Preparation and application of mitochondrion-oriented peptide complex for ischemic myocardium targeting

Abstract

The present application relates to a kind of preparation and application of mitochondrial targeting peptide complex of ischemic myocardium, belong to the field of biological medicine technology.The mitochondrial recognition molecule and ischemic myocardial cell targeting peptide are combined, and form mitochondrial-TPP+CSTSMLKAC complex with active mitochondria response, and ischemic myocardial injury is treated by intravenous transplantation;Guiding peptide CSTSMLKAC can guide mitochondria to myocardial ischemic area, and damaged myocardial cell is enhanced by uptake of exogenous mitochondria Energy metabolism, improve myocardial energy-dependent myocardial cell's mechanical contraction and diastolic function, inhibit the damage caused by myocardial cell apoptosis, delay the occurrence of heart failure;Mitochondrial-TPP+CSTSMLKAC complex can relieve cardiac dysfunction after intravenous transplantation.By intravenous transplantation of mitochondria, mitochondrial transplantation pathway can be significantly simplified, and the cost of mitochondrial transplantation application can be reduced, which has important clinical popularization and application value.

Description

Technical Field

[0001] This invention relates to the preparation and application of a mitochondrial-guided peptide complex targeting ischemic myocardium, belonging to the field of biomedical technology. Background Technology

[0002] Mitochondria play a crucial role in maintaining cellular homeostasis and function. As a high-oxygen-consuming organelle, mitochondrial dysfunction directly impacts cellular function, ultimately leading to organ damage. The heart, as a high-energy-demand organ, is even more dependent on mitochondria. The fact that mitochondria constitute 30% of cardiomyocytes underscores their importance from a developmental and evolutionary perspective. Mitochondria produce 30 kg of ATP daily to maintain normal cardiac mechanical function. They also ensure the contractile and relaxation functions of cardiomyocytes through oxidative phosphorylation. Mitochondrial structural and functional disorders are primarily manifested as mitochondrial volume expansion, increased cristae area, and increased mitochondrial matrix volume. Furthermore, decreased activity of the mitochondrial electron transport chain complex and increased calcium accumulation within mitochondria lead to a decline in mitochondrial DNA transcription regulation. Therefore, timely replenishment and functional repair of mitochondria after myocardial injury can alleviate the myocardial damage caused by these factors.

[0003] It has been confirmed that aldehyde dehydrogenase 2 (ALDH2), a mitochondrial protective molecule, plays a crucial role in mitochondrial transplantation therapy. Targeted mitochondrial migration is a key challenge in advancing the clinical application of mitochondrial transplantation. Blood is the best medium for enabling cardiomyocytes to more effectively take up exogenous mitochondria, making intravenous injection the optimal choice. The question then arises: can exogenous mitochondria survive in the blood and directly reach their target sites? Triphenylphosphine (TPP+) is a mitochondrial-specific targeting molecule. TPP+ can effectively deliver probes, antioxidants, and drug groups to mitochondria. In biological systems, the TPP+ binding group possesses both hydrophilic and lipophilic properties, and its purification and synthesis are simple, with minimal chemical reactions with intracellular components. Therefore, modifications to TPP+ can accelerate mitochondrial targeting in this research context. Thus, TPP+ can be considered an effective mitochondrial recognition molecule. The question then becomes how to achieve targeted delivery of mitochondria to cardiomyocytes after intravenous injection. Studies have shown that the guide peptide CSTSMLKAC sequence can effectively recognize ischemic cardiac tissue and can serve as a novel molecular tool. By linking with TPP+, it can bind to mitochondria, allowing for the delivery of bioactive mitochondria to damaged myocardium via systemic intravenous injection. By using biomaterials to target and move mitochondria, heart failure can be treated from the perspective of energy metabolism. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problem of how to enable cardiomyocytes to more effectively take up exogenous mitochondria.

[0005] To achieve the goal of solving the above problems, the technical solution adopted by the present invention is to provide a TPP. + The chemical structure of the targeting peptide that binds to the guide peptide CSTSMLKAC is shown in Formula (I).

[0006]

[0007] This invention provides a mitochondrial-guided peptide complex comprising TPP. + Target peptides CSTSMLAKAC and mitochondria; TPP + The guide peptide CSTSMLKAC binds to mitochondria to form mitochondrial-TPP. + +CSTSMLKAC guided peptide complex.

[0008] This invention provides a method for preparing a mitochondrial-guided peptide complex, comprising the following steps:

[0009] Step 1: TPP is chemically synthesized by combining TPPh3 (5-carboxypentyl-triphenylphosphine bromide) with the myocardial guiding peptide CSTSMLKAC. + +CSTSMLKAC guided peptide;

[0010] Step 2: Isolate and purify mitochondria using a tissue mitochondrial isolation kit;

[0011] Step 3: Dissolve the TPP + +CSTSMLKAC guide peptide resuspended the purified mitochondrial precipitate and incubated on a shaker at 4°C in the dark for 20 minutes, followed by static incubation at 4°C for 20 minutes to allow the mitochondria to react with the TPP. + The CSTSMLKAC guide peptide binds fully to form a mitochondrial-TPP-CSTSMLKAC guide peptide complex. After centrifugation, the complex is resuspended in phosphate buffer to a working concentration of 7.5-10 × 10⁻⁶. 4 Mitochondrial suspension per ml, kept on ice until ready for use.

[0012] This invention provides a TPP + Application of targeting peptides that bind to the guide peptide CSTSMLKAC.

[0013] This invention provides a TPP + Application of targeting peptides that bind to the guide peptide CSTSMLKAC in mitochondrial targeted migration.

[0014] This invention provides the application of a mitochondrial-TPP-CSTSMLKAC guide peptide complex in the preparation of drugs for treating and preventing mitochondrial dysfunction.

[0015] This invention provides the application of a mitochondrial-TPP-CSTSMLKAC guiding peptide complex in the preparation of drugs for the treatment and prevention of myocardial injury and heart failure, wherein the myocardial injury includes ischemic myocardial injury.

[0016] This invention provides the application of a mitochondrial-TPP-CSTSMLKAC guided peptide complex in the preparation of a drug that alleviates cardiac dysfunction and increases myocardial energy.

[0017] Preferably, the formulation of the drug includes sprays, drops, and injections.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention uses TPP + A novel ischemic myocardial targeting tool, specifically designed to bind to mitochondria, was synthesized by linking the targeting peptide CSTSMLKAC. The mitochondria were then in vitro bound to this targeting molecule to form a mitochondrial-TPP-CSTSMLKAC polymer. Through intravenous injection of the packaged mitochondrial complex, the CSTSMLKAC targeting peptide delivers mitochondria to the ischemic myocardium. Damaged cardiomyocytes enhance their own energy metabolism by taking up exogenous mitochondria, thereby improving their mechanical contractile and diastolic functions, inhibiting damage caused by cardiomyocyte apoptosis, and delaying the onset of heart failure. Mitochondrial dysfunction is a primary initiating factor in myocardial injury. Implementing a targeted mitochondrial transplantation strategy can effectively compensate for and enhance cardiomyocyte metabolic function, thereby increasing mitochondrial energy production, reducing myocardial reactive oxygen species production, and inhibiting myocardial metabolic transformation, thus effectively enhancing ventricular systolic function and slowing the onset of heart failure.

[0020] This invention combines mitochondrial recognition molecules with ischemic cardiomyocyte-targeting peptides, and reacts with active mitochondria to form a mitochondrial-TPP+CSTSMLKAC complex. This complex is then transplanted intravenously to treat ischemic myocardial injury. Intravenous transplantation of the mitochondrial-TPP+CSTSMLKAC complex effectively alleviates cardiac dysfunction and increases the effectiveness of myocardial energy-dependent systolic and diastolic functions. Intravenous mitochondrial transplantation significantly simplifies the mitochondrial transplantation route and reduces the cost of mitochondrial transplantation, making it of significant clinical application value. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the technical route for treating ischemic myocardial injury via intravenous transplantation of the mitochondrial-TPP+CSTSMLKAC complex of the present invention.

[0022] Figure 2 This is a schematic diagram of the TPP+CSTSMLKAC targeted peptide synthesis.

[0023] Figure 3This image shows the purity analysis of mouse myocardial mitochondria extracted and detected by transmission electron microscopy.

[0024] Figure 4 To detect the effect of echocardiography on the improvement of cardiac function in mice with ischemia-reperfusion injury after mitochondrial transplantation.

[0025] Figure A shows mouse echocardiograms: Sham group (sham-operated group), IR group (ischemia-reperfusion injury mouse group), and IR+PEP-MIO (mitochondrial transplantation intervention group). Figure B shows the significant increase in left ventricular ejection fraction (EF) after mitochondrial transplantation in mice with ischemia-reperfusion injury (EF increased by 17.17%). Figure C shows the significant increase in short-axis shortening (FS) after mitochondrial transplantation in mice with ischemia-reperfusion injury (FS increased by 10.87%). *p<0.05, **p<0.01.

[0026] Figure 5 Evans blue / TTC staining is used to assess the infarct area of ​​the heart in mice with ischemia-reperfusion injury after mitochondrial transplantation.

[0027] Mitochondrial transplantation significantly reduced the infarct size in the heart of mice with ischemia-reperfusion injury.

[0028] White represents myocardial infarction areas (cannot be stained by Evans blue and TTC staining solutions), red represents myocardial ischemia areas (cannot be stained by Evans blue but can be stained by TTC), and blue represents non-ischemic areas (stained by Evans blue and TTC staining solutions).

[0029] Figure 6 This is an immunofluorescence analysis image of a frozen section.

[0030] Green fluorescence was detected in mitochondria. The results confirm that mitochondria can effectively localize to ischemic myocardium. DAPI: localizes to the cell nucleus; GFP: mitochondrial-specific green fluorescent protein; cTnT: cardiac troponin T.

[0031] Figure 7 This image shows the protein analysis of myocardial apoptosis in mice with ischemia-reperfusion injury caused by mitochondrial vein transplantation.

[0032] The expression of the apoptosis protein C-C3 in the myocardium of mice with ischemia-reperfusion injury was significantly upregulated, and intravenous transplantation of mitochondrial guide peptides significantly reduced the expression of C-C3 in the myocardium of mice with ischemia-reperfusion injury. In the figure, C-C3: cleaved-caspase 3 (activated apoptosis protein); β-Actin: internal reference protein. Detailed Implementation

[0033] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0034] This invention provides a TPP + The chemical structure of the targeting peptide that binds to the guide peptide CSTSMLKAC is shown in Formula (I).

[0035]

[0036] This invention provides a mitochondrial-guided peptide complex comprising TPP. + Target peptides CSTSMLAKAC and mitochondria; TPP + The guide peptide CSTSMLKAC binds to mitochondria to form mitochondrial-TPP. + +CSTSMLKAC guided peptide complex.

[0037] This invention provides a method for preparing a mitochondrial-guided peptide complex, comprising the following steps:

[0038] Step 1: TPP is chemically synthesized by combining TPPh3 (5-carboxypentyl-triphenylphosphine bromide) with the myocardial guiding peptide CSTSMLKAC. + +CSTSMLKAC guided peptide;

[0039] Step 2: Isolate and purify mitochondria using a tissue mitochondrial isolation kit;

[0040] Step 3: Dissolve the TPP + +CSTSMLKAC guide peptide resuspended the purified mitochondrial precipitate and incubated on a shaker at 4°C in the dark for 20 minutes, followed by static incubation at 4°C for 20 minutes to allow the mitochondria to react with the TPP. + The CSTSMLKAC guide peptide binds fully to form a mitochondrial-TPP-CSTSMLKAC guide peptide complex. After centrifugation, the complex is resuspended in phosphate buffer to a working concentration of 7.5-10 × 10⁻⁶. 4 Mitochondrial suspension per ml, kept on ice until ready for use.

[0041] This invention provides a TPP + Application of targeting peptides that bind to the guide peptide CSTSMLKAC.

[0042] This invention provides a TPP + Application of targeting peptides that bind to the guide peptide CSTSMLKAC in mitochondrial targeted migration.

[0043] This invention provides the application of a mitochondrial-TPP-CSTSMLKAC guide peptide complex in the preparation of drugs for treating and preventing mitochondrial dysfunction.

[0044] This invention provides the application of a mitochondrial-TPP-CSTSMLKAC guiding peptide complex in the preparation of drugs for the treatment and prevention of myocardial injury and heart failure, wherein the myocardial injury includes ischemic myocardial injury.

[0045] This invention provides the application of a mitochondrial-TPP-CSTSMLKAC guided peptide complex in the preparation of a drug that alleviates cardiac dysfunction and increases myocardial energy.

[0046] The formulations of the aforementioned drugs include sprays, drops, and injections.

[0047] The purpose of this invention is to combine mitochondria with myocardial targeting peptides in vitro to form a mitochondrial-TPP-CSTSMLKAC polymer. After packaging, the mitochondrial transplant is performed by intravenous injection. The targeting peptide CSTSMLKAC can guide mitochondria to the ischemic area of ​​myocardium, where they can be taken up by myocardial cells, thereby improving the energy metabolism and mechanical contraction and relaxation function of ischemic myocardial cells and delaying the onset of heart failure.

[0048] Experimental verification process:

[0049] This invention utilizes a mitochondrial isolation kit (Beyotime Biotechnology Co., Ltd., C3606) to enzymatically digest cardiac muscle, resuspend the mitochondrial isolates in the mitochondrial isolation reagent, grind appropriately, centrifuge at 600g at 4℃ for 10 minutes, collect the supernatant, transfer it to a new EP tube, and continue centrifuging (11000g, 10min) to obtain purified mitochondria for later use.

[0050] The TPP+CSTSMLKAC guide peptide complex was dissolved in phosphate buffer at a concentration of 10 mg / ml. The purified mitochondrial precipitate was resuspended in the dissolved TPP+CSTSMLKAC guide peptide and incubated on a shaker at 4°C for 20 minutes in the dark, followed by incubation at 4°C for 20 minutes to allow the mitochondria to fully bind with the TPP+CSTSMLKAC guide peptide, forming a mitochondrial-TPP-CSTSMLKAC guide peptide complex. After centrifugation, the complex was resuspended in phosphate buffer to the working concentration (7.5-10×10⁻⁶). 4 (each / ml), for later use.

[0051] Finally, a mouse model of myocardial ischemia-reperfusion injury was established (ischemia 45 min, reperfusion 24 h). This model was compared with a mouse model of myocardial ischemia-reperfusion injury model established by tail vein injection of 200 μL of mitochondrial-TPP-CSTSMLKAC guide peptide complex (7.5-10 × 10⁻⁶) during ischemia. 4 The ischemia-reperfusion injury group received only the same volume of PBS (number of transplanted mice per ml), while the control group underwent sham surgery. Echocardiography was performed 24 hours after perfusion to assess cardiac function, immunofluorescence was used to detect the localization of transplanted mitochondria, and Evans blue / TTC staining was used to assess the infarct area of ​​the mouse myocardium.

[0052] Example

[0053] As attached Figure 1 This is a schematic diagram of the technical route for treating ischemic myocardial injury via intravenous transplantation of the mitochondrial-TPP+CSTSMLKAC complex of the present invention.

[0054] 1. TPPh3 (TPPh3 = 5-carboxypentyl-triphenylphosphine bromide) was combined with the myocardial guiding peptide CSTSMLKAC to chemically synthesize TPP+CSTSMLKAC guiding peptide, and a TPP+CSTSMLKAC guiding peptide suspension was prepared by dissolving it in PBS at a concentration of 10 mg / ml.

[0055] 2. Mitochondrial-specific green fluorescent mouse myocardium was obtained. Mitochondria were isolated and purified using a tissue mitochondrial isolation kit (Beyotime Biotechnology Co., Ltd., C3606). The purified mitochondrial precipitate was resuspended in dissolved TPP+CSTSMLKAC guide peptide and incubated on a shaker at 4°C for 20 minutes in the dark, followed by static incubation at 4°C for 20 minutes to allow the mitochondria to fully bind with the TPP+CSTSMLKAC guide peptide to form a mitochondrial-TPP-CSTSMLKAC guide peptide complex. After centrifugation, the complex was resuspended in phosphate buffer to a working concentration of 7.5-10×10⁻⁶. 4 Mitochondrial suspension (number of mitochondria per ml) was kept on ice until use. All procedures were performed under aseptic conditions.

[0056] 3. After transplanting 200 μL of mitochondrial-TPP-CSTSMLKAC guiding peptide complex suspension via the tail vein, adult C57BL / 6 mice with shaved chests were anesthetized with 2% isoflurane gas. The left thorax was opened, and the pectoralis major and pectoralis minor muscles were separated using hemostatic forceps to expose the heart. The heart was excised, and the left anterior descending branch was ligated with a live knot using 6-0 silk suture for 45 minutes to induce ischemia. The ligation was then released, and myocardial reperfusion was performed for 24 hours. 24 hours after reperfusion, echocardiography was performed to assess cardiac function, histopathological examination was conducted to examine changes in myocardial structure, immunofluorescence was used to detect the localization of transplanted mitochondria, and Evans blue / TTC staining was used to assess the infarct area of ​​the myocardium.

[0057] like Figure 1 The diagram shows a schematic of the technical route for treating ischemic myocardial injury via intravenous transplantation of the mitochondrial-TPP+CSTSMLKAC complex of the present invention.

[0058] like Figure 2 The diagram shown illustrates the synthesis of the TPP+CSTSMLKAC targeted peptide.

[0059] like Figure 3 The image shown is a transmission electron microscopy (TEM) analysis of the purity of mouse myocardial mitochondria after extraction.

[0060] like Figure 4The figures show the effect of echocardiography on the improvement of cardiac function in mice with ischemia-reperfusion injury after mitochondrial transplantation. Figure A shows the echocardiograms of mice: Sham group (sham-operated group), IR group (mice with ischemia-reperfusion injury), and IR+PEP-MIO group (mitochondrial transplantation intervention group). Figure B shows the significant increase in left ventricular ejection fraction (EF) after mitochondrial transplantation in mice with ischemia-reperfusion injury (EF increased by 17.17%). Figure C shows the significant increase in short-axis shortening (FS) after mitochondrial transplantation in mice with ischemia-reperfusion injury (FS increased by 10.87%). *p<0.05, **p<0.01.

[0061] like Figure 5 The image shows the effect of mitochondrial transplantation on the infarct area of ​​the heart in mice with ischemia-reperfusion injury, as assessed by Evans blue / TTC staining. Mitochondrial transplantation significantly reduced the infarct area of ​​the heart in mice with ischemia-reperfusion injury; white represents myocardial infarction areas (not stained by Evans blue and TTC), red represents myocardial ischemia areas (not stained by Evans blue but stained by TTC), and blue represents non-ischemic areas (stained by Evans blue and TTC).

[0062] like Figure 6 The image shown is an immunofluorescence analysis of a frozen section. Green fluorescence from mitochondria was detected. The results confirm that mitochondria can effectively localize to ischemic myocardium. In the image, DAPI represents the nucleus; GFP represents mitochondrial-specific green fluorescent protein; and cTnT represents cardiac troponin T.

[0063] like Figure 7 The figure shows the analysis of apoptosis proteins in the myocardium of mice with ischemia-reperfusion injury after mitochondrial intravenous transplantation. The expression of the apoptosis protein C-C3 in the myocardium of mice with ischemia-reperfusion injury was significantly upregulated, and the expression of C-C3 in the myocardium of mice with ischemia-reperfusion injury was significantly reduced after intravenous transplantation of mitochondrial guide peptides. In the figure, C-C3 represents cleaved-caspase 3 (activated apoptosis protein); β-Actin represents an internal reference protein.

[0064] Mitochondrial myocardial targeted intravenous transplantation can ensure myocardial energy supply and inhibit myocardial cell damage caused by mitochondrial dysfunction in patients with ischemic cardiovascular disease, thereby improving myocardial systolic and diastolic function, reducing reperfusion injury, and inhibiting the occurrence of heart failure.

[0065] Mitochondria play a vital role in maintaining cellular homeostasis and function. Ischemia leads to a certain degree of mitochondrial damage, causing the accumulation of mitochondrial metabolites and activating the production of large amounts of reactive oxygen species during reperfusion, further impairing mitochondrial function. As a high-oxygen-consuming and high-energy-producing organelle, mitochondrial dysfunction directly affects normal cardiac mechanical function. Furthermore, mitochondria can also cause cardiomyocyte damage and death by regulating ion concentrations, through mitophagy, and by generating mitochondrial-related apoptotic signals.

[0066] Therefore, mitochondrial dysfunction-induced functional deficiencies are a major target for signal regulation in various aspects of myocardial injury. Based on this, this invention effectively alleviates ischemia-reperfusion myocardial injury through exogenous mitochondrial supplementation. This invention relates to the preparation and intravenous transplantation of the mitochondrial-TPP+CSTSMLKAC complex. The method is authentic and reliable, and the research results have confirmed that intravenous transplantation of the mitochondrial-TPP+CSTSMLKAC complex can indeed effectively target the myocardium and improve ischemia-reperfusion injury. The mitochondrial-TPP+CSTSMLKAC complex provided by this invention has mature preparation conditions, and the improvement of myocardial function after mitochondrial transplantation is significant, which has important clinical translational significance.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A mitochondrial-guided peptide complex, characterized in that, Including TPP + Target peptides CSTSMLAKAC and mitochondria; TPP + The guide peptide CSTSMLKAC binds to mitochondria to form mitochondrial-TPP. + +CSTSMLKAC guided peptide complex; wherein... TPP + It binds to the guide peptide CSTSMLKAC to form a targeting peptide with a chemical structure as shown in formula (I): Formula (I).

2. A method for preparing a mitochondrial-guided peptide complex, characterized in that, Includes the following steps: Step 1: TPP is chemically synthesized by combining TPPh3 (5-carboxypentyl-triphenylphosphine bromide) with the myocardial guiding peptide CSTSMLKAC. + +CSTSMLKAC guided peptide; Step 2: Isolate and purify mitochondria using a tissue mitochondrial isolation kit; Step 3: Use the dissolved TPP + +CSTSMLKAC guide peptide resuspended the purified mitochondrial precipitate and incubated on a shaker at 4°C in the dark for 20 minutes, followed by static incubation at 4°C for 20 minutes to allow the mitochondria to react with the TPP. + The CSTSMLKAC guide peptide binds fully to form a mitochondrial-TPP-CSTSMLKAC guide peptide complex. After centrifugation, the complex is resuspended in phosphate buffer to a working concentration of 7.5-10 × 10⁻⁶. 4 Mitochondrial suspension per ml, kept on ice until ready for use.

3. The use of the mitochondrial guide peptide complex as described in claim 1 in the preparation of drugs for treating and preventing ischemic myocardial injury and heart failure.

4. The application of the mitochondrial guide peptide complex as described in claim 3, characterized in that: The drug is formulated as a spray, drops, or injection.

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