Preparation method and application of novel nano motor for treating myocardial ischemia reperfusion injury

Abstract

The invention discloses a preparation method and application of a novel nano motor for treating myocardial ischemia reperfusion injury, and belongs to the crossing field of biomedical technology and nano materials. The invention particularly relates to a lipid motor LNMs-P obtained by connecting PSGL1 (P-selectin ligand) and lipase through modified mesoporous silica, and ferroptosis is inhibited by improving the mitochondrial function, so that ischemia reperfusion injury is relieved. According to the invention, two key problems are solved: firstly, a therapeutic agent is effectively and directly conveyed to injured myocardial tissues; 2, the most important cell death pathway of ischemia reperfusion injury, namely ferroptosis, is solved; the medicine disclosed by the invention can permeate myocardial tissues in a targeted manner under the action of PSGL1 and lipase on the surface of the medicine, so that a receptor PPARalpha is activated. PPARalpha is combined with fatty acid and translocated to enter a cell nucleus to promote transcription of a mitochondrial transcription factor A, so that synthesis of mitochondria is promoted, the cell lipid content is reduced, ferroptosis is inhibited, and then ischemia reperfusion injury is relieved.

Description

Technical Field

[0001] The present invention belongs to the cross field of biomedical technology and nanomaterials, and specifically relates to a preparation method and application of a novel nanomotor for treating myocardial ischemia-reperfusion injury. Background Art

[0002] Myocardial ischemia-reperfusion (I / R) injury is one of the major challenges in the field of cardiovascular disease, which has a serious impact on the quality of life and survival rate of patients. Although significant progress has been made in new treatment strategies such as drug intervention, gene therapy, and cell therapy in recent years, how to effectively and directly deliver therapeutic agents to damaged myocardial tissue and how to find effective therapeutic targets in the complex pathophysiological process are still the main obstacles limiting the clinical application of these innovative treatments.

[0003] The pathophysiological process of myocardial ischemia-reperfusion injury is complex and involves the interaction of multiple cell death pathways, especially the ferroptosis mechanism, which has attracted much attention in recent years. Ferroptosis is an iron-dependent programmed cell death, which is mainly characterized by the accumulation of lipid peroxides derived from fatty acids and triglycerides in cells, leading to cell damage. This form of death is regulated by a variety of biomarkers, including upregulation of Ptgs2 mRNA expression, decreased NADPH levels, and increased lipid peroxidation. In addition, there is a close connection between ferroptosis and mitochondrial dynamics. Mitochondrial dysfunction and iron ion homeostasis imbalance may trigger the ferroptosis process.

[0004] Mitochondrial fatty acid β-oxidation (FAO) is a key link in cardiomyocyte energy metabolism and is essential for maintaining normal heart function. However, during myocardial ischemia-reperfusion, the FAO process may be disturbed, leading to insufficient energy supply and cardiomyocyte damage. Therefore, regulating the FAO process and the activity of the nuclear hormone receptor PPARα associated with it may provide new ideas for the treatment of myocardial ischemia-reperfusion injury.

[0005] As a new type of biomedical tool, nanomotors have the ability to convert various forms of energy into kinetic energy and penetrate damaged tissues, so they have broad application prospects in the treatment of myocardial ischemia-reperfusion injury. By modifying nanomotors, targeted delivery and penetration of damaged myocardial tissue can be achieved, thereby improving the bioavailability and therapeutic effect of therapeutic agents. However, the synthesis and application of nanomotors still face many challenges, such as how to achieve targeting and permeability at the same time, and how to ensure their safety and stability in the body. Summary of the invention

[0006] The purpose of the present invention is to solve the application problem of nanomotors in damaged myocardial tissue and provide a preparation method and application of a novel nanomotors for treating myocardial ischemia-reperfusion injury. The method obtains lipase-powered PSGL1-loaded mesoporous silica nanomotors (LNMs-P) ​​by modifying mesoporous silica to connect P-selectin ligand (P-Selectin Glycoprotein Ligand 1, PSGL1) and lipase, and inhibits ferroptosis by improving mitochondrial function, thereby reducing ischemia-reperfusion injury.

[0007] The present invention uses mesoporous silica nanoparticles (MSNPs) modified with PSGL1 and lipase as nanomotors to achieve effective treatment of myocardial ischemia-reperfusion injury. PSGL1, as a high-affinity ligand of P-selectin, can specifically recognize and target damaged myocardial tissue; while lipase can hydrolyze fatty acids, thereby regulating the FAO process and the activity of PPARα. Through this modification method, it is possible to achieve precise treatment of damaged myocardium, reduce the degree of myocardial ischemia-reperfusion injury, and improve the quality of life and survival rate of patients.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a novel nanomotor for treating myocardial ischemia-reperfusion injury, the method comprising:

[0010] 1. Synthesis of mesoporous silica nanoparticles (MSNPs);

[0011] Second, amino functionalization of MSNPs;

[0012] 3. Functional modification of MSNP-NH2 using lipase and PSGL1: dissolve the MSNP-NH2 precipitate in PBS solution, ultrasonicate it, add glutaraldehyde to the mixture, and stir it evenly; suspend the glutaraldehyde GA-activated nanoparticles in PBS containing lipase and PSGL1, and stir it evenly.

[0013] Furthermore, the step 1 is specifically as follows: CTAB, TEOA and water are mixed to prepare a mixed solution, the solution is heated in an oil bath, the temperature is maintained at 95°C, and stirring is continued for 30 minutes, tetraethyl silicate (TEOS) is added dropwise to the heated and stirred solution, and the reaction is continued to stir at 95°C for 2 hours, thereby forming a mesoporous silica matrix around the CTAB template; the mixing ratio of CTAB, TEOA, water and tetraethyl silicate is 570g:35000g:20g:0.1mol.

[0014] Further, in step 1, the post-treatment step of MSNPs is specifically as follows: the obtained MSNPs are separated by centrifugation and washed three times with EtOH ethanol, and then subjected to 10 min of ultrasonic treatment between each washing cycle to remove any unreacted precursors or byproducts; they are resuspended in a mixture of MeOH and HCl (the ratio of the mixture is 10:0.6 v / v, and the total volume is 30 ml), and refluxed at 80 ° C for 24 h to eliminate the surfactant CTAB in the internal pores of MSNPs; after reflux, a new round of centrifugation and cleaning is performed. Finally, a 0.5 mL aliquot is collected, centrifuged, and air-dried to determine the concentration of the synthesized MSNPs suspension and its subsequent application or characterization.

[0015] Furthermore, the step 2 is specifically as follows: resuspending the MSNPs in ethanol at a concentration of 2 mg / mL, and then ultrasonically treating for 10 min; subsequently adding APTES (3-aminopropyltriethoxysilane) to the solution at an amount of 10 μL per mg of MSNPs; and vigorously stirring the resulting mixture using an end-rotating oscillator for 24 hours.

[0016] Furthermore, the step 2 further comprises: separating the modified particles by centrifugation and washing them thoroughly, and finally, carefully collecting 0.5 mL aliquots from the suspension, centrifuging them, and drying them in air.

[0017] Furthermore, the step three is specifically as follows: MSNP-NH2 is initially centrifuged at 1340 g / s for 10 min, and then the precipitate is redissolved in 900 μL PBS solution and subjected to ultrasonic treatment for 20 min; 100 μL glutaraldehyde is added to the mixture, and then rapidly vortexed for 30 s; the glutaraldehyde-treated suspension is vigorously stirred using a vortex oscillator for 3 h at room temperature; during this period, the solution turns pink, indicating successful activation; the activated particles are washed three times with PBS solution, with an interval of 20 min ultrasonic treatment between each wash; then, the glutaraldehyde is removed. GA-activated nanoparticles were suspended in PBS containing 8 mg / ml lipase and 5 mg / mL PSGL1; these suspensions were placed on a vortex shaker and left overnight at room temperature with a stirring speed maintained at 1500 r / min; the resulting nanomotors (lipase and PSGL1-coated MSNP-NH2) were subjected to another set of rigorous washings: centrifugation at 1340 g for 10 min with PBS and three washes for 10 min each; between each wash, an additional 1 min of vortexing was performed, followed by 3 min of ultrasonic treatment to ensure thorough cleaning of the nanomotors.

[0018] Application of a novel nanomotor prepared by the above preparation method in a drug for treating myocardial ischemia-reperfusion injury.

[0019] The beneficial effects of the present invention compared to the prior art are:

[0020] 1. Strong myocardial targeting and permeability: Modification with PSGL1 and lipase enables the nanomotor to specifically target and penetrate damaged myocardial tissue.

[0021] 2. Significant therapeutic effect: By upregulating PPARα, ferroptosis is reduced, thereby effectively alleviating I / R damage, protecting the myocardium, and significantly improving the therapeutic effect.

[0022] 3. Good biocompatibility: Nanomotors use mesoporous silica nanoparticles with good biocompatibility as substrates, reducing the side effects of drugs on the human body.

[0023] 4. The nanomotor provided by the present invention has broad application prospects in the treatment of myocardial ischemia-reperfusion injury, and is expected to provide a new and effective means for the treatment of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is an overall diagram of the therapeutic mechanism of artificially constructed nanomotors (LNMs-P) ​​to alleviate ischemia-reperfusion injury in Example 1.

[0025] Figure 2 The DLS results show the size schematic diagrams of LNMs-P.

[0026] Figure 3 SEM microscopy images of MSNPs modified with lipase and PSGL1.

[0027] Figure 4 TEM microscopy images of MSNPs modified with lipase and PSGL1.

[0028] Figure 5 Representative traces of LNMs-P with different triacetin concentrations of 0 mM (black), 1 mM (red), 10 mM (green), and 100 mM (blue).

[0029] Figure 6 The mean square displacement (MSD; n+20, error bars represent SE) of LNMs-P with different triacetin concentrations of 0 mM (black), 1 mM (red), 10 mM (green) and 100 mM (blue).

[0030] Figure 7 Figure 3 is the effective diffusion coefficient obtained by analyzing the MSD of different triacetin concentrations.

[0031] Figure 8 Schematic diagram of LNMs-P targeting and adsorption to the HUVEC cell surface through the close interaction between P-selectin and PSGL1.

[0032] Fig. 9 Schematic diagram of the movement and detachment process of LNMs-P after activation.

[0033] Fig.10 The figure compares the movement of LNMs-P before (red) and after (blue) activation.

[0034] Fig.11 This is a comparison of the permeability of LNMs-P and MSNP in the transwell system.

[0035] Fig.12 This is a diagram showing the lipid-lowering study of the artificially constructed nanomotors (LNMs-P) ​​in Example 1.

[0036] Fig.13 Schematic diagram of Western blotting for detecting PSGL1 protein in the infection site of MI or I / R mice.

[0037] Fig.14 Schematic diagram of an echocardiogram performed to measure heart function.

[0038] Fig.15 Schematic diagram of left ventricular ejection fraction (EF) measurement using echocardiography.

[0039] Fig.16 Schematic diagram of left ventricular fractional shortening (FS) measured using echocardiography.

[0040] Fig.17 Schematic diagram of left ventricular internal diameter systolic duration (LVESD) measured using echocardiography.

[0041] Fig.18 Schematic diagram of heart rate (HR) measured by electrocardiogram.

[0042] Fig.19 Whole cell protein expression profiles of PPARα, TFAM, VDAC1, TOMM20, and NDUFB8 were measured by Western blotting.

[0043] Fig. 20 The mRNA levels of Pparα, Tfam, Vdac1, Tomm20, and Ndufb8 were confirmed by TR-qPCR.

[0044] Fig.21 The nuclear protein expression of PPARα was measured by Western blotting.

[0045] Fig. 22 The cytoplasmic protein expression of PPARα was measured by Western blotting.

[0046] Fig.23 Schematic diagram of lipid determination using C11 BODIPY 581 / 591 fluorescent probe.

[0047] Fig.24 Schematic diagram of detecting ATP concentration using an ATP detection kit.

[0048] Fig.25 Schematic diagram of ChIP analysis of PPARα enrichment on the Tfam gene promoter.

[0049] Fig.26 Schematic diagram of measuring fatty acid concentration in the cell nucleus using the Amplex Red free fatty acid detection kit.

[0050] Fig. 27 Schematic diagram of whole-cell protein expression of PPARα, TFAM, VDAC1, TOMM20 and NDUFB8 measured and confirmed by Western blotting.

[0051] Fig.28 Schematic diagram of the confirmation of mRNA levels of PPARα, TFAM, VDAC1, TOMM20 and NDUFB8 by TR-q PCR.

[0052] Fig.29Schematic diagram of measuring and confirming the nuclear protein expression of PPARα by Western blotting.

[0053] Fig.30 Schematic diagram of the cytoplasmic protein expression of PPARα measured by Western blotting.

[0054] Fig.31 Schematic diagram of detecting ATP concentration using an ATP detection kit.

[0055] Fig.32 Schematic diagram of whole cell protein expression of PPARα, TFAM, VDAC1, TOMM20, and NDUFB8 measured by Western blotting.

[0056] Fig.33 Schematic diagram of the mRNA levels of PPARα, TFAM, VDAC1, TOMM20 and NDUFB8 confirmed by TR-qPCR.

[0057] Fig.34 Schematic diagram of the measurement of nuclear protein expression of PPARα by Western blotting.

[0058] Fig.35 Schematic diagram of the confirmation of cytoplasmic protein expression of PPARα measured by Western blotting.

[0059] Fig.36 Schematic diagram of detecting ATP concentration using an ATP detection kit. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0061] The present invention obtains the lipid motor LNMs-P by modifying mesoporous silica to connect PSGL1 (P-selectin ligand) and lipase, and inhibits ferroptosis by improving mitochondrial function, thereby reducing ischemia-reperfusion injury. The present invention solves two key problems: achieving effective delivery of therapeutic agents directly to injured myocardial tissue; solving the most important cell death pathway of ischemia-reperfusion injury-ferroptosis. The drug of the present invention can target and penetrate myocardial tissue and activate the receptor PPARα through the action of PSGL1 and lipase on its surface. PPARα binds to fatty acids, translocates into the cell nucleus, promotes the transcription of mitochondrial transcription factor A, thereby promoting mitochondrial synthesis, reducing cellular lipid content, inhibiting ferroptosis, and then reducing ischemia-reperfusion injury.

[0062] Embodiment 1:

[0063] 1. Synthesis of mesoporous silica nanoparticles (MSNPs):

[0064] The synthesis was carried out by sol-gel method involving the use of hexadecyltrimethylammonium bromide (CTAB) as a structure directing agent and triethanolamine (TEOA) as a base catalyst to achieve the desired porous structure. 570 mg of CTAB, 35 g of TEOA and 20 ml of water were mixed to make a mixture. The solution was heated in a silicone oil bath and the temperature was maintained at 95 °C while stirring for 30 min. 0.1 mol of tetraethyl silicate (TEOS) was added dropwise to the heated and stirred solution. The reaction was continued with stirring at 95 °C for 2 h, thereby forming a mesoporous silica matrix around the CTAB template. The resulting MSNPs were separated by centrifugation and washed three times with EtOH followed by 10 min of sonication between each washing cycle to remove any unreacted precursors or byproducts. They were resuspended in a mixture of MeOH and HCl (the ratio of the mixture was 10:0.6 v / v, and the total volume was 30 ml) and refluxed at 80 °C for 24 h to eliminate the surfactant CTAB in the internal pores of the MSNPs. After reflux, a new round of centrifugation and cleaning was performed. Finally, a 0.5 mL aliquot was collected, centrifuged, and air-dried to determine the concentration of the synthesized MSNPs suspension and its subsequent application or characterization.

[0065] 2. Amino functionalization of MSNPs

[0066] The synthesized MSNPs were resuspended in ethanol at a concentration of 2 mg / mL and then sonicated for 10 min. APTES (3-aminopropyltriethoxysilane) was then added to the solution at 10 μL per mg of MSNP. The resulting mixture was vigorously stirred for 24 h using an end-rotating oscillator. After completing this functionalization step, the modified particles were separated by centrifugation and thoroughly washed. Finally, 0.5 mL aliquots were carefully collected from the suspension, centrifuged, and allowed to dry in air.

[0067] 3. Functional modification of MSNP-NH2 using lipase and PSGL1:

[0068] MSNP-NH2 was initially centrifuged at 1340 g / s for 10 min. The precipitate was then redissolved in 900 μL PBS solution and sonicated for 20 min. 100 μL glutaraldehyde was added to the mixture, followed by rapid vortexing for 30 s. The glutaraldehyde-treated suspension was vigorously stirred for 3 h at room temperature using a vortex oscillator. During this period, the solution turned pink, indicating successful activation. The activated particles were washed three times with PBS solution, with 20 min of sonication between each wash. Subsequently, the glutaraldehyde GA-activated nanoparticles were suspended in PBS containing 8 mg / ml lipase and 5 mg / mL PSGL1. These suspensions were placed on a vortex oscillator and left overnight at room temperature with the stirring speed maintained at 1500 r / min. The resulting nanomotors (lipase and PSGL1-coated MSNP-NH2) were subjected to another set of rigorous washes: centrifuged at 1340 g for 10 min with PBS, washed for 10 min each time, and washed three times. Between each wash, an additional 1 min of vortexing followed by 3 min of sonication was performed to ensure thorough cleaning of the nanomotors.

[0069] 4. Functional Study of Nanomotor LNMs-P

[0070] After the left coronary artery of the selected mice was ligated, LNMs-P (50 μg / kg) was immediately injected into the mice through the tail vein and allowed to rest for 1 hour. After 1 hour of ischemia, high-fat gavage (HF) was given, followed by 3 hours of ischemia and 48 hours of reperfusion. The results showed that the serum triglyceride level of mice receiving high-fat gavage (16.73±3.03mmol / L) was significantly higher than that of the control group mice (2.11±0.12mmol / L). The effective diffusion coefficient of LNMs-P in the serum of HF mice (1.62±0.12μm 2 / s) was higher than the effective diffusion coefficient in control mice (0.23 ± 0.07 μm 2 / s). The biological effect of LNMs-P in myocardial ischemia-reperfusion was verified.

[0071] To further evaluate the permeability of LNMs-P in arteries, we ligated a 6-cm long ventral segment of rabbit aorta at its midpoint in vitro. The left side of the ligation was filled with high-fat treated mouse serum containing LNMs-P, while the right side contained only high-fat treated mouse serum. As a result, we found a large number of LNMs-P on the right side of the ligation. This proved that LNMs-P used triglycerides as fuel and passed through the ligation site.

[0072] Furthermore, LNMs-P could cross the erythrocyte membrane in the blood of high-fat-fed mice. Notably, high-fat gavage significantly enhanced the migration of LNMs-P across tissue barriers and further upregulated the level of P-selectin glycoprotein ligand (PSGL1) in the infarct area of ​​myocardial infarction and ischemia-reperfusion mice.

[0073] Rat cardiomyocytes (H92c cells) were pretreated with LNMs-P and then exposed to oleic acid (OA) to examine the biological effects of LNMs-P. LNMs-P showed a stronger degradation ability than lipase, which was attributed to the tendency of free lipase to form bimolecular aggregates, which partially blocked its active center and reduced its activity. Similarly, the improved performance of LNMs-P in degrading intracellular lipids compared with free lipase may be attributed to the superior dispersibility of lipase, which remained well dispersed when immobilized on the mesoporous structure of LNMs-P, with a considerable surface area to volume ratio, facilitating enzyme-substrate contact and improving catalytic efficiency.

[0074] PPARs are key lipid-sensing transcription factors that coordinate whole-body energy metabolism. These nuclear hormone receptors, which consist of PPARa, PPARδ, and PPARy isoforms, regulate gene expression when activated by specific ligands. Numerous studies have used various experimental animal models of I / R injury to show the cardioprotective effects of PPARa activation, as manifested by a reduction in myocardial infarction size. Our results revealed that LNMs-P+HF treatment promoted the nuclear translocation of PPARa in vivo and in vitro, indicating its upregulation. Considering that the improvement in mitochondrial function alleviated I / R injury, it is noteworthy that LNMs-P+HF treatment resulted in healthier mitochondrial morphology. The data showed increased expression levels of key mitochondrial proteins TFAM, VDAC1, TOMM20, and NDUFB8 in vivo and in vitro, and increased ATP concentrations after LNMs-P treatment, confirming enhanced mitochondrial function. Overall, LNMs-P treatment increased mitochondrial biogenesis and promoted the translocation of PPARa into the nucleus. Subsequent experiments were designed to inhibit PPARa activity using MK886 (a novel and potent 5-lipoxygenase-activating protein inhibitor and leukotriene biosynthesis inhibitor). After inhibition, the expression of mitochondrial proteins in LNMs-P-treated cells was significantly decreased, indicating that LNMs-P indeed enhanced mitochondrial biogenesis by upregulating PPARa.

[0075] ChIP analysis of the enriched fractions revealed that PPARa acts as a transcription factor for TFAM (methyl trifluoroacetate), indicating that PPARa plays a role in mitochondrial biogenesis through its effects on key mitochondrial target genes. The role of TFAM in ferroptosis was investigated by gene knockout and overexpression experiments. The results showed that low TFAM protein levels promoted ferroptosis in cells treated with Fe-SP (a well-established inducer of ferroptosis) and LNMs-P. In contrast, overexpression of TFAM prevented ferroptosis under similar conditions. LNMs-P upregulated mitochondrial biogenesis through the PPARa / TFAM pathway, demonstrating a protective effect against ferroptosis in myocardial I / R injury.

[0076] Figure 1 This is a general diagram of the therapeutic mechanism of artificially constructed nanomotors (LNMs-P) ​​to reduce ischemia-reperfusion injury in Example 1. It can be seen from the figure that the drug of the present invention has targeting, permeability and lipid-lowering properties. Through the action of PSGL1 and lipase on its surface, it can target and penetrate myocardial tissue and activate the receptor PPARα. PPARα binds to fatty acids, translocates into the cell nucleus, promotes the transcription of mitochondrial transcription factor A, thereby promoting mitochondrial synthesis, reducing cellular lipid content, inhibiting ferroptosis, and thus reducing ischemia-reperfusion injury.

[0077] Figure 2 The DLS results of particle size distribution coefficient show the size of LNMs-P respectively.

[0078] Figure 3 SEM microscopy images of MSNPs modified with lipase and PSGL1.

[0079] Figure 4 TEM microscopy images of MSNPs modified with lipase and PSGL1.

[0080] Figure 5 Representative traces of LNMs-P with different triacetin concentrations of 0 mM (black), 1 mM (red), 10 mM (green), and 100 mM (blue).

[0081] Figure 6 The mean square displacement (MSD; n+20, error bars represent SE) of LNMs-P with different triacetin concentrations of 0 mM (black), 1 mM (red), 10 mM (green) and 100 mM (blue).

[0082] Figure 7 Schematic diagram of the effective diffusion coefficient obtained by analyzing the MSD of different triacetin concentrations.

[0083] from Figure 5 to Figure 7It can be seen that as the triglyceride concentration increases, LNMs-P becomes more and more dispersed. It can also be seen that the effect of concentration above 10 mm on the dispersion coefficient becomes smaller (there is not much difference between 10 mm and 100 mm).

[0084] Figure 8 LNMs-P was targeted and adsorbed to the HUVEC cell surface through the close interaction between P-selectin and PSGL1.

[0085] Fig. 9 The movement and detachment process of LNMs-P after activation. After adding 10mM triacetin to activate LNMs-P, it was observed that LNMs-P began to show mobility. Initially, most LNMs-P were still tightly adsorbed on HUVEC cells. Over time (about one hour later), LNMs-P gradually detached from the cells and moved with higher degrees of freedom, indicating that LNMs-P has excellent permeability.

[0086] Fig.10 The permeability of LNMs-P was further verified by comparing their movement before (red) and after (blue) activation. After activation, LNMs-P were able to move more freely between cells, demonstrating their potential application in drug delivery.

[0087] Fig.11 The permeability comparison of LNMs-P and MSNP in the transwell system. The experimental results show that after TG degradation, the number of LNMs-P (FITC) that migrated to the HUVEC cell layer and reached the H9c2 cells was significantly greater than that of MSNP (FITC). This result strongly proves that LNMs-P has superior permeability and targeted delivery capabilities compared to traditional nanodrug carriers (such as MSNP). By utilizing targeting and self-propulsion mechanisms, LNMs-P can effectively deliver drugs to the infarcted myocardium, providing new possibilities for the treatment of cardiovascular diseases such as myocardial infarction.

[0088] Fig.12 This is a study on the lipid-lowering properties of the artificially constructed nanomotors (LNMs-P) ​​of Example 1. H9c2 cells were pretreated with 1μg / mL LNMs-P, 1μg / mL PMSNs or 0.5μg / mL lipase for 2 hours, and then incubated with 30μM OA for 12 hours. Intracellular lipid droplets were evaluated using the fluorescent probe BODIPY 493 / 503 staining kit. Green fluorescence indicates an increase in intracellular lipid droplets. LNMs-P exhibited a stronger degradation ability than lipase, and the performance of LNMs-P in degrading intracellular lipids was improved. Balb / c mice were injected with 50μg (body weight) LNMs-P via the tail vein and left to rest for 1 hour. After 1 hour of ischemia, a high-fat diet was orally administered by gavage, followed by 3 hours of ischemia and 48 hours of reperfusion ( Figures 14 to 17 ).

[0089] Fig.13 Western blotting was used to detect PSGL1 protein in the infected area of ​​MI or I / R mice. n = 3. Mice were gavaged with HF, and the results showed that their serum TG was significantly higher than that of control mice. LNMs-P have the ability to effectively penetrate the myocardium.

[0090] Fig.14 Echocardiography was performed to measure cardiac function. n=7.

[0091] Fig.15 For Fig.14 Corresponding statistical graph of left ventricular ejection fraction (EF) measured by echocardiography.

[0092] Fig.16 For Fig.14 Corresponding statistical graph of left ventricular fractional shortening (FS) measured by echocardiography.

[0093] Fig.17 For Fig.14 The corresponding left ventricular internal diameter systolic period (LVESD) was measured by echocardiography. The echocardiographic results at the end of reperfusion showed that the ejection fraction (EF) and fractional shortening (FS) of I / R mice treated with LNMs-P were significantly increased, and the left ventricular internal diameter systolic period (LVIDS) was decreased compared with untreated I / R mice. Figures 15 to 17 ).

[0094] Fig.18 Heart rate (HR) was measured by electrocardiogram. Heart rate (HR) decreased. Overall, these findings suggest that LNMs-P treatment effectively improves cardiac function. H9c2 cells were pretreated with 1 μM MK886 for 24 hours and then treated with 1 μg / mL LNMs-P for 18 hours. ( Figures 19 to 26 ).

[0095] Fig.19 Whole cell protein expression of PPARα, TFAM, VDAC1, TOMM20, and NDUFB8 was measured by Western blotting.

[0096] Fig. 20 The mRNA levels of Pparα, Tfam, Vdac1, Tomm20, and Ndufb8 were confirmed by TR-qPCR. After inhibition, the expression of mitochondrial proteins was significantly decreased in LNMs-P-treated cells, indicating that LNMs-P indeed enhanced mitochondrial biogenesis by upregulating PPARα.

[0097] Fig.21 Nuclear protein expression of PPARα was measured by Western blotting.

[0098] Fig. 22 The cytoplasmic protein expression of PPARα was measured by Western blotting. The results suggest that LNMs-P stimulated PPARα to translocate into the nucleus, possibly together with its lipid ligands, and triggered downstream events that ultimately enhanced mitochondrial function and energy production in I / R-injured cardiac cells.

[0099] Fig.23 The C11 BODIPY 581 / 591 fluorescent probe is used to measure lipids. Red: lipids are not peroxidized; blue arrows indicate lipids entering the nucleus. The control group has less lipids entering the nucleus, but it increases after adding LNMs-P. At the same time, the addition of PPARα blocker reduces the amount of lipids entering the nucleus, proving that PPARα is related to lipid entry into the nucleus.

[0100] Fig.24 ATP concentration was detected using an ATP detection kit.

[0101] Fig.25 ChIP analysis of PPARα enrichment at the Tfam gene promoter. IgG was used as a negative control. The results of these experiments indicate that PPARα is a transcription factor for Tfam.

[0102] Fig.26 The concentration of fatty acids in the nucleus was measured using the Amplex Red free fatty acid detection kit. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. The results showed that the concentration of FAs in the digestive fluid of LNMs-P-treated cells increased, and MK886 effectively blocked this increase. Balb / c mice were injected with 50 μg (body weight) of LNMs-P via the tail vein and left to rest for 1 h. After 1 h of ischemia, a high-fat diet was orally administered by gavage, followed by 3 h of ischemia and 48 h of reperfusion ( Figures 27-31 )

[0103] Fig. 27 To measure and confirm the whole-cell protein expression of PPARα, TFAM, VDAC1, TOMM20, and NDUFB8 by Western blotting.

[0104] Fig.28 To confirm the mRNA levels of PPARα, TFAM, VDAC1, TOMM20, and NDUFB8 by TR-q PCR.

[0105] Fig.29 To measure and confirm the nuclear protein expression of PPARα by Western blotting.

[0106] Fig.30 The cytoplasmic protein expression of PPARα was measured by Western blotting.

[0107] Fig.31 ATP concentration was detected by ATP detection kit. n = 7.ns, no significant difference **P < 0.01,

[0108] ***P<0.001. H9c2 cells were pretreated with 1μg / mL LNMs-P for 2h and then incubated with 2μM Fe-SP for 16h. ( Figures 32-36 ).

[0109] Fig.32 Whole cell protein expression of PPARα, TFAM, VDAC1, TOMM20, and NDUFB8 was measured by Western blotting.

[0110] Fig.33 The mRNA levels of PPARα, TFAM, VDAC1, TOMM20, and NDUFB8 were confirmed by TR-qPCR.

[0111] Fig.34 Nuclear protein expression of PPARα was measured by Western blotting.

[0112] Fig.35 The cytoplasmic protein expression of PPARα was confirmed by Western blotting.

[0113] Fig.36 ATP concentration was detected by ATP detection kit. n = 3.ns, no significance **P < 0.01,

[0114] ***P<0.001. The data showed that the expression levels of key mitochondrial proteins TFAM, VDAC1, TOMM20, and NDUFB8 were increased in vivo and in vitro, which enhanced mitochondrial function and made mitochondria healthier. Finally, ATP concentration increased after LNMs-P treatment in vivo and in vitro, confirming the enhanced mitochondrial function. Overall, LNMs-P treatment increased mitochondrial biogenesis and promoted the translocation of PPARα to the nucleus in vivo and in vitro.

Claims

1. A method for preparing a novel nanomotor for treating myocardial ischemia-reperfusion injury. Features: The method is:

1. Synthesis of fully mesoporous silica nanoparticles; Second, amino functionalization of MSNPs; 3. Using lipase and PSGL1 to synthesize MSNP-NH 2 Functional modification: MSNP-NH 2 The precipitate was dissolved in PBS solution and sonicated, and glutaraldehyde was added to the mixture and stirred evenly; the glutaraldehyde GA-activated nanoparticles were suspended in PBS containing lipase and PSGL1 and stirred evenly.

2. A method for preparing a novel nanomotor for treating myocardial ischemia-reperfusion injury according to claim 1, Features: The step 1 is specifically as follows: CTAB, TEOA and water are mixed to prepare a mixed solution, the solution is placed in an oil bath and heated, the temperature is maintained at 95° C., and the solution is continuously stirred for 30 minutes, tetraethyl silicate (TEOS) is added dropwise to the heated and stirred solution, and the reaction is continued to be stirred at 95° C. for 2 hours; the mixing ratio of CTAB, TEOA, water and tetraethyl silicate is 570 g: 35000 g: 20 g: 0.1 mol.

3. A method for preparing a novel nanomotor for treating myocardial ischemia-reperfusion injury according to claim 1 or 2, Features: In step 1, the post-treatment steps of MSNPs are specifically as follows: the obtained MSNPs are separated by centrifugation and washed three times with EtOH ethanol, followed by 10 min of ultrasonic treatment between each washing cycle to remove any unreacted precursors or by-products; they are resuspended in a mixture of MeOH and HCl (the ratio of the mixture is 10:0.6 v / v, and the total volume is 30 ml), and refluxed at 80°C for 24 h to eliminate the surfactant CTAB in the internal pores of MSNPs; after reflux, a new round of centrifugation and cleaning is performed.

4. A method for preparing a novel nanomotor for treating myocardial ischemia-reperfusion injury according to any one of claims 1 to 3, Features: The step 2 is specifically as follows: resuspending the MSNPs in ethanol at a concentration of 2 mg / mL, and then ultrasonically treating for 10 min; subsequently adding APTES (3-aminopropyltriethoxysilane) to the solution at an amount of 10 μL per mg of MSNPs; and vigorously stirring the resulting mixture using an end-over-end oscillator for 24 hours.

5. A method for preparing a novel nanomotor for treating myocardial ischemia-reperfusion injury according to claim 4, Features: The step 2 also includes: separating the modified particles by centrifugation and washing them thoroughly, and finally, carefully collecting a 0.5 mL aliquot from the suspension, centrifuging it, and drying it in air.

6. A method for preparing a novel nanomotor for treating myocardial ischemia-reperfusion injury according to claim 1 or 4, Features: The step three is specifically as follows: MSNP-NH 2 After initial centrifugation at 1340 g / s for 10 min, the pellet was redissolved in 900 μL PBS solution and sonicated for 20 min; 100 μL of glutaraldehyde was added to the mixture, which was then rapidly vortexed for 30 s. The glutaraldehyde-treated suspension was vigorously stirred using a vortex oscillator for 3 h at room temperature. During this period, the solution turned pink, indicating successful activation. The activated particles were washed three times with PBS solution, with 20 min of ultrasonic treatment between each wash. Subsequently, the glutaraldehyde-GA-activated nanoparticles were suspended in PBS containing 8 mg / ml lipase and 5 mg / mL PSGL1. These suspensions were placed on a vortex oscillator and left overnight at room temperature with a stirring speed of 1500 r / min. 2 ) Another set of stringent washes was performed: three washes of 10 min each with PBS followed by centrifugation at 1340 g for 10 min; between each wash, an additional 1 min of vortexing was performed, followed by 3 min of sonication to ensure thorough cleaning of the nanomotors.

7. Use of a novel nanomotor prepared by the preparation method according to any one of claims 1 to 6 in a drug for treating myocardial ischemia-reperfusion injury.

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