Mitochondrial compound with acidic targeting effect as well as preparation method and application of mitochondrial compound

Abstract

The invention discloses a mitochondrial compound with an acidic targeting effect and a preparation method and application thereof. The mitochondrial compound is prepared according to the following steps: (1) synthesis of pHLIP-PEG-TPP polypeptide: the polypeptide pHLIP and triphenylphosphine-polyethylene glycol-active ester (TPP-PEG1000-NHS) are subjected to an amide reaction to form pHLIP-PEG-TPP; (2) separating and purifying mitochondria (Mito) with complete structure and function by using a mechanical crushing and gradient centrifugation method; and (3) carrying out co-incubation on the mitochondria and the pHLIP-PEG-TPP polypeptide to form a mitochondria complex (PPT-Mito). The polypeptide pHLIP with acidic targeting ability is conjugated with exogenous mitochondria on the basis that the glycolytic function of the M1 type macrophage is enhanced to cause H < + > accumulation on the surface of the cell, so that the targeting affinity of the polypeptide pHLIP to the M1 type macrophage at an inflammation part is optimized, the efficiency of exogenous mitochondrial transfer is improved, the mitochondrial dysfunction of the M1 type macrophage is improved, and the function of the M1 type macrophage is improved. The macrophages are promoted to be converted to M2 type, so that inflammation is relieved, and tissue repair is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a mitochondrial complex with acidic targeting effect and a preparation method and application thereof. Background Art

[0002] Mitochondrial dysfunction is an important feature of the development of inflammation. Mitochondria are semi-autonomous, highly dynamic organelles that play an important role in energy metabolism. They are involved in aerobic respiration, cell proliferation and differentiation, signal transduction, etc. Therefore, mitochondrial dysfunction is considered to be the cause of many human chronic diseases. Macrophages, as sentinel cells, play a vital role in the process of inflammation and tissue repair. Macrophages are plastic cells. Mitochondrial and metabolic dysfunction are closely related to the phenotypic conversion of macrophages. M1 macrophages with pro-inflammatory functions are mainly glycolytic, while M2 macrophages with repair and anti-inflammatory functions are mainly mitochondrial oxidative phosphorylation (OXPHOS). Macrophage phenotypes can be converted to each other, but it is easy for M2 to convert to M1, while it is difficult for M1 to convert to M2, mainly because it is difficult to convert the metabolic mode of glycolysis to oxidative phosphorylation because of mitochondrial dysfunction.

[0003] Mitochondrial transplantation refers to the transfer of exogenous healthy mitochondria to damaged tissues to save cells and tissues. This technology is based on the theory of endosymbiosis, that is, eukaryotic cells engulf free-living aerobic bacteria, which evolve into mitochondria to improve energy efficiency. Mitochondrial transfer also occurs between cells, indicating its ability to survive in recipient cells, providing a theoretical basis for mitochondrial transplantation. At present, this method has been tried in the treatment of myocardial infarction, nerve damage and other diseases and has achieved good experimental results, but there are problems such as lack of targeting, low efficiency, and easy inactivation, which limit its clinical application. Therefore, for inflammatory diseases, it is a very promising strategy to construct a mitochondrial complex that can preferentially target M1 macrophages and facilitate cell internalization. When inflammation occurs, M1 macrophages, which are mainly glycolytic, will produce a large amount of lactic acid, and it needs to be pumped out in time to ensure that the intracellular pH is as close to neutral as possible to maintain normal cell life activities, resulting in the accumulation of hydrogen ions on the cell surface, and the lowest pH, which is an ideal pathological sign. Therefore, it is urgent to develop an active targeted mitochondrial complex that can achieve pH-driven for the treatment of inflammatory diseases.

[0004] The pH Low Insertion Peptides (pHLIPs) are a class of moderately hydrophobic and pH-sensitive peptides, which are composed of two flanks and a transmembrane portion in the middle (TM is essential for interaction with the membrane), and can be inserted into the cell membrane at weakly acidic pH to achieve acidic targeting. Therefore, the synthesis of pHLIP-exogenous mitochondrial conjugates has great prospects for inflammatory diseases mediated by mitochondrial dysfunction. Summary of the invention

[0005] In order to solve the above technical problems, the first purpose of the present invention is to provide a method for preparing a mitochondrial complex with acidic targeting effect, the second purpose is to provide a mitochondrial complex with acidic targeting effect, and the third purpose is to provide its application, by targeting M1 macrophages with acidic cell surface in inflammatory environment, improving mitochondrial transfer efficiency, alleviating mitochondrial dysfunction of M1 macrophages, thereby providing a unique treatment plan for the treatment of diseases related to mitochondrial dysfunction.

[0006] In order to achieve the above first purpose, the technical solution of the present invention is: a method for preparing a mitochondrial complex with acidic targeting effect, characterized in that: the preparation is carried out according to the following steps:

[0007] (1) Synthesizing pHLIP-PEG-TPP polypeptide, the polypeptide pHLIP is subjected to a condensation reaction with triphenylphosphine-polyethylene glycol-active ester (TPP-PEG1000-NHS) to form a pHLIP-PEG-TPP composite polypeptide, wherein the sequence of the polypeptide pHLI is ACDDQNPWRAYLDLLFPTDTLLLDLLWK;

[0008] (2) Use mechanical disruption and gradient centrifugation to isolate and purify mitochondria with intact structure and function;

[0009] (3) The purified mitochondria were co-incubated with pHLIP-PEG-TPP polypeptide to form a mitochondrial complex PPT-Mito.

[0010] In the above scheme: in step (1), during the reaction, the mass ratio of the added polypeptide pHLIP to TPP-PEG1000-NHS is 2:1.

[0011] In the above scheme, the operation of step (2) is as follows: CRL-12424 mouse bone marrow mesenchymal stem cells are cultured, and when the cell density is about 80%, the cells are washed with PBS solution, the cells are digested with trypsin, and the cell pellet is collected; the cell pellet is gently resuspended with pre-cooled PBS solution, a small amount of cells are taken for counting, and the remaining cells are centrifuged to collect the cell pellet; each 1×10 7Add 2 ml of mitochondrial isolation reagent to the cells and let them stand on ice. Then use a glass homogenizer to homogenize them and centrifuge at 4°C to remove unbroken cells, cell nuclei and large cell fragments. Discard the precipitate and transfer the supernatant to 4°C. Centrifuge again and discard the supernatant to obtain the mitochondrial precipitate.

[0012] In the above scheme: in step (2), the culture medium is Gibco modified Eagle medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0013] In the above scheme: the specific operation in step (3) is that the pHLIP-PEG-TPP polypeptide is first dissolved with DMSO and diluted with mitochondrial storage solution to obtain pHLIP-PEG-TPP working solution; then, the mitochondrial precipitate is resuspended with the pHLIP-PEG-TPP working solution, shaken in the dark at 4°C, and then allowed to stand, and finally centrifuged at 4°C to obtain the mitochondrial complex PPT-Mito.

[0014] In the above scheme: in step (3), the mass ratio of pHLIP-PEG-TPP to mitochondria is 1:50.

[0015] Step (2) of the present invention uses mechanical disruption and differential centrifugation to separate and purify mitochondria. The disruption conditions are mild to avoid damaging the mitochondrial structure. The entire operation is carried out at 4°C to avoid a decrease in mitochondrial activity. Mitochondrial storage solution (Biyuntian Biotechnology) is used to maintain mitochondrial integrity.

[0016] A mitochondrial complex with acidic targeting effect is prepared by the method for preparing the mitochondrial complex with acidic targeting effect.

[0017] The mitochondrial complex with acidic targeting effect prepared by the present invention has the effects of improving mitochondrial transfer efficiency, restoring mitochondrial dysfunction of M1 macrophages, promoting the conversion of M1 macrophages to M2 macrophages, and finally inhibiting inflammatory response.

[0018] Application of a mitochondrial complex with acidic targeting effect in the preparation of drugs for treating mitochondrial dysfunction and anti-inflammatory drugs.

[0019] The disease is chronic periodontitis.

[0020] The present invention aims to target M1 macrophages in the inflammatory area. The polypeptide with acidic targeting ability is conjugated to mitochondria to optimize its targeting affinity for M1 macrophages in the inflammatory area. By targeting the acidic cell surface, the mitochondrial transfer efficiency is improved, mitochondrial dysfunction is alleviated, and mitochondrial function is restored. Compared with simple mitochondria, the mitochondrial complex PPT-Mito with acidic targeting effect of the present invention can restore mitochondrial membrane potential of M1 macrophages, increase ATP generation, reduce mitochondrial ROS production and other mitochondrial functions, and weaken the pro-inflammatory effect of macrophages. The results of in vivo animal experiments show that the alveolar bone resorption of periodontitis mice is alleviated, thereby providing a new treatment plan for the treatment of chronic periodontitis and other diseases related to mitochondrial dysfunction.

[0021] Beneficial effects:

[0022] 1) The affinity of the mitochondrial complex for the acidic pH surface of macrophages can significantly improve the ability of mitochondrial transfer, and the polypeptide can be separated from the mitochondria during the transfer into the cell without affecting the activity and function of the mitochondria;

[0023] (2) M1 macrophages efficiently internalize exogenous mitochondria, which can promote the recovery of their mitochondrial function and then transform into M2 macrophages, inhibiting inflammatory responses and promoting tissue repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the synthesis of PPT-Mito mitochondrial complex.

[0025] Figure 2 HPLC chart of pHLIP.

[0026] Figure 3 Mass spectrum of pHLIP

[0027] Figure 4 For pHLIP-PEG-TPP 1 H NMR spectra.

[0028] Figure 5 This is a graph showing the cytotoxicity test of pHLIP-PEG-TPP on RAW264.7 cells.

[0029] Figure 6 Transmission electron microscopy images of purified mitochondrial Mito and PPT-Mito.

[0030] Figure 7 This is the fluorescence co-localization image of pHLIP and Mito in PPT-Mito.

[0031] Figure 8 Mitochondrial membrane potential of purified mitochondria Mito and PPT-Mito.

[0032] Fig. 9 This is a fluorescence image showing that pHLIP can separate from Mito during the membrane penetration process of PPT-Mito.

[0033] Fig.10 This is a fluorescence image of the acidic targeting ability of PPT-Mito.

[0034] Fig.11 PPT-Mito restores mitochondrial function of M1 macrophages.

[0035] Fig.12 PPT-Mito promotes the conversion of M1 macrophages to M2 macrophages.

[0036] Fig.13 PPT-Mito inhibits macrophage inflammatory response.

[0037] Fig.14 PPT-Mito can alleviate periodontal inflammatory bone resorption in mice.

[0038] Fig.15 PPT-M ito can promote the conversion of M1 macrophages to M2 in periodontal tissue in vivo. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0040] Example 1

[0041] A method for preparing a mitochondrial complex with acidic targeting effect is prepared according to the following steps:

[0042] (1) Synthesize pHLIP-PEG-TPP peptide. The terminal amino acid A of pHLIP peptide with sequence ACDDQNPWRAYLDLLFPTDTLLLDLLWK is connected with fluorescent group 5-TMARA to facilitate subsequent experimental observation. The mass spectrometry and HPLC results are shown in Figure 2 ; The synthesis of ACDDQNPWRAYLDLLFPTDTLLLDLLWK is an existing technology, using a solid phase synthesis method. Subsequently, 2% hydrazine hydrate is used to remove the side chain protecting group DDE of K, and then a condensation reaction is performed with triphenylphosphine-polyethylene glycol-active ester (TPP-PEG1000-NHS) to form a pHLIP-PEG-TPP composite polypeptide, the mass ratio of the polypeptide pHLIP to TPP-PEG1000-NHS is 2:1, and it is cut from the resin and purified to obtain a pHLIP-PEG-TPP polypeptide.

[0043] (2) Mitochondrial extraction and purification. CRL-12424 mouse bone marrow mesenchymal stem cells (MSCs) were purchased from ATCC and used as the cell source for mitochondria. An appropriate amount of MSCs were cultured in Gibco modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin. When the cell density was about 80%, the cells were washed with PBS solution, digested with trypsin, and the cell pellets were collected; the cell pellets were gently resuspended with pre-cooled PBS solution, a small amount of cells were taken for counting, and the remaining cells were centrifuged to collect the cell pellets. A mitochondrial isolation kit (Beyotime) was used. Each 1×10 7 Add 2 ml of mitochondrial isolation reagent to the cells and let them stand on ice for 10 min. Then use a 5 ml glass homogenizer to homogenize 15 times, centrifuge at 1000 g for 10 min at 4°C, remove unbroken cells, cell nuclei and large cell fragments, transfer the supernatant, centrifuge at 3500 g for 10 min at 4°C, discard the supernatant to obtain the mitochondrial precipitate for later use, or resuspend the mitochondria in 200 ul of mitochondrial storage solution for subsequent comparative experiments.

[0044] (3) Synthesis of mitochondrial complex PPT-Mito: pHLIP-PEG-TPP was first dissolved in DMSO to a 10 mg / ml stock solution, and then diluted with mitochondrial stock solution to a 50 ug / ml working solution; then, 0.5 mg of mitochondrial precipitate was resuspended in 200 ul of pHLIP-PEG-TPP working solution, shaken for 20 min at 4°C in the dark, and then allowed to stand for 20 min. Finally, PPT-Mito was obtained by centrifugation at 4°C, 3500 g for 10 min.

[0045] Study Results

[0046] 1. Synthesis and characterization of PPT-Mito:

[0047] First, the acidic peptide pHLIP with fluorescent label 5-TMARA was synthesized by solid phase synthesis. The mass spectrometry and HPLC results are shown in Figure 2 and Figure 3 . Then, TPP-PEG-NHS was modified on the amino acid (K) at the end of pHLIP by amide reaction to synthesize pHLIP-PEG-TPP. H NMR spectrum showed that characteristic peaks of TPP, PEG and pHLIP appeared on the synthesized pHLIP-PEG-TPP ( Figure 4 ), indicating that pHLIP-PEG-TPP was successfully synthesized. CCK-8 assay detected the cytotoxicity of pHLIP-PEG-TPP to RAW264.7 cells, and the results showed that pHLIP-PEG-TPP had no obvious toxicity to cells at all concentrations ( Figure 5 ).

[0048] Subsequently, mouse bone marrow mesenchymal stem cells CRL-12424 were used as mitochondrial donors to isolate healthy free mitochondria. TEM and JC-1 results showed that the free mitochondria had normal mitochondrial cristae structure and good mitochondrial membrane potential ( Figure 6 and Figure 8 ), indicating that healthy mitochondria were successfully extracted. Subsequently, the isolated healthy mitochondria were incubated with pHLIP-PEG-TPP at 4°C to finally obtain the PPT-Mito complex (PPT-Mito)( Figure 1 ), TEM results showed that PPT-MITO also had normal mitochondrial cristae structure ( Figure 6 ), the membrane potential is close to that of simple mitochondria ( Figure 8 ), the fluorescence co-localization results showed that pHLIP labeled with 5-TAMRA fluorescence and mitochondria labeled with Mitotracker-Deep Red had good co-localization ( Figure 7 ), the above results indicate the successful synthesis of mitochondrial complexes and show that the polypeptide will not affect the structure and normal activity of mitochondria.

[0049] 2. Acidic targeting ability of PPT-Mito

[0050] CLSM observation showed that the peptides in LPS-stimulated macrophages treated with PPT-Mito showed obvious ring-shaped results around the cytoplasm, that is, the peptides tended to be inserted into the cell membrane and could be separated from the transferred mitochondria, making the mitochondria more effectively internalized by M1 macrophages ( Fig. 9 ). Subsequently, the difference in the internalization ability of M1 and M2 macrophages on mitochondria in PPT-Mito was compared. The mitochondria in PPT-Mito were first labeled with Mitotracker-Deep Red and then co-cultured with M1 and M2 macrophages. Confocal observation results showed that M1 macrophages could internalize more mitochondrial complexes than M2 macrophages ( Fig.10 a), indicating that pHLIP can better target M1 macrophages with acidic cell surfaces. In order to explore whether the internalization efficiency of mitochondria by M1 macrophages is assisted by the acidic peptide pHLIP, Mitotracker-Deep Red was used to label simple free mitochondria Mito and mitochondrial complex PPT-Mito, and then incubated with M1 macrophages for 24 hours. The confocal results showed that the efficiency of mitochondrial transfer of PPT-Mito was significantly higher than that of the simple mitochondria group. The above results indicate that the acidic targeting of pHLIP is very conducive to the efficient internalization of mitochondria by M1 macrophages ( Fig.10 b).

[0051] 3. PPT-Mito restores mitochondrial function in M1 macrophages

[0052] Whether PPT-Mito can restore mitochondrial function was tested. Mitotracker-Deep Red is a membrane potential-dependent dye that focuses more in cells with higher membrane potential, thereby indirectly indicating the functional state of mitochondria. Simple mitochondria and mitochondrial complexes stained with Mitotracker-Deep Red were first incubated with Raw264.7 cells for 24 hours, and then 100ng / ml LPS was added for stimulation. Confocal images showed that LPS stimulation can lead to a significant decrease in the area of ​​Mitotracker-Deep Red fluorescent staining, indicating that inflammation can lead to mitochondrial dysfunction. At the same time, the transfer of simple mitochondria cannot play a significant role in restoring mitochondrial dysfunction, but the connection with mitochondrial complexes with acidic targeting ability can significantly increase the staining area of ​​Mitotracker-Deep Red ( Fig.11 A), which shows that it has a rescue effect on mitochondrial dysfunction caused by LPS stimulation. These mitochondria may come from the healthy mitochondria of the donor or the functional recovery of damaged mitochondria of the recipient. JC-1 experiments also found that mitochondrial complexes can restore the membrane potential of Raw264.7 cells ( Fig.11 B). Mitochondrial ROS (mROS) was detected by Mitosox staining, and PPT-Mito effectively reduced the mitochondrial oxidative stress level ( Fig.11 C). Next, the mitochondrial function was further evaluated, and the ATP test kit was used to detect the ability of cells to produce ATP. The results showed that LPS stimulation would weaken the ability of Raw264.7 cells to produce ATP, while mitochondrial transplantation could offset the inhibitory effect of LPS to a certain extent. At the same time, PPT-Mito treatment could restore the APT production ability of Raw264.7 cells to the greatest extent ( Fig.11 D). As mentioned above, lactic acid is one of the main products of glycolysis. Therefore, in order to determine whether the changes in ATP synthesis in Raw264.7 cells are accompanied by changes in cellular respiration, the amount of lactic acid produced by cells was tested. The results showed that LPS stimulation promoted the production of lactic acid, indicating that inflammatory macrophages mainly use glycolysis as the respiration mode, and after receiving exogenous mitochondria, PPT-Mito treatment significantly reduced it ( Fig.11 E).

[0053] In summary, the mitochondrial complex PPT-Mito can restore mitochondrial dysfunction by increasing the mitochondrial number, membrane potential and ATP production in Raw264.7 cells, reducing ROS and lactate production.

[0054] 4. PPT-Mito promotes macrophage reprogramming and inhibits inflammatory response

[0055] PPT-Mito can promote the recovery of mitochondrial function. In order to determine whether the recovery of mitochondrial function can promote macrophage reprogramming, we first detected the effect of PPT-Mito on macrophage surface markers under LPS stimulation by immunofluorescence. The results showed that CD86 was significantly downregulated and CD206 was significantly upregulated ( Fig.12 In addition, the DCFH results showed that PPT-Mito could also significantly reduce the level of intracellular ROS, while the transplantation of mitochondria alone had no significant effect ( Fig.13 ). q-PCR results also showed that the expression of TNF-α, IL-1β, and IL-6 pro-inflammatory genes was significantly downregulated. In summary, PPT-Mito can promote the reprogramming of M1 macrophages into M2 macrophages by effectively restoring the mitochondrial dysfunction of M1 macrophages, thereby achieving the purpose of inhibiting inflammation.

[0056] 5. The therapeutic effect of PPT-Mito on silk ligature periodontitis in mice

[0057] Finally, a chronic periodontitis model was constructed by ligating the maxillary second molars of mice with silk thread for one week to explore the biological function of PPT-Mito in vivo. First, the results of Micro CT showed that PPT-Mito effectively restored periodontitis-induced bone resorption ( Fig.14 ), the results of immunohistochemistry showed that the M1 in LIP periodontal tissue increased significantly, but PPT-Mito effectively reduced the M1 macrophages and upregulated the M2 macrophages ( Fig.15 ), indicating that PPT-Mito can also effectively reprogram M1 to M2 in vivo, thereby alleviating periodontal inflammatory bone resorption and promoting tissue repair.

[0058] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a mitochondrial complex with acidic targeting effect, characterized in that: Prepare according to the following steps: (1) Synthesizing pHLIP-PEG-TPP polypeptide, condensing the polypeptide pHLIP with triphenylphosphine-polyethylene glycol-active ester (TPP-PEG1000-NHS) to form a pHLIP-PEG-TPP composite polypeptide, wherein the sequence of the polypeptide pHLIP is ACDDQNPWRAYLDLLFPTDTLLLDLLWK; (2) Use mechanical disruption and gradient centrifugation to isolate and purify mitochondria with intact structure and function; (3) The purified mitochondria are co-incubated with pHLIP-PEG-TPP polypeptide to form a mitochondrial complex PPT-Mito.

2. The method for preparing the mitochondrial complex with acidic targeting effect according to claim 1, characterized in that: In step (1), during the reaction, the mass ratio of the added polypeptide pHLIP to TPP-PEG1000-NHS is 2:

1.

3. The method for preparing the mitochondrial complex with acidic targeting effect according to claim 1 or 2, characterized in that: The operation of step (2) is as follows: CRL-12424 mouse bone marrow mesenchymal stem cells are cultured, and when the cell density is about 80%, the cells are washed with PBS solution, the cells are digested with trypsin, and the cell pellet is collected; the cell pellet is gently resuspended with pre-cooled PBS solution, a small amount of cells are taken for counting, and the remaining cells are centrifuged to collect the cell pellet; each 1×10 7 Add 2 ml of mitochondrial isolation reagent to the cells and let them stand on ice. Then use a glass homogenizer to homogenize them and centrifuge at 4°C to remove unbroken cells, cell nuclei and large cell fragments. Discard the precipitate and transfer the supernatant to 4°C. Centrifuge again and discard the supernatant to obtain the mitochondrial precipitate.

4. The method for preparing the mitochondrial complex with acidic targeting effect according to claim 3, characterized in that: In step (2), the culture medium is Gibco modified Eagle medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

5. The method for preparing the mitochondrial complex with acidic targeting effect according to claim 4, characterized in that: The specific operation in step (3) is that the pHLIP-PEG-TPP polypeptide is first dissolved with DMSO and diluted with mitochondrial storage solution to obtain pHLIP-PEG-TPP working solution; then, the mitochondrial precipitate is resuspended with the pHLIP-PEG-TPP working solution, shaken in the dark at 4°C, and then allowed to stand, and finally centrifuged at 4°C to obtain the mitochondrial complex PPT-Mito.

6. The method for preparing the mitochondrial complex with acidic targeting effect according to claim 5, characterized in that: In step (3), the mass ratio of pHLIP-PEG-TPP to mitochondria is 1:

50.

7. A mitochondrial complex with acidic targeting effect prepared by the method for preparing a mitochondrial complex with acidic targeting effect according to any one of claims 1 to 6.

8. Use of the mitochondrial complex with acidic targeting effect as claimed in claim 7 in the preparation of drugs for treating mitochondrial dysfunction and anti-inflammatory drugs.

9. The use according to claim 8, characterized in that: The disease is chronic periodontitis.

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