Guidant polypeptide for mitochoral transplantation

Abstract

The invention relates to a guiding polypeptide for mitochondrial transplantation for improving penetration rate and targeting property. The specific amino acid sequence of the guiding polypeptide is as shown in SEQ ID No.1. The guiding polypeptide can serve as a penetrating peptide and a targeting peptide, the tumor targeting property of mitochondria is improved by combining with the mitochondria, the mitochondria with biological activity is accurately and efficiently conveyed into tumor cells in the mitochondria transplantation process, and the penetrating efficiency and targeting property of the mitochondria are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular genetics and relates to a polypeptide for modifying mitochondria. The mitochondria modified with the polypeptide of the present invention can improve the targeting and penetration efficiency of mitochondria to recipient tumor cells during mitochondrial transplantation. Background Art

[0002] Malignant tumors, as a major killer endangering human health, have surpassed cardiovascular and cerebrovascular diseases to become the leading cause of death. Among them, lung cancer is the cancer with the fastest growing incidence and mortality rate among malignant tumors, and it poses the greatest threat to human health and life. In the treatment of advanced lung cancer, the commonly used radiotherapy and chemotherapy methods not only cause adverse reactions such as bone marrow suppression, anorexia and vomiting, but immunotherapy also often has many side effects such as inflammatory storms. Moreover, with the accumulation of radiotherapy and chemotherapy times and targeted treatment time, drug resistance treatment bottlenecks and problems often appear in clinical practice, which is also the main cause of death in cancer patients.

[0003] Researchers have recently detected mitochondrial loss in various tumor cells, including liver cancer AS-30D, human breast cancer MDA-MB-231, and liver cancer and breast cancer tissues. Tumor cells with mitochondrial loss are often accompanied by reduced sensitivity to radiotherapy and chemotherapy.

[0004] In recent years, with the development of metabolomics technology and the discovery of tumor metabolites, more and more evidence has shown that tumors are a metabolic disease. Therefore, tumor treatment strategies should be adjusted accordingly, and tumor metabolic regulation therapy should be used as a new treatment method.

[0005] Mitochondria are the most important organelles in human cells, responsible for important life activities such as cellular energy metabolism and apoptosis. The adenosine triphosphate (ATP) produced by mitochondria provides more than 90% of the energy needs of cells to meet human activities.

[0006] Since mitochondria provide most of the energy for cellular life activities, their dysfunction is related to human diseases, especially the occurrence and development of tumors, which are closely related to mitochondrial imbalance and dysfunction. Once mitochondrial dysfunction occurs, it will lead to a decrease in ATP energy synthesis. With the further dysfunction of mitochondria, the further decline of energy synthesis will have a great impact on the further deterioration of tumors.

[0007] Mitochondrial transplantation is a new mitochondrial targeted therapeutic intervention technology that separates exogenous viable healthy mitochondria from normal tissue cells and directly injects them into the site of mitochondrial damage or loss, thereby replacing damaged mitochondria, saving damaged cells and restoring organ function. Existing studies have shown that mitochondrial transplantation has emerged in the treatment of a few diseases related to mitochondrial defects and dysfunctions, opening up new avenues in the fields of cardiovascular ischemia-reperfusion and neuroprotection.

[0008] Mitochondrial transplantation has a wide range of potential clinical applications, and its unique treatment regimen is in line with the precision medicine concept of "minimizing iatrogenic damage and maximizing patient benefits." However, so far, there are no known and approved reports on the treatment of tumors through mitochondrial transplantation.

[0009] Although most cells have the ability to internalize mitochondria, how to accurately transport mitochondria to target cells and improve their efficiency in entering cells (being internalized) remains a difficult problem and primary issue that plagues mitochondrial transplantation.

[0010] The booming development of nanomaterials has brought new opportunities to improve the efficiency and targeting of mitochondrial transplantation. Nanomaterials are located inside or on the surface of mitochondria through encapsulation and adsorption, thereby accurately transporting mitochondria to target cells and improving their efficiency of endocytosis. At present, nanomaterials that have shown potential for biological applications usually include nanoparticles, nanospheres, and nanoliposomes. Among them, peptides are increasingly used in the field of nanobiology because of their good biocompatibility.

[0011] Chang JC et al (Mitochondrial transplantation regulates antitumouractivity, chemoresistance and mitochondrial dynamics in breast cancer. J Exp Clin Cancer Res . 2019, 38(1): 30.) used peptide labeling to achieve transcellular mitochondrial transplantation. The study found that after incubating free mitochondria with the cell membrane penetrating peptide Pep-1 at 37°C, a mitochondria-Pep-1 complex could be formed. After the complex was co-cultured with the recipient cells for 48 hours, exogenous mitochondria could be observed to enter the recipient cells under the mediation of Pep-1. Compared with the conventional co-culture method, the transfer efficiency of this peptide labeling transplantation scheme can be greatly improved. However, the bottleneck problem of how to stably anchor mitochondria and how to accurately transport mitochondria to target cells has not been effectively solved.

[0012] Currently, the commonly used method of anchoring mitochondria is based on the higher membrane potential of mitochondria, and mitochondrial targeting is achieved through TPP (triphenylphosphine) cation modification, but it often faces the problem of poor water solubility. Kuang X et al. (SS-31 peptide enables mitochondrial targeting drug delivery: a promising therapeutic alteration to prevent hair cell damage from aminoglycosides. Drug Deliv .2017, 24(1): 1750-1761.) reported that peptides that are electropositive in physiological solutions, such as SS-31, can avoid the water solubility problem of TPP and be enriched in mitochondria to achieve targeted anchoring to mitochondria. However, the sequence of SS-31 is short and is not suitable for promoting mitochondrial transplantation and penetration.

[0013] In terms of accurately transporting mitochondria to target cells, RGD sequence peptides are a class of short peptides containing arginine-glycine-aspartic acid (Arg-Gly-Asp). As the recognition site for the interaction between integrins and their ligands, they can bind to integrin avp3 specifically expressed by tumor cells or new blood vessels, thereby targeting the therapeutic effector molecules into the tumor site and achieving targeting of tumor cells.

[0014] To date, there are no known and approved reports on the treatment of tumors from the perspective of energy metabolism through the targeted transplantation of mitochondria using biomaterials. Summary of the invention

[0015] The purpose of the present invention is to provide a guide polypeptide for mitochondrial transplantation for improving penetration rate and targeting in view of the problems existing in the prior art.

[0016] To achieve the above-mentioned purpose, the present invention first designs a guide polypeptide constructed by linking the following amino acids:

[0017] Arg-Gly-Asp-Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Asp-Ala-Asp-Leu-Pro

[0018] The guiding polypeptide designed by the present invention is named Pep-144, which is synthesized with arginine as the amino terminus (N terminus) and proline as the carboxyl terminus (C terminus), and the specific amino acid sequence is shown in SEQ ID No.1.

[0019] The guide polypeptide of the present invention can adopt the conventional solid phase peptide synthesis (SPPS) method, using the amino group at the C-terminus of the polypeptide as the starting point of synthesis, and reacting with the activated carboxyl groups of other amino acids to form peptide bonds, and after continuous repetition, finally synthesizing the polypeptide composed of the amino acid links.

[0020] The guiding peptide Pep-144 designed by the present invention includes tumor targeting peptide RGD, mitochondrial targeting peptide DADLP and cell membrane penetrating peptide. Specifically, in Pep-144, the RGD sequence can specifically bind to the integrin receptors highly expressed on the surface of various tumor cells, targeting tumor tissues, and the mitochondrial targeting sequence can anchor mitochondria. The further functionalized cell penetrating peptides of the two can promote the distribution of mitochondria in tumor tissues.

[0021] Therefore, the guiding polypeptide designed by the present invention can be used as a penetrating peptide and a targeting peptide to improve the mitochondrial penetration efficiency and tumor targeting during mitochondrial transplantation.

[0022] The guide polypeptide designed by the present invention can enhance the tumor targeting of mitochondria by binding to mitochondria, and can be used as a new molecular tool to accurately and efficiently deliver biologically active mitochondria into tumor cells.

[0023] Furthermore, the present invention also provides a polypeptide mitochondrial complex, wherein the polypeptide mitochondrial complex comprises the guiding polypeptide Pep-144 designed by the present invention and mitochondria with biological activity obtained by in vitro purification.

[0024] Furthermore, the polypeptide mitochondrial complex of the present invention can be prepared by the following method:

[0025] The dissolved guide polypeptide Pep-144 was mixed with the mitochondrial precipitate isolated in vitro, incubated in a light-proof 4°C shaker for 20 min, and then incubated at 4°C for 20 min to allow the mitochondria and the guide polypeptide to fully combine to form a polypeptide-mitochondrial complex. After centrifugation, the mixture was resuspended in phosphate buffer and placed on ice.

[0026] More specifically, it is preferred that the mitochondrial precipitate is mixed with the guiding polypeptide Pep-144 at a mass ratio of 1:(1.2-1.4).

[0027] The guiding polypeptide Pep-144 constructed by the present invention is a new type of tumor-targeted mitochondrial penetrating peptide, which integrates tumor-targeting polypeptide, mitochondrial-targeting polypeptide and cell membrane-penetrating polypeptide. In biological systems, the guiding polypeptide of the present invention has both hydrophilicity and lipophilicity, and has the characteristics of high biocompatibility, high tumor targeting, good cell membrane penetration, etc., and is not easy to react chemically with intracellular components. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the effect of different peptides on mitochondrial performance and receptor cell performance.

[0029] Among them, a is the change of ATP content in different polypeptide mitochondrial complexes, b is the change of membrane potential in different polypeptide mitochondrial complexes, and c is the effect of different polypeptides on the proliferation ability of recipient tumor cells.

[0030] Figure 2 Circular dichroism spectra of different peptides.

[0031] Figure 3 It is a comparison of the transplantation efficiency of different polypeptide mitochondrial complexes with mitochondria.

[0032] Figure 4 This is a Transwell experiment after different polypeptide mitochondrial complexes were transplanted into recipient tumor cells.

[0033] Figure 5 These are fluorescent photos taken 6 hours after transplantation of Pep-144 mitochondrial complexes into normal cells and tumor cells.

[0034] Figure 6 It is the ATP production after different polypeptide mitochondrial complexes are transplanted into recipient normal or tumor cells. Implementation

[0035] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solution of the present invention so that those skilled in the art can well understand and utilize the present invention, rather than limiting the scope of protection of the present invention.

[0036] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0037] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0038] The mitochondria used in the embodiments of the present invention were extracted from AC16, 293T and other cells by using a cell mitochondrial extraction kit produced by Bio-Tech using differential gradient centrifugation.

[0039] Specifically, 1-2.5 ml of mitochondrial separation reagent is added to 20-50 million cells, the cells are gently suspended, placed in an ice bath for 10-15 minutes, transferred to a glass homogenizer and homogenized for 20-35 seconds, and purified by differential centrifugation at 4°C to finally obtain mitochondria.

[0040] The receptor cells used in the embodiments of the present invention include tumor cells, human large cell lung cancer cells H460, and normal cells, human lung (bronchial) epithelial cells BEAS-2B.

[0041] The recipient cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (P / S) and incubated in a constant temperature incubator at 37°C with 5% CO2.

[0042] In order to solve the problems of mitochondrial transplantation penetration efficiency and tumor targeting, the present invention designed and synthesized three different polypeptides, namely Pep-7 including a cell membrane penetrating peptide and a mitochondrial targeting sequence, Pep-10 including a tumor targeting peptide and a mitochondrial targeting sequence, and a novel polypeptide Pep-144 that fused a tumor targeting polypeptide / mitochondrial targeting sequence and a cell membrane penetrating peptide. By measuring the Zeta potential of each polypeptide, it was found that Pep-10 was not suitable for anchoring mitochondria due to the high proportion of RGD in the entire polypeptide sequence and the negative charge. Furthermore, by comparing the differences between Pep-7 and Pep-144 in enhancing the mitochondrial transplantation efficiency, it was found that Pep-144 was more effective. Example

[0043] Example 1

[0044] In order to solve the problems of penetration efficiency and tumor targeting during mitochondrial transplantation, this embodiment designed and constructed a novel mitochondrial guiding polypeptide Pep-144 with a unique structure based on the relevant mechanism and combined with the structural characteristics of mitochondria. The specific amino acid sequence is shown in SEQ ID No. 1:

[0045] H2N-Arg-Gly-Asp-Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Asp-Ala-Asp-Leu-Pro-COOH

[0046] The guide peptide Pep-144 of the above sequence integrates the tumor-targeting peptide RGD, the mitochondrial-targeting peptide DADLP and the cell membrane-penetrating peptide. Among them, the RGD sequence can specifically bind to the integrin receptors highly expressed on the surface of various tumor cells and target tumor tissues; the mitochondrial-targeting sequence can target the mitochondrial inner membrane and anchor mitochondria. The further functionalized cell-penetrating peptides of the two can promote the distribution of mitochondria in tumor tissues.

[0047] As a control, different peptides Pep-7 and Pep-10 were also designed in this example.

[0048] Among them, the polypeptide Pep-7 contains a cell membrane penetrating peptide and a mitochondrial targeting peptide, and its amino acid sequence is shown in SEQ ID No. 2:

[0049] H2N-Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Cys-Asp-Ala-Asp-Leu-Pro-COOH

[0050] The polypeptide Pep-10 includes a tumor targeting polypeptide and a mitochondrial targeting polypeptide, and its amino acid sequence is shown in SEQ ID No. 3:

[0051] H2N-Arg-Gly-Asp-Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-COOH.

[0052] The above polypeptides were synthesized using a solid phase polypeptide synthesizer according to the designed sequence, with a purity of 98%.

[0053] Example 2

[0054] The synthesized guide polypeptide Pep-144 was dissolved in PBS buffer to prepare a 1 mg / ml stock solution, which was stored in aliquots at -20°C.

[0055] The purified mitochondrial precipitate resuspended in fresh PBS was mixed with the dissolved guide polypeptide Pep-144 at a mass ratio of 1:(1.2-1.4), incubated in a light-proof 4°C shaking incubator for 20 min, and then incubated at 4°C for another 20 min to allow the mitochondria and Pep-144 to fully combine to form a polypeptide mitochondrial complex. After centrifugation at 3500g for 5 min at 4°C, the mixture was resuspended in phosphate buffer to the working concentration to obtain the Pep-144 mitochondrial complex, and placed on ice for use.

[0056] Example 3

[0057] The Pep-7 mitochondrial complex was prepared by the same method as in Example 2 using the synthetic polypeptide Pep-7 and placed on ice for later use.

[0058] Example 4

[0059] The polypeptide Pep-10 synthesized in Example 1 was used to prepare the Pep-10 mitochondrial complex in the same manner as in Example 2 and placed on ice for later use.

[0060] Example 5

[0061] The guide polypeptide used to complex with mitochondria to form a polypeptide-mitochondrial complex should not damage the mitochondrial structure and should not affect the activity of the recipient cells.

[0062] In this example, the extracted mitochondria were used as blank controls to test the effects of the guiding polypeptide in the polypeptide-mitochondrial complex on the mitochondrial ATP content and membrane potential, as well as the effects of the guiding polypeptide on the recipient tumor cells.

[0063] The purified mitochondria, Pep-144 mitochondrial complex, Pep-7 mitochondrial complex and Pep-10 mitochondrial complex were lysed with ATP lysis buffer, centrifuged at 10,000 rpm for 5 min at 4°C, and incubated in a black plate with ATP detection buffer to detect ATP content. The results are as follows: Figure 1 As shown in (a), the three peptides had no significant effect on the mitochondrial ATP content.

[0064] Using Yisheng JC-10 membrane potential kit, the purified mitochondria, Pep-144 mitochondrial complex, Pep-7 mitochondrial complex and Pep-10 mitochondrial complex were centrifuged at 10000rpm for 5min at 4℃, and JC-10 detection solution was added. After mixing, the mixture was incubated at room temperature. The fluorescence value at 490nm / 525nm was read by microplate reader, and the red / green fluorescence ratio (R / G) was calculated. The results are as follows: Figure 1 As shown in (b), compared with the mitochondrial blank control, the three peptides have no significant effect on the mitochondrial membrane potential and will not destroy the mitochondrial membrane structure.

[0065] H460 cells were plated in a 96-well plate at a concentration of 4000-5000 cells / well. After the cells were completely attached and the cell confluence reached 70-80%, the culture medium was discarded, and culture medium containing polypeptides Pep-144, Pep-7 and Pep-10 were added respectively. After culturing for 24 hours, the culture medium was discarded, PBS and CCK-8 were added and incubated for 1-2 hours, the A450 value was detected by microplate reader, and the proliferation inhibition rate was calculated.

[0066] Figure 1 Figure c) shows the effect of peptides Pep-144, Pep-7 and Pep-10 intervention on the proliferation ability of recipient cells. The results show that the peptides have no significant effect on the proliferation of recipient tumor cells.

[0067] The above test results show that the guiding polypeptide designed by the present invention will not destroy the structure and function of mitochondria, has no obvious killing effect, and will not affect the growth state of recipient tumor cells.

[0068] Example 6

[0069] This example explores the structure of the designed guide polypeptide. Figure 2 The corresponding circular dichroism characterizations of peptides Pep-144, Pep-7 and Pep-10 are given. The results show that the three designed and constructed peptides all have random coil secondary structures, there are no regular peptide conformations in the peptide chains, and their structures are relatively loose, which is not easy to cause occlusion of sequence functional regions.

[0070] Furthermore, the charge characteristics of the constructed peptides Pep-144, Pep-7 and Pep-10 were tested, and their Zeta potentials were 14.7mV, 6.69mV and -19mV respectively. The results showed that only the Zeta potential of Pep-10 was negative, and it could not anchor mitochondria well to form a peptide-mitochondria complex.

[0071] Based on this, in order to achieve peptide modification to enhance mitochondrial transplantation efficiency, Pep-7 or Pep-144 should be used.

[0072] Example 7

[0073] In this example, H460 cells were used as recipient cells to further verify the effects of polypeptides Pep-144 and Pep-7 on improving the ability of mitochondria to enter recipient tumor cells and enhancing the efficiency of mitochondrial transplantation.

[0074] The mitochondrial-specific plasmid pCMV-myc-mito-GFP with a GFP fluorescent label was transferred into 293T cells and cultured at 37°C for 24 h. The mitochondrial fluorescence expression in 293T cells was observed under a fluorescence microscope. The cells with high expression were used for mitochondrial extraction, and the green fluorescent labeled polypeptide mitochondrial complex was prepared according to the method of Example 2.

[0075] One day before transplantation, 4 × 10 4 H460 cells were plated in a 12-well plate at a density of 10 cells / well. The culture medium was replaced and the cells were starved in serum-free culture medium for 2 h. Then the corresponding mitochondria or polypeptide mitochondrial complexes were added and the cells were intervened in low-serum culture medium containing 1% serum for 6 h. The transplantation of green fluorescent mitochondria into the recipient tumor cells H460 was observed under a fluorescence microscope. The green fluorescence intensity was counted by image-J and the mitochondrial transplantation efficiency was compared.

[0076] Figure 3 A) and b) provide the immunofluorescence effect and its corresponding quantitative results, respectively.

[0077] Among them, the appearance of GFP green fluorescence in a), combined with bright field cell positioning, indicates that the in vitro transplanted mitochondria have entered the recipient tumor cells. The green fluorescence intensity and proportion were counted by image-J to obtain the mitochondrial transplantation efficiency shown in b). By comparing the relative fluorescence areas of purified mitochondria, Pep-144 mitochondrial complexes, and Pep-7 mitochondrial complexes in b), the relative fluorescence areas of Pep-144 mitochondrial complexes and Pep-7 mitochondrial complexes were significantly higher than those of mitochondria, indicating that the guiding polypeptide can significantly enhance the ability of in vitro transplanted mitochondria to enter recipient tumor cells. And the fluorescence proportion of Pep-144 mitochondrial complexes is significantly higher than that of Pep-7 mitochondrial complexes, indicating that the composite polypeptide Pep-144 is the best at improving the efficiency of mitochondrial transplantation, which is consistent with the design expectations.

[0078] Furthermore, the phenotypic effect of guide peptide modification on mitochondrial transplantation efficiency was further verified through Transwell experiments.

[0079] like Figure 4 As shown, compared with the H460 blank control group, the crystal violet staining area in the mitochondrial transplantation group was reduced, indicating that mitochondrial transplantation can effectively inhibit the metastasis and invasion ability of tumor cells H460.

[0080] Furthermore, the crystal violet staining area of ​​Pep-144 mitochondrial complex and Pep-7 mitochondrial complex was lower than that of the mitochondrial group, proving that compared with single mitochondria, Pep-144 mitochondrial complex and Pep-7 mitochondrial complex have stronger ability to inhibit the metastasis and invasion of tumor cells H460, among which the crystal violet staining area of ​​Pep-144 mitochondrial complex was the lowest.

[0081] Example 8

[0082] More importantly, further experiments have shown that although the novel and uniquely structured Pep-144 mitochondrial complex of the present invention can significantly enhance the transplantation efficiency of mitochondria in tumor cells, it does not affect the transplantation efficiency of mitochondria in normal cells, and has significant tumor cell mitochondrial transplantation targeting.

[0083] Figure 5 The green fluorescent labeled Pep-144 mitochondrial complex prepared in Example 7 was transplanted into tumor cells H460 and normal cells BEAS-2B for immunofluorescence effect control.

[0084] The transplantation of Pep-144 mitochondrial complex in tumor cells H460 and normal human lung (bronchial) epithelial cells BEAS-2B was compared by GFP green fluorescence statistics. The results showed that the green fluorescence of Pep-144 mitochondrial complex in H460 was significantly higher than that in BEAS-2B, and almost no green fluorescence appeared in BEAS-2B, indicating that Pep-144 mitochondrial complex can enhance the transplantation efficiency of mitochondria in tumor cells without affecting the transplantation efficiency of mitochondria in normal cells, and has significant tumor targeting of mitochondrial transplantation.

[0085] Example 9

[0086] The present invention also measured the ATP production in the recipient cells after mitochondrial transplantation by Seahorse, and further compared the effects of the polypeptide mitochondrial complex on improving the ATP production capacity (energy metabolism) of mitochondrial transplantation in tumor cells and normal cells.

[0087] Figure 6 In the normal cells BEAS-2B, there was no significant difference in ATP production between the Pep-144 mitochondrial complex and the Pep-7 mitochondrial complex compared with the mitochondrial group; while in the tumor cells H460, compared with the mitochondrial group and the Pep-7 mitochondrial complex, the Pep-144 mitochondrial complex could significantly reduce ATP production and enhance the effect of mitochondrial transplantation on energy metabolism.

[0088] The above results indicate that the RGD sequence plays a tumor cell targeting role in Pep-144. Pep-144 can not only enhance the efficiency of mitochondrial transplantation, but also improve the tumor targeting of mitochondrial transplantation.

[0089] The above embodiments of the present invention do not describe all the details in detail, nor limit the present invention to the above embodiments. Various changes, modifications, substitutions and variations made by ordinary technicians in this field without departing from the principles and purpose of the present invention should be included in the protection scope of the present invention.

Claims

1. A guide polypeptide for mitochondrial transplantation for improving penetration rate and targeting, wherein the guide polypeptide has the amino acid sequence shown below: Arg-Gly-Asp-Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Asp-Ala-Asp-Leu-Pro.

2. Use of the guiding polypeptide according to claim 1 in the preparation of penetrating peptides and targeting peptides for improving mitochondrial penetration efficiency and tumor targeting during mitochondrial transplantation.

3. A polypeptide-mitochondrial complex, comprising the guiding polypeptide according to claim 1 and mitochondria purified in vitro.

4. The method for preparing the polypeptide mitochondrial complex according to claim 3 is to mix the dissolved guiding polypeptide according to claim 1 with the mitochondrial precipitate separated in vitro, incubate in a light-proof 4°C shaking incubator for 20 minutes, incubate at 4°C statically for 20 minutes, so that the mitochondria and the guiding polypeptide are fully combined to form a polypeptide mitochondrial complex, and resuspend in phosphate buffer after centrifugation.

5. The method for preparing the polypeptide mitochondrial complex according to claim 4, characterized in that The mitochondrial precipitate and the guide polypeptide were mixed at a mass ratio of 1:(1.2-1.4).

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