres, comprising the following steps:
[0054] 1. Isolation and extraction of mitochondria
[0055] The specific steps are as follows:
[0056] (1) Take rat adipose-derived mesenchymal stem cells that have been subcultured to P4-P5, and use cell digestion solution to digest the rat adipose-derived mesenchymal stem cells to detach them from the cell culture bottle. Add mitochondria extraction reagent to resuspend, ice bath for 10-15 min, and use a glass homogenizer to homogenize 10-30 times, with the positive rate of trypan blue staining > 50% as the standard, and the homogenization should not be excessive;
[0057] (2) Take the homogenate in (1) in a 1.5 mL EP tube, centrifuge at 500 x g for 10 min at 4℃;
[0058] (3) Take the supernatant after centrifugation in (2) in a 1.5 mL EP tube, centrifuge at 13000 x g for 10 min at 4℃, to obtain the separated mitochondria with biological activity.
[0059] 2. Preparation of hydrogel microspheres for mitochondrial delivery
[0060] The specific steps are as follows:
[0061] (1) Dissolve lecithin (15 mg / mL) and cholesterol in a mass ratio of 2 mg: 1 mg in 1 mL of chloroform, and after volatilizing the solvent, add 2 mL of phosphate buffer to hydrate at 37℃. Dissolve 0.1 g of GelMA in 2 mL of phosphate buffer, add 0.01% of blue light initiator, and mix thoroughly to obtain a mixed system.
[0062] (2) Use a syringe to draw the mixed system obtained in step (1), and add the separated mitochondria with biological activity described above. Connect the syringe to a liposome extruder with a filter membrane with a pore size of 3 μm, and then connect an empty syringe to the other end of the liposome extruder for receiving the material passing through the filter membrane.
[0063] (3) Pass the mixed system through the membrane 5 times, i.e., without stopping in the initial syringe, dilute the collected liquid, and irradiate it with ultraviolet light (405 nm; 30 mW / cm 2 ) at a distance of 5 cm from the sample for 10 minutes at room temperature. The added photo-crosslinking agent is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate salt, to obtain the desired micro-nano-sized hydrogel microspheres for mitochondrial delivery.
[0064] Example 2
[0065] A method for preparing mitochondria-loaded micro-nano-sized hydrogel microspheres, comprising the following steps:
[0066] 1. Isolation and extraction of mitochondria
[0067] The specific steps are as follows:
[0068] (1) Take the rat adipose-derived mesenchymal stem cells that have been passaged to P4-P5, and use cell digestion solution to digest the rat adipose-derived mesenchymal stem cells to detach them from the cell culture bottle. Add mitochondrial extraction reagent to resuspend, ice bath for 10-15 min, and use a glass homogenizer to homogenize 10-30 times, with the standard being that the positive rate of trypan blue staining is > 50%, and the homogenization should not be excessive.
[0069] (2) Take the homogenate in (1) in a 1.5 mL EP tube, centrifuge at 500 x g for 10 min at 4°C;
[0070] (3) Take the supernatant after centrifugation in (2) in a 1.5 mL EP tube, centrifuge at 13000 x g for 10 min at 4°C, to obtain the separated mitochondria with biological activity.
[0071] 2. Preparation of hydrogel microspheres for mitochondrial delivery
[0072] The specific steps are as follows:
[0073] (1) Dissolve lecithin (15 mg / mL) and cholesterol in a mass ratio of 2 mg: 1 mg in 1 mL of chloroform, and after volatilizing the solvent, add 2 mL of phosphate buffer to hydrate at 37°C. Resuspend the mitochondria obtained in step 1 with 8 mL of PBS, then weigh 0.2 g of calcium-containing sodium alginate powder (calcium content of 4.42%) at room temperature, dissolve it in the above-mentioned 8 mL of PBS containing mitochondria, and mix well to obtain a mixed system.
[0074] (2) Use a syringe to draw the mixed system obtained in step (1). Connect the syringe to a liposome extruder with a filter membrane with a pore size of 0.5 μm, and then connect an empty syringe to the other end of the liposome extruder for receiving the material passing through the filter membrane.
[0075] (3) Pass the mixed system through the membrane 5 times, i.e. without staying in the initial syringe, dilute the collected liquid, and use mechanical stirring at 300 rpm for 10 min at room temperature. The added chemical crosslinking agent is calcium ions, and the desired micro-nano-sized hydrogel microspheres for delivering mitochondria are obtained.
[0076] Example 3
[0077] A method for preparing mitochondria-loaded micro-nano-sized hydrogel microspheres, comprising the following steps:
[0078] 1. Collection of rat red blood cell membranes
[0079] The specific steps are as follows:
[0080] (1) Take the mouse whole blood in a centrifuge tube with heparin phosphate buffer, and centrifuge at 2500 rpm for 5 min in a low-speed centrifuge at 4°C.
[0081] (2) Remove the upper plasma, leaving the dark red packed red blood cells, and wash the lower red blood cells with phosphate buffer 3 times, and centrifuge at low speed to obtain packed red blood cells.
[0082] (3) The red blood cells are placed in three volumes of 0.25x PBS for hypotonic treatment on ice for 40-60 min, and centrifuged at 13000 r / min in a high-speed centrifuge at 4°C for 15 min, and the precipitate in the lower layer is taken.
[0083] (4) The red blood cells are washed with 0.25x PBS for 2-5 times until the supernatant is colorless, and pink red blood cell membranes are obtained, which are resuspended with a PBS solution and stored at 4°C.
[0084] 2. Isolation and extraction of mitochondria
[0085] The specific steps are as follows:
[0086] (1) The rat adipose mesenchymal stem cells passaged to P4-P5 are taken, and the cell digestion solution is used to digest the rat adipose mesenchymal stem cells to make them detach from the cell culture bottle. Mitochondrial extraction reagent is added for resuspension, ice bath for 10-15 min, and glass homogenizer is used for homogenization for 10-30 times. The positive rate of trypan blue staining is >50% as the standard, and the homogenization should not be excessive;
[0087] (2) The homogenized system in (1) is taken in a 1.5 mL EP tube, centrifuged at 500xg for 10 min at 4°C;
[0088] (3) The supernatant after centrifugation in (2) is taken in a 1.5 mL EP tube, centrifuged at 13000xg for 10 min at 4°C, and the isolated mitochondria with biological activity are obtained.
[0089] 3. Preparation of hydrogel microspheres for mitochondrial delivery
[0090] The specific steps are as follows:
[0091] (1) The obtained red blood cell membranes are centrifuged and precipitated, GelMA is dissolved in a phosphate buffer solution, blue light initiator is added and mixed thoroughly to obtain a mixed system.
[0092] (2) The mixed system obtained in step (1) is taken by using a syringe, and the mitochondria with biological activity separated in the second step are added. The syringe is connected with a liposome extruder clamped with a filter membrane with a pore size of 5 μm, and then an empty syringe is connected at the other end of the liposome extruder for receiving the material passing through the filter membrane.
[0093] (3) The mixed system is passed through the membrane for 10 times, i.e. without staying in the initial syringe, and the collected liquid is diluted and irradiated with ultraviolet light (365 nm; 100 mW / cm 2 ) at a distance of 5 cm from the sample at room temperature for 10 minutes. The added photo-crosslinking agent is 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, and the required micro-nano hydrogel microspheres for delivering mitochondria are obtained.
[0094] Performance characterization of the mitochondria-delivering hydrogel microspheres prepared in Example 1 above
[0095] 1. Gelation verification
[0096] The hydrogel microspheres prepared based on different pore size filters (0.4 pm, 1.0 pm and 3.0 pm) and different precursor concentrations (2%, 5% and 10%) were mixed with Triton X-100 to obtain the lipid membrane-removed hydrogel microspheres. The used dyes were Dil (cell membrane red fluorescent probe) and FITC (green dispersion dye in the hydrogel component).
[0097] The specific staining operation was as follows: Dil was diluted with PBS at a ratio of 1:200, the resuspended lipid membrane after hydration was incubated at 37°C in the dark for 30 min, and then centrifuged at 3000 x g for 10 min to obtain the stained lipid membrane; FITC was added to the GelMA solution at a concentration of 10 pg / mL for labeling.
[0098] The results are shown in Figure 1 By observing the immunofluorescence staining images of the micron-sized hydrogel microspheres obtained by different pore size filters (0.4 pm, 1.0 pm and 3.0 pm), it can be found that the size of the gel microspheres gradually increased, and the green fluorescent gel core could still be observed after removing the lipid membrane by Triton X-100. By observing the immunofluorescence staining images of the micron-sized hydrogel microspheres obtained by different precursor concentrations (2%, 5% and 10%), it can be found that they have similar phenomena. These results show that micron-sized hydrogel microspheres have been successfully prepared, and micron-sized hydrogel microspheres can be prepared based on different pore size filters and different precursor concentrations.
[0099] 2. Particle size
[0100] The hydrogel microspheres prepared by different pore size filters (0.4 pm, 1.0 pm and 3.0 pm) were compared with flow cytometry particle size calibration microspheres (particle size of 1.0 pm and 4.0 pm, purchased from ThermoFisher), and the particle size differences between the samples were compared by the peak value of FSC in the flow cytometry results.
[0101] The results show that there is a certain FSC signal shift between the peaks of the three sizes of micron-sized hydrogel microspheres, and the micron-sized hydrogel microspheres prepared based on different pore size filters are compared with flow cytometry particle size calibration microspheres, and the actual size of the micron-sized hydrogel microspheres basically corresponds to the expected size. The 0.4 pm and 1.0 pm micron-sized hydrogel microspheres are more concentrated, while the 3.0 pm micron-sized hydrogel microspheres are mainly concentrated between 1-4 pm.
[0102] 3. Fluorescent staining
[0103] Take the mitochondria-containing hydrogel microspheres prepared based on different pore size filter membranes (0.4 μm, 1.0 μm and 3.0 μm), and use fluorescent staining method to dye them, wherein the used dye is FITC (green disperse dye in the hydrogel component), TMRM (mitochondrial membrane potential red fluorescent probe).
[0104] The specific staining operation is as follows: dilute TMRM with PBS according to the ratio of 1:200, resuspend the separated mitochondria with the staining working solution, incubate at 37°C in the dark for 30 min, centrifuge at 13000 x g for 10 min to obtain the dyed lipid membrane; add FITC in the GelMA solution at a concentration of 10 μg / ml for labeling.
[0105] The results are shown in Table 1. Figure 2 It can be found that the FITC-labeled GelMA hydrogel and the TMRM-labeled active mitochondria appear co-localization, that is, the mitochondria are successfully encapsulated in the hydrogel microspheres, and have biological activity. And there are active mitochondria in the hydrogel microspheres of different sizes.
[0106] 4. Proportion characterization of mitochondria encapsulated in hydrogel microspheres
[0107] Label the mitochondria with TMRM, and label the hydrogel component with FITC, and use flow cytometry to characterize the proportion of mitochondria encapsulated in the mitochondria-containing hydrogel microspheres prepared based on different pore size filter membranes (0.4 μm, 1.0 μm and 3.0 μm).
[0108] The results are shown in Table 2. Figure 3 It can be found that about 82.3% of the mitochondria-containing hydrogel microspheres prepared by the 3.0 μm pore size filter membrane encapsulate mitochondria, about 73.6% of the mitochondria-containing hydrogel microspheres prepared by the 1.0 μm pore size filter membrane encapsulate mitochondria, and about 73.0% of the mitochondria-containing hydrogel microspheres prepared by the 0.4 μm pore size filter membrane encapsulate mitochondria.
[0109] Use effect of mitochondria-containing hydrogel microspheres
[0110] 1. Endocytosis of cells to the hydrogel microspheres prepared in Example 1
[0111] Inoculate H9C2 / HUVEC cells in the well plate, and according to the particle concentration, the mitochondria-containing hydrogel microspheres with DiI-labeled lipid membrane are respectively added at 4 x 10 6 particles / ml, 1 x 10 7 particles / ml, 2 x 10 7 particles / ml, 4 x 107 Add particles / ml and co-incubate with cells for 36 hours. Then perform confocal chromatography or use the group without hydrogel microspheres as a blank control for flow cytometry detection.
[0112] The results are as follows Figure 4 As shown in the typical image, DiI-labeled hydrogel microspheres exhibit red fluorescence and are engulfed by cells. Flow cytometry analysis was performed, and the endocytosis efficiency of the hydrogel microspheres in cells was obtained based on the positive ratio. It was found that the endocytosis efficiency of hydrogel microspheres of different sizes in different cell types increased with increasing particle concentration.
[0113] 2. Biocompatibility of the hydrogel microspheres prepared in Example 1
[0114] H9C2 / HUVEC cells were seeded in well plates. 4 × 10⁻⁶ hydrogel microspheres containing mitochondria were added to each experimental group according to particle concentration. 6 Cells were treated with mitochondrial-containing hydrogel microspheres with a particle size of 1 μm, while the control group received no treatment. Cell proliferation activity was detected using CCK8 reagent.
[0115] The results are as follows Figure 5 As shown in Example 1, H9C2 or HUVEC cells were treated with the hydrogel microspheres prepared using the method described above. Changes in cell proliferation activity were detected, and it was found that the hydrogel microspheres had no significant effect on cell proliferation and exhibited good biocompatibility.
[0116] 3. Mitochondrial fusion effect after delivery of hydrogel microspheres prepared in Example 1
[0117] H9C2 or HUVEC cells were seeded in well plates, and hydrogel microspheres containing TMRM-labeled mitochondria were added according to particle concentration at a ratio of 4 × 10⁻⁶. 6 Particles / ml were added and co-incubated with cells for 36 hours. Subsequently, confocal imaging was performed using the group without hydrogel microspheres as a blank control.
[0118] The results are as follows Figure 6 As shown, endogenous mitochondria are marked in green by Mito-Tracker Green, while delivered mitochondria are marked in red by TMRM. Although they are not co-localized, they form a certain connection, indicating that the delivered mitochondria have fused with the cell's original mitochondria and are biologically active.
[0119] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.
Claims
1. A type of mitochondrial-loaded micro / nanoscale hydrogel microsphere, characterized in that, The mitochondrial-loaded micro / nano-scale hydrogel microspheres are micro / nano-scale hydrogel microspheres containing active mitochondria encapsulated by a lipid membrane; the particle size of the micro / nano-scale hydrogel microspheres is 0.4 μm to 3 μm. The method for preparing the mitochondrial-loaded micro / nano-scale hydrogel microspheres includes the following steps: Mitochondria were isolated from cells and mixed with a hydrogel precursor to obtain a gel microsphere content system. After adding a lipid membrane, the mixture was repeatedly extruded using an extrusion device with a specific pore structure. Subsequently, cross-linking was performed to obtain the micro / nano-scale hydrogel microspheres carrying mitochondria. The size of the specific pore structure was 0.4 μm to 3 μm. The extrusion device with a specific filter pore structure is a liposome extruder with a filter membrane; the number of extrusions is 3-10. The crosslinking method is either ultraviolet light crosslinking or chemical crosslinking. Specifically, the uniformly mixed system with added photocrosslinking agent is placed under ultraviolet light for direct irradiation. The ultraviolet light crosslinking temperature is room temperature, and the time is 5-10 minutes. Alternatively, the uniformly mixed system with added chemical crosslinking agent is placed in a mechanical stirrer for stirring. The chemical crosslinking temperature is room temperature, and the time is 10 minutes.
2. The mitochondrial-loaded micro / nanoscale hydrogel microspheres according to claim 1, characterized in that, The active mitochondria are cellular mitochondria extracted from fresh cells, and the cell sources include human cells and non-human mammalian cells.
3. The mitochondrial-loaded micro / nanoscale hydrogel microspheres according to claim 1, characterized in that, The lipid membrane includes natural lipid membranes, artificial lipid membranes, and other lipid membranes that have been functionalized.
4. The mitochondrial-loaded micro / nanoscale hydrogel microspheres according to claim 1, characterized in that, The hydrogel comprises methacrylamide gelatin, hyaluronic acid methacrylate, polyvinyl alcohol, polymethacrylamide, sodium alginate, chitosan, and polyethylene glycol acrylate.
5. The application of the mitochondrial-loaded micro / nano-scale hydrogel microspheres according to any one of claims 1 to 4 in the following (1) to (3); (1) Preparation of drugs for delivering mitochondria into cells; (2) Prepare drugs to repair mitochondrial damage in cells or tissues; (3) Prepare drugs for the prevention and treatment of diseases related to mitochondrial dysfunction.