Mitochondria-loaded cell membrane vesicle preparation as well as design method, preparation method and application thereof

Abstract

The invention discloses a design and preparation method of a cell membrane vesicle preparation loaded with mitochondria. The design method comprises the following steps: loading the mitochondria into an artificially constructed cell membrane bionic vesicle, and constructing a nano-scale vesicle preparation loaded with the mitochondria inside and a cell membrane outside; the preparation method comprises the following steps: preparing cells into a cell suspension; repeatedly extruding the cell suspension through an extrusion device provided with a filter membrane with a specific aperture; and carrying out differential centrifugation on the extruded liquid to prepare the cell membrane vesicle preparation loaded with mitochondria. The invention also discloses the vesicle preparation obtained by the method and application of the vesicle preparation in preparation of drugs for treating mitochondrial damage related diseases. According to the method, rapid and efficient loading of mitochondria in cells can be realized, and the problems of low yield of mitochondria vesicles from natural sources, limited loading efficiency and the like are solved. The prepared vesicle preparation can effectively protect the physiological function of mitochondria, delay rapid inactivation of mitochondria in the using process and achieve efficient mitochondrial transplantation of damaged cells.

Description

Technical Field

[0001] The present invention relates to the fields of pharmaceutical preparations and biological technology, and in particular to a cell membrane vesicle preparation carrying mitochondria, and a design method, a preparation method and an application thereof. Background Art

[0002] Mitochondria are important organelles in cells. They mainly synthesize adenosine triphosphate (ATP) through the process of oxidative phosphorylation to provide cells with the energy they need and maintain normal cell metabolic activities. More and more studies have pointed out that the occurrence and development of various diseases are closely related to mitochondrial function damage, such as decreased efficiency of electron transfer in the respiratory chain, excessive accumulation of reactive oxygen species (ROS), and damage to mitochondrial DNA. These abnormalities will aggravate intracellular oxidative stress and energy metabolism disorders, further exacerbating the progression of the disease. Delivering healthy mitochondria to damaged cells to replace damaged mitochondrial function is a new treatment strategy proposed in recent years. Through this exogenous supplement of healthy mitochondria, the efficiency of the electron transport chain can be restored, the energy supply in damaged cells can be improved, and the oxidative stress homeostasis can be maintained, thereby repairing damaged cells to slow down or even stop the continued development of the disease.

[0003] Currently, in most mitochondrial transplantation cases, free mitochondria are separated and purified from healthy cells and then injected into the disease site for treatment. However, this treatment strategy faces many problems: the mitochondrial separation and purification process will damage its activity and thus affect the efficacy; free mitochondria are easily interfered by calcium and magnesium ions and proteases in the in vivo and in vitro environment, which damages mitochondrial activity; free mitochondria lack the ability to selectively transport damaged cells, and the delivery efficiency to recipient cells is limited. These problems limit the efficacy of the mitochondrial transplantation strategy.

[0004] By encapsulating mitochondria to construct mitochondrial preparations through reasonable formulation design, the adverse effects of the extracellular environment on the physiological function of mitochondria can be shielded, the activity stability can be improved, and the in vivo delivery of mitochondria can be achieved. It is expected to become a potential mitochondrial preparation product to solve the above-mentioned problems in mitochondrial transplantation therapy. However, there are currently few reports on the design of mitochondrial preparations and related preparation methods, and there is a lack of relevant patents. Some studies have shown that some cells naturally secrete extracellular vesicles containing mitochondrial components, and by separating and purifying the cell supernatant, extracellular vesicles containing mitochondria can be obtained. For example, Chinese patent CN115068502A proposes a mitochondrial delivery system based on natural cell vesicle suspension, which can deliver active mitochondria to recipient cells, tissues or organs through natural vesicles containing mitochondria, reflecting the feasibility of vesicle systems as mitochondrial delivery carriers. However, mitochondrial vesicles secreted by natural cells have problems such as low yield and limited mitochondrial loading efficiency. Therefore, this mitochondrial delivery strategy based on natural vesicles has not yet completely solved the problem of mitochondrial preparation construction in mitochondrial transplantation therapy, and it is difficult to meet the needs of future clinical treatment.

[0005] In summary, there is currently a lack of good preparations that can be used for mitochondrial transplantation therapy, and there is also a lack of relevant preparation methods that can achieve efficient encapsulation, effective protection and targeted delivery of mitochondria outside cells. Breakthroughs in new preparation designs and preparation methods are urgently needed to solve the mitochondrial preparation problems faced in mitochondrial transplantation therapy. Summary of the invention

[0006] The purpose of the present invention is to provide a cell membrane vesicle preparation loaded with mitochondria and its design method and preparation method. The design method provided by the present invention is to load mitochondria into an artificially constructed vesicle preparation to achieve good protection and efficient delivery of mitochondria; the preparation method provided by the present invention can achieve rapid and effective loading of mitochondria in donor cells; the present invention also provides a vesicle preparation loaded with mitochondria obtained by the above preparation method and its application in the treatment of diseases related to mitochondrial damage, and the prepared vesicle preparation can protect the physiological function of mitochondria therein and improve the transplantation efficiency of mitochondria.

[0007] The present invention provides the following technical solutions to solve the above technical problems:

[0008] The present invention provides a design method for a cell membrane vesicle preparation loaded with mitochondria, wherein the design method is to load mitochondria into artificially constructed cell membrane bionic vesicles to construct a nanoscale vesicle preparation with mitochondria loaded inside and cell membrane outside. The preparation can achieve good protection and efficient delivery of mitochondria and is used for the treatment of related diseases.

[0009] In the present invention, the cell membrane vesicle preparation carrying mitochondria may be referred to as the vesicle preparation carrying mitochondria.

[0010] The present invention provides a method for preparing a cell membrane vesicle preparation carrying mitochondria, the preparation method comprising the following steps:

[0011] (1) preparing cells into a cell suspension;

[0012] (2) The cell suspension is repeatedly extruded through an extrusion device equipped with a filter membrane with a specific pore size so that the cell membrane is fully loaded with intracellular mitochondria;

[0013] (3) The extrudate is subjected to differential centrifugation, the precipitate is collected, and a cell membrane vesicle preparation containing mitochondria is prepared.

[0014] In step (1), the cell is any type of cell containing mitochondria. In the present invention, any eukaryotic cell containing mitochondria can be used, so the present invention is not specifically limited.

[0015] In step (1), cells are resuspended in a buffer system, which is non-toxic to cells, does not affect the basic functions of cells, and does not damage the physiological functions of mitochondria.

[0016] In step (2), the pore size of the filter pores of the extrusion device is 2-20 μm, and the number of extrusions is 2-30 times.

[0017] The present invention achieves that the mitochondria in the donor cells are fully loaded with vesicles by regulating the pore size of the filter pores, and avoids damage to the mitochondrial structure and function caused by excessive and repeated extrusion by regulating the number of extrusions.

[0018] Preferably, the pore size of the filter is 4-10 μm, and the number of repeated extrusions is preferably 4-8 times. The vesicle preparation prepared by the above extrusion method has the highest mitochondrial loading efficiency.

[0019] In step (3), the differential centrifugation includes two centrifugation steps, and the centrifugation conditions are: centrifugal force of 800-2000g, centrifugation time of 8-15min; centrifugal force of 7500-25000g, centrifugation time of 10-30min. The purpose of the first centrifugation step is to separate cell debris and apoptotic bodies, and the purpose of the second centrifugation step is to purify and collect vesicles containing mitochondria.

[0020] Furthermore, the differential centrifugation needs to be performed under low temperature conditions to protect the physiological function of mitochondria, preferably at 4°C.

[0021] The present invention also provides a vesicle preparation containing mitochondria obtained by the above preparation method. The vesicle preparation can protect the physiological function of mitochondria therein and improve the transplantation efficiency of mitochondria.

[0022] The size of the mitochondrial vesicle preparation is 100-1000 nm.

[0023] The present invention also provides an application of the above-mentioned mitochondria-loaded vesicle preparation in treating diseases related to mitochondrial damage.

[0024] The diseases associated with mitochondrial damage include pulmonary fibrosis, myocardial ischemia, obesity, Alzheimer's disease and Parkinson's disease.

[0025] The administration methods of the mitochondria-loaded vesicle preparation include intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, atomization inhalation, nasal administration and oral administration.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) The mitochondria-loaded vesicle preparation technology provided by the present invention can achieve rapid and efficient loading of mitochondria into cells, is applicable to a wide range of cell types, has a simple preparation method, and a fast and convenient process. It can overcome the problems of low yield and limited loading efficiency of mitochondrial vesicles from natural sources, and is expected to meet the needs of future clinical treatments.

[0028] (2) The mitochondria-loaded vesicle preparation technology provided by the present invention avoids the cumbersome free mitochondria extraction process in the early stage of existing mitochondrial transplantation, greatly reducing the adverse effects of the preparation process on mitochondrial activity;

[0029] (3) The mitochondria-loaded vesicle preparation provided by the present invention can better protect the physiological functions of mitochondria and avoid the rapid inactivation of mitochondria during transplantation. At the same time, it can also enhance the selective mitochondrial delivery to damaged cells and improve the efficiency of mitochondrial transplantation. This new mitochondrial vesicle preparation can solve the problems faced by current mitochondrial transplantation therapy, such as low efficiency of free mitochondrial transplantation and lack of targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the design and preparation of the mitochondria-loaded vesicle preparation constructed in the present invention.

[0031] Figure 2 is the loading efficiency of the vesicle preparation in Example 1 on mitochondria.

[0032] Figure 3 This is a transmission electron micrograph of the mitochondrial vesicle preparation prepared in Example 2.

[0033] Figure 4 The particle size and potential of the mitochondrial vesicle preparation prepared in Example 2.

[0034] Figure 5 The loading efficiency of the vesicle preparations prepared in Examples 2 and 3 into mitochondria.

[0035] Figure 6 The single-shot yield and mitochondrial loading capacity of the mitochondrial vesicle preparation prepared in Example 2.

[0036] Figure 7 The figure shows the protective effect of the vesicle preparation prepared in Example 2 on the mitochondria under different storage conditions.

[0037] Figure 8 Fluorescence images of mitochondrial transplantation of the mitochondrial vesicle preparation prepared in Example 1 into healthy and bleomycin (BLM)-induced damaged lung epithelial cells.

[0038] Fig. 9 This is a fluorescent image of the distribution of the mitochondrial vesicle preparation prepared in Application Example 2 in the lungs of fibrotic mice after tracheal instillation.

[0039] Fig.10 This is the effect of the mitochondrial vesicle preparation of the present invention in Application Example 3 on the proliferation ability, intracellular ROS level and transforming growth factor β (TGF-β) of damaged alveolar epithelial cells.

[0040] ns in the figure means no significant difference; *P<0.05; **P<0.01; ***P<0.001;

[0041] ****P<0.0001. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments, application examples and accompanying drawings.

[0043] Example 1: Effect of filter membrane pore size on mitochondrial loading efficiency

[0044] Taking human umbilical cord-derived mesenchymal stem cells (hMSCs) as an example, Figure 1 The mitochondria-loaded vesicle preparation is prepared by the preparation method shown, and the specific steps are as follows:

[0045] 1) hMSCs were cultured to a cell density of 70-80%, the cells were digested with trypsin, and the cells were resuspended in phosphate buffered saline (PBS) without calcium and magnesium ions.

[0046] 2) The cell suspension is extruded using an extrusion device with a specific filter pore structure. The groups are set as: passing through 2μm, 5μm, and 10μm pore size filters separately, or passing through 10μm, 5μm, and 2μm pore size filters in sequence.

[0047] 3) Mitochondria and membrane structures were marked with Mitotracker Green (MTG, excitation wavelength 490 nm, emission wavelength 516 nm) and cell membrane dye DID (excitation wavelength 644 nm, emission wavelength 665 nm), respectively. Nanoflow cytometry was used to analyze the proportion of double-positive vesicles of the above two staining reagents in the total vesicle population to examine the loading efficiency of mitochondria.

[0048] Depend on Figure 2 It can be seen that when the cell suspension is extruded only through 2μm, 5μm, and 10μm pore size filters, the nanoflow cytometry results show that the preparation method provided by the present invention can successfully prepare mitochondria-loaded vesicle preparations, wherein when the filter membrane pore size is 10μm, the mitochondrial loading efficiency is the highest. After the cell suspension is extruded through 10μm, 5μm, and 2μm pore size filters in turn, the mitochondrial loading efficiency is the lowest.

[0049] Example 2: Preparation of mitochondria-loaded vesicle preparations

[0050] Taking hMSCs as an example, Figure 1 The preparation method shown is used to prepare a vesicle preparation loaded with mitochondria, and the specific steps are as follows:

[0051] 1) hMSCs were cultured to a cell density of 70-80%, the cells were digested with trypsin, and the cells were resuspended in PBS without calcium and magnesium ions.

[0052] 2) The cell suspension was repeatedly extruded 6 times using an extrusion device equipped with a filter membrane with a specific pore size (pore size: 10 μm).

[0053] 3) Collect the extrudate, centrifuge at 1000 g for 10 min at 4°C, and discard the precipitate.

[0054] 4) Take the supernatant, centrifuge at 16000g for 10 min, and collect the precipitate to obtain the mitochondrial-loaded vesicle preparation.

[0055] Example 3: Preparation of mitochondria-loaded vesicle preparations

[0056] Taking mouse melanoma cells (B16F10) as an example, the Figure 1 The mitochondria-loaded vesicle preparation is prepared by the preparation method shown, and the specific steps are as follows:

[0057] 1) B16F10 cells were cultured to a density of 70-80%, and after trypsin digestion, the cells were resuspended in PBS without calcium and magnesium ions.

[0058] 2) The cell suspension was repeatedly extruded 6 times using an extrusion device with a specific pore structure (pore size: 10 μm).

[0059] 3) The extrudate was collected and centrifuged at 1000 g for 15 min at 4°C, and the precipitate was discarded to remove cell debris and apoptotic body components.

[0060] 4) Take the supernatant, centrifuge at 16000g for 10 min, and precipitate the vesicle preparation containing mitochondria.

[0061] The mitochondria-loaded vesicle preparation prepared in Example 2 or 3 was characterized, specifically including:

[0062] (1) Transmission electron microscopy images of mitochondrial vesicle preparations

[0063] Take the mitochondria-loaded vesicles prepared in Example 2, and quickly put them into 2.5% glutaraldehyde fixative for 2-4 hours. Rinse with 0.1M phosphate buffer (pH 7.0) 3 times, 15 minutes each time. Fix with 1% tris(IV) acid (pH 7.0) for 1-3 hours. Wash with 0.1M phosphate buffer (pH 7.0) 3 times, 15 minutes each time. After dehydration and infiltration treatment, embed in an embedding agent overnight. After drying, slice, double stain with uranium-lead, and dry at room temperature overnight. After sample preparation, place the slices under a transmission electron microscope to observe and collect pictures.

[0064] Depend on Figure 3 It can be seen that the mitochondrial structure can be clearly observed in the vesicle preparation with a single-layer membrane under an electron microscope, which proves that the present invention can achieve successful loading of mitochondria.

[0065] Depend on Figure 4 It can be seen that the particle size and potential of the vesicle preparation loaded with mitochondria were measured by a dynamic light scattering instrument, and the particle size was 291.5±10.66nm and the potential was -12.4±1.2mV.

[0066] (2) The proportion of mitochondria-loaded vesicles in the total vesicle population prepared

[0067] Take the mitochondria-loaded vesicle preparation prepared in Example 2 or 3, and use the mitochondria-specific staining reagent MTG and the cell membrane dye DID to mark the mitochondria and membrane structure respectively. Use nanoflow cytometry to analyze the proportion of the double-positive vesicle population of the above two staining reagents in the total vesicle population to examine the mitochondria loading efficiency.

[0068] Depend on Figure 5 It can be seen that the mitochondrial loading efficiency of the vesicle preparation prepared by extrusion of hMSCs is more than 50%, and the mitochondrial loading efficiency of the vesicle preparation prepared by extrusion of B16F10 cells is more than 40%, indicating that the preparation method of the mitochondrial vesicle preparation provided by the present invention is applicable to different types of eukaryotic cells containing mitochondria.

[0069] (3) Single preparation yield and mitochondrial loading capacity of mitochondrial vesicle preparations

[0070] The extracellular vesicle components naturally secreted by cells in the cell culture supernatant were collected according to the differential centrifugation separation and purification scheme described in Example 2 as control group 1. The collection scheme for natural mitochondrial biological vesicles has been reported in the reference (PMID: 33632482), and the natural extracellular vesicle components in the cell culture supernatant were collected as control group 2. The protein level in the prepared vesicle preparation was analyzed using a protein quantitative detection kit (Biyuntian, P0012). The number of prepared vesicles was analyzed using a nanoflow analyzer. The mitochondrial components were labeled using the mitochondrial-specific staining reagent MTG.

[0071] Depend on Figure 6 It can be seen that the natural biological vesicles containing mitochondria prepared in Example 2 have disadvantages such as low yield and limited mitochondrial loading efficiency when compared with the control group 1 and the control group 2. The artificial extrusion preparation method proposed in the present invention can realize convenient and efficient loading of mitochondria in cells.

[0072] (4) Protective effect of mitochondrial vesicle preparations on mitochondria

[0073] Free mitochondria were extracted from hMSCs using a commercially available mitochondrial extraction kit (such as Solebold, SM0020). Vesicle preparations containing mitochondria derived from hMSCs were prepared using Example 2. Free mitochondria and mitochondrial vesicle preparations were stored at 4°C and 25°C, respectively, and the changes in ATP levels in the samples were detected every 6 hours using an ATP detection kit to evaluate the protective effect of the vesicle preparations prepared by the present invention on the mitochondria therein.

[0074] Depend on Figure 7 It can be seen that regardless of the storage conditions at 4°C or 25°C, the rate of ATP decline in the mitochondrial vesicle preparation was significantly reduced, indicating that the vesicle preparation has a protective effect on the mitochondria therein and prolongs the duration of mitochondrial activity.

[0075] Application Example 1: Selective mitochondrial transplantation into damaged cells using mitochondrial vesicle preparations

[0076] The mitochondria-loaded vesicle preparation prepared in Example 2 was taken and the mitochondria therein were labeled with the mitochondria-specific staining reagent MTG. After labeling, the mitochondria were inoculated into a 24-well plate (2×10 4 The well plate was placed in the Incucyte live cell real-time imaging analyzer, and the culture condition was set to 5% CO 2, 37° C., and fluorescence images of the cells in the wells were taken every 5 h to analyze the transport efficiency of the mitochondria-loaded vesicle preparation of the present invention to the mitochondria of damaged cells.

[0077] Depend on Figure 8 It can be seen that the mitochondrial vesicle preparation prepared by the present invention is more easily taken up by damaged cells and has a certain selectivity for damaged cells.

[0078] Application Example 2: Mitochondrial vesicle preparations can significantly prolong the residence of active mitochondria in mouse lungs

[0079] Taking the BLM-induced mouse pulmonary fibrosis model as an example, the residence of the mitochondrial vesicle preparation prepared in Example 2 in the lungs of fibrotic mice was investigated. A pulmonary fibrosis mouse model (male C57BL / 6 mouse) was constructed by intratracheal instillation of BLM at a dose of 0.8 mg / kg. The mitochondrial-specific staining reagent Mitotracker deep red (MTDR, excitation wavelength 644 nm, emission wavelength 665 nm) was used to mark the mitochondria with physiological functions in the free mitochondria and mitochondrial vesicle preparations, respectively. After labeling, the mitochondrial vesicle preparations loaded with mitochondria were administered to fibrotic mice stimulated with BLM for 24 h by tracheal instillation at a dose of 5 μg. Free mitochondria were intratracheally instilled at a dose of 1.25 μg (the MTDR fluorescence intensity level was consistent with that in the mitochondrial vesicle preparation group loaded with mitochondria at this dose). At 4h and 8h after administration, the mice were killed by cervical dislocation, the lung tissue of the mice was removed, and the MTDR signal intensity in the lungs of each group of mice was analyzed using a small animal in vivo imaging system to determine the distribution of active mitochondria.

[0080] Depend on Fig. 9 It can be seen that 8 hours after tracheal instillation, the active mitochondrial signal in the lungs of mice in the free mitochondria group was significantly reduced and could not be detected, while there was still an obvious active mitochondrial signal in the lungs of mice in the group given the mitochondria-loaded vesicle preparation, indicating that the mitochondria-loaded vesicle preparation provided by the present invention can effectively prolong the residence time of active mitochondria in the lungs of fibrotic mice.

[0081] Application Example 3: Using mitochondrial vesicle preparations to perform mitochondrial transplantation to repair damaged cells

[0082] Taking BLM-induced damaged mouse TC-1 cells as an example, the mitochondrial vesicle preparation prepared in Example 2 was used for mitochondrial transplantation therapy.

[0083] 1) TC-1 cells were cultured at 2×10 4 The cells were seeded at a density of 10 cells / well in a 24-well plate. After the cells adhered to the wall, they were treated with 10 μg / mL BLM for 24 h to induce mitochondrial damage in TC-1 cells.

[0084] 2) The mitochondrial vesicle preparation prepared in Example 2 was inoculated into the culture system of the damaged TC-1 cells and incubated in 5% CO 2 , and incubated at 37°C for 24 h.

[0085] 3) A cell proliferation detection kit (Biyuntian, C0071S) and flow cytometry were used to detect the proliferation efficiency of damaged TC-1 cells treated with BLM after mitochondrial transplantation therapy.

[0086] 4) The ROS level in TC-1 cells was detected using a ROS detection kit (Biyuntian, S0033S).

[0087] 5) The level of TGF-β in the supernatant was detected using an enzyme-linked immunosorbent assay kit (Beline, MU30574).

[0088] Depend on Fig.10 It can be seen that the mitochondrial vesicle preparation provided by the present invention can effectively restore the proliferation ability of damaged TC-1 cells, significantly improve the intracellular ROS level, and inhibit the secretion of fibrosis factor TGF-β by damaged cells. This shows that the mitochondrial vesicle preparation provided by the present invention can be used to treat diseases related to mitochondrial damage.

Claims

1. A method for designing a cell membrane vesicle preparation carrying mitochondria, characterized in that: The design method is to load mitochondria into artificially constructed cell membrane bionic vesicles to construct a nano-scale vesicle preparation with mitochondria loaded inside and cell membrane on the outside.

2. A method for preparing the cell membrane vesicle preparation containing mitochondria according to claim 1, characterized in that: The preparation method comprises the following steps: (1) preparing cells into a cell suspension; (2) The cell suspension is repeatedly extruded through an extrusion device equipped with a filter membrane with a specific pore size so that the cell membrane is fully loaded with intracellular mitochondria; (3) The extrudate is subjected to differential centrifugation, the precipitate is collected, and a cell membrane vesicle preparation containing mitochondria is prepared.

3. The method for preparing the cell membrane vesicle preparation containing mitochondria according to claim 2, characterized in that: The cell is a eukaryotic cell containing mitochondria.

4. The method for preparing the cell membrane vesicle preparation containing mitochondria according to claim 2, characterized in that: In step (2), the pore size of the filter holes of the extrusion device is 2-20 μm, and the number of extrusions is 2-30 times.

5. The method for preparing the cell membrane vesicle preparation containing mitochondria according to claim 2, characterized in that: In step (3), the differential centrifugation includes two centrifugation steps, and the centrifugation conditions are: centrifugal force of 800-2000g, centrifugation time of 8-15min; centrifugal force of 7500-25000g, centrifugation time of 10-30min.

6. A cell membrane vesicle preparation containing mitochondria obtained according to the preparation method according to any one of claims 1 to 5.

7. The cell membrane vesicle preparation carrying mitochondria according to claim 6, characterized in that: The particle size of the cell membrane vesicle preparation carrying mitochondria is 100-1000 nm.

8. Use of the cell membrane vesicle preparation carrying mitochondria according to claim 6 in the preparation of drugs for treating diseases related to mitochondrial damage.

9. The use according to claim 6, characterized in that: The diseases associated with mitochondrial damage include pulmonary fibrosis, myocardial ischemia, obesity, Alzheimer's disease and Parkinson's disease.

10. The use according to claim 6, characterized in that: The administration methods of the cell membrane vesicle preparation carrying mitochondria include intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, atomization inhalation and oral administration.

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