Superparamagnetic nanoparticle-exosome compound for preparing medicine for treating myocardial infarction and preparation method of superparamagnetic nanoparticle-exosome compound

Abstract

The invention provides a superparamagnetic nanoparticle-exosome compound for preparing a medicine for treating myocardial infarction and a preparation method of the superparamagnetic nanoparticle-exosome compound. The superparamagnetic nanoparticle-exosome compound comprises a human umbilical cord mesenchymal stem cell exosome, superparamagnetic iron oxide nanoparticles and a chemical coupling agent for connecting the human umbilical cord mesenchymal stem cell exosome and the superparamagnetic iron oxide nanoparticles. The application of the compound in preparation of targeted therapeutic species for treating myocardial infarction is realized through the following steps: separating and purifying exosome from mesenchymal stem cells (MSCs); superparamagnetic iron oxide nanoparticles with carboxylated surfaces; the superparamagnetic iron oxide nanoparticles are combined with exosome membrane surface protein through chemical coupling (an EDC / NHS cross-linking agent); after intravenous injection, a gradient magnetic field is applied in vitro to guide the compound to be enriched to a myocardial infarction area. The surface-carboxylated superparamagnetic iron oxide nanoparticle composition cooperates with the exosome, the enrichment efficiency of the exosome in an infarct region is improved under the guidance of a magnetic field, myocardial cell apoptosis is inhibited, angiogenesis is promoted, and immune response is regulated.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cells, and specifically relates to a treatment system and method for myocardial repair by forming a complex formed by combining superparamagnetic nanoparticles with exosomes, which is guided by an external magnetic field and targetedly delivered to the myocardial infarction area. Background Art

[0002] Traditional treatments for myocardial infarction mainly include drug therapy, interventional therapy, surgery, and cardiac rehabilitation. Although these methods have significantly reduced the mortality rate of myocardial infarction, there are still key obstacles and limitations. The main obstacles are time sensitivity, irreversible myocardial damage, side effects of treatment, lack of individualization, and uneven resource allocation. Although existing methods have greatly improved prognosis, myocardial regeneration and microcirculatory repair remain unsolved problems. In the future, it is necessary to combine regenerative medicine, precision medicine, and technological innovation to break through the current bottleneck. Cell therapy, as a new treatment for myocardial infarction, has shown repair potential in animal models and early clinical trials, but it still has low survival rate, unclear mechanism, high heterogeneity, and cost barriers. Traditional drugs or stem cell therapy are difficult to achieve the multi-effect synergistic effects of anti-apoptosis, promoting angiogenesis, and inhibiting fibrosis at the same time.

[0003] Exosomes are a type of extracellular vesicle (EV) actively secreted by stem cells. These membrane-bound vesicles, approximately 30-150 nm in size, are secreted by cells via exocytosis and are widely involved in intercellular communication, disease progression, and physiological regulation. Stem cell exosomes have shown great potential in promoting cardiac repair after myocardial infarction by improving cardiomyocyte survival and angiogenesis, but they still face numerous technical and biological limitations. Natural exosomes have low in vivo delivery efficiency, are easily cleared by the mononuclear phagocytic system, and have insufficient accumulation in areas of cardiac injury. Low exosome doses may not be sufficient to initiate repair signaling, while high doses may induce immune responses or promote fibrosis (e.g., excessive TGF-β). Natural exosomes rely on surface integrins (e.g., integrin α6β4) or chemokines for passive targeting, but the complex microenvironment of the infarcted area (e.g., inflammation and fibrosis) can hinder exosome penetration. Existing magnetic targeting carriers (such as liposomes and synthetic nanoparticles) have poor biocompatibility and lack the bioactive components of natural exosomes (such as miRNA and functional proteins), which may trigger a response from the immune system, leading to inflammatory or allergic reactions, thereby affecting the therapeutic effect and increasing the health risks of patients. Summary of the Invention

[0004] In view of the current state of the art, the present invention provides a superparamagnetic nanoparticle-exosome complex for preparing a drug for treating myocardial infarction and a preparation method thereof, which overcomes the problems of the existing technology such as low enrichment rate of exosomes in the infarct area, high dose-induced fibrosis, and poor biocompatibility leading to the risk of immune rejection.

[0005] In a first aspect, the present invention provides a superparamagnetic nanoparticle-exosome complex for preparing a drug for treating myocardial infarction, wherein the complex comprises human umbilical cord mesenchymal stem cell exosomes, superparamagnetic iron oxide nanoparticles, and EDC and NHS cross-linking agents connecting the two, wherein the molar mass ratio of the cross-linking agent is 5:2.

[0006] The superparamagnetic iron oxide nanoparticles include one or a combination of Fe3O4, γ-Fe2O3, Co-Fe2O4;

[0007] Optionally, the superparamagnetic iron oxide nanoparticles are preferably a superparamagnetic iron oxide nanoparticle composition of Fe3O4, γ-Fe2O3, and Co-Fe2O4, and the weight ratio of the superparamagnetic iron oxide nanoparticle composition Fe3O4:γ-Fe2O3:Co-Fe2O4 is (2-6):(3-5):(1-5); the weight ratio of the superparamagnetic iron oxide nanoparticle composition Fe3O4:γ-Fe2O3:Co-Fe2O4 is preferably 6:3:1.

[0008] In a second aspect, the present invention provides a method for preparing a superparamagnetic nanoparticle-exosome complex for preparing a drug for treating myocardial infarction, comprising the following steps:

[0009] (1) Exosomes were extracted from mesenchymal stem cells by differential centrifugation at 300×g, 2000×g, and 10,000×g for 6 minutes each time; then ultracentrifugation was performed at 100,000×g for 30 minutes. After centrifugation, the supernatant was discarded and the precipitate was harvested; the exosomes were purified by ultrafiltration using a 100 kDa membrane to obtain human umbilical cord mesenchymal stem cell exosomes.

[0010] (2) Modifying nanoparticles, dissolving a superparamagnetic iron oxide nanoparticle composition in citric acid, performing sonication followed by constant temperature water bath and stirring, cooling and centrifuging, and finally washing to remove free citric acid; the superparamagnetic iron oxide nanoparticle composition comprises Fe3O4, γ-Fe2O3, and Co-Fe2O4, and the weight ratio thereof is (2-6): (3-5): (1-5); the preferred weight ratio is 6:3:1;

[0011] Optionally, the concentration of the superparamagnetic iron oxide nanoparticle composition is 1 mg / mL, the citric acid concentration is 0.1 moL / L, and the pH is 5-6; the ultrasonic power is 100 W, and the ultrasonic time is 5 min; the constant temperature water bath temperature is 60±2°C, the stirring speed is 600 rpm, and the stirring time is 6 hours; the centrifugal speed is 12,000 rpm, and the centrifugation is 15 min.

[0012] STEP 3: Constructing a complex, mixing the carboxylated superparamagnetic iron oxide nanoparticles with exosomes, adding an EDC / NHS crosslinker, and removing free carboxylated superparamagnetic iron oxide nanoparticles by sucrose density gradient centrifugation to obtain a carboxylated nanoparticle-Exo complex;

[0013] Optionally, in the complex formation step, the mass ratio of carboxylated superparamagnetic iron oxide nanoparticles to exosomes is 1:5, and the molar mass ratio of the EDC / NHS crosslinker is 5:2. The sucrose density gradient is 60% (w / v), 45% (w / v), 30% (w / v), and 8% (w / v). The centrifugation conditions are 4°C, 100,000 × g, and 2 hours. After centrifugation, the target layer (30%-45% interface) is slowly aspirated with a pipette and transferred to a new centrifuge tube. The mixture is diluted to 10 mL with PBS and centrifuged at 100,000 × g for 1 hour to precipitate the complex. The supernatant is discarded, and the complex is resuspended in 1 mL of PBS and sterilized by filtration through a 0.22 μm filter.

[0014] In a third aspect, the present invention provides a use of the complex in preparing a drug for treating myocardial infarction, wherein targeted treatment of myocardial infarction is achieved by the following steps:

[0015] 1. Exosome extraction: Isolation and purification of exosomes from mesenchymal stem cells (MSCs);

[0016] 2. Nanoparticle modification: surface carboxylation of superparamagnetic iron oxide nanoparticles;

[0017] 3. Complex construction: Superparamagnetic iron oxide nanoparticles are bound to exosome surface proteins through chemical coupling (EDC / NHS crosslinker);

[0018] 4. Magnetic field-guided delivery: After intravenous injection, a gradient magnetic field (1.0T) is applied in vitro to guide the complex to the infarcted area;

[0019] 5. Synergistic repair mechanism: Surface carboxylated superparamagnetic iron oxide nanoparticles cooperate with exosomes. The magnetic field guides the exosomes to increase their enrichment efficiency in the infarcted area, inhibiting myocardial cell apoptosis, promoting angiogenesis, and regulating the immune response.

[0020] The superparamagnetic iron oxide-human umbilical cord mesenchymal stem cell exosome complex provided by the present invention can promote the formation and differentiation of blood vessels, and has the effect of significantly enhancing the proliferation of vascular endothelial cells and reducing the infarct area.

[0021] In summary, the present invention has the following beneficial effects:

[0022] 1. Enhanced targeting: Through the magnetic responsiveness of carboxylated superparamagnetic iron oxide nanoparticles, combined with an external magnetic field, exosomes are precisely guided to the ischemic area of the heart, and the enrichment efficiency is increased by 3-5 times compared to natural exosomes.

[0023] 2. Functional synergy: The carboxylated superparamagnetic iron oxide nanoparticle composition works synergistically with exosomes, generating localized warmth (40-42°C) under an alternating magnetic field, promoting the release of exosome contents and vasodilation. The infarct area was reduced by 55.4% compared to native exosomes, and the vascular density was 2.4 times that of native exosomes.

[0024] 3. High safety: The carboxylated superparamagnetic iron oxide nanoparticle-exosome complex of the present invention is biodegradable, and the exosomes have no risk of immune rejection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Flowchart for the preparation of SPION-Exo complexes;

[0026] Figure 2 : Blood vessel density was observed by CD31 immunofluorescence staining. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are described in detail below. The embodiments provided by the present invention are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0028] Human umbilical cord mesenchymal stem cells of the present invention were purchased from American type culture collection, ATCC; magnetic Fe3O4, γ-Fe2O3, and Co-Fe2O4 nanoparticles were purchased from Beijing Zhongke Keyou Nanotechnology Co., Ltd.; and the remaining reagents, solvents, and other experimental materials were all commercially available.

[0029] Example 1 Preparation of exosomes

[0030] 1.1 Cell culture

[0031] Human umbilical cord mesenchymal stem cells (hUC-MSCs) were seeded in DMEM medium at a density of 0.3×106 / mL for subculture. After subculture to the third generation of human mesenchymal stem cells, after 2 days of culture, when the cell density reached 80%, the medium was replaced with basal medium and cultured in a 5% volume CO2, 37°C incubator.

[0032] 1.2 Exosome extraction

[0033] The human umbilical cord mesenchymal stem cells in step 1.1 were starved for 36 hours, and the supernatant was collected and subjected to differential centrifugation. The supernatant was centrifuged at 300×g for 6 minutes to remove cell debris and other precipitates, and the supernatant was taken; the supernatant was then centrifuged at 2000×g for 6 minutes to remove the precipitate and the supernatant was taken; the supernatant was further centrifuged at 10,000×g for 6 minutes to remove the precipitate and the supernatant was taken; finally, the supernatant was ultracentrifuged at 100,000×g for 30 minutes. After centrifugation, the supernatant was discarded, the precipitate was harvested, and the exosomes were purified by ultrafiltration using a 100 kDa membrane to obtain human umbilical cord mesenchymal stem cell exosomes.

[0034] Example 2 Preparation of carboxyl-rich superparamagnetic nanoparticles

[0035] The commercially available nanoparticles were dissolved in 0.1 mol / L citric acid (pH adjusted to 5.0-6.0) to a final concentration of 1 mg / mL.

[0036]

[0037] Ultrasonication at 100 W was performed for 5 minutes to uniformly disperse the nanoparticles in the citric acid. The mixed solution was transferred to a three-necked flask and placed in a water bath at 60 ± 2°C with magnetic stirring at 600 rpm for 6 hours. A nitrogen atmosphere was used throughout the reaction to prevent oxidation of the nanoparticles. After the reaction, the mixture was cooled to room temperature. Carboxylated nanoparticles were obtained by centrifugation at 12,000 rpm for 15 minutes. The mixture was washed three times with deionized water to remove free citric acid.

[0038] The carboxylated nanoparticles were dispersed in deionized water and stored at 4°C until use.

[0039] Example 3 Construction of a covalently coupled carboxylated nanoparticle-exosome complex

[0040] The carboxylated superparamagnetic nanoparticles prepared in Example 2 (including Preparation Examples 1-6) were mixed with exosomes at a mass ratio of 1:5, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) crosslinker was added (PBS, pH 7.4, 4°C for 2 h). Free carboxylated nanoparticles were removed by sucrose density gradient centrifugation to obtain carboxylated nanoparticle-Exo complexes.

[0041] 3.1 Covalent Coupling of Carboxylated Nanoparticles and Exosomes: Disperse 1 mg / mL of the carboxylated nanoparticles prepared in Example 2 (including those in Preparation Examples 1-6) in pH 7.4 PBS. Add the crosslinker at a molar ratio of EDC:NHS of 5:2. Vortex mix for 5 minutes and pre-cool to 4°C in an ice bath. Place the mixed solution on a magnetic stirrer at 4°C and stir at 200 rpm for 30 minutes. Add 0.1 M glycine (final concentration 10 mM) to quench unreacted EDC / NHS, and incubate on ice for 15 minutes.

[0042] 3.2 Sucrose Density Gradient Centrifugation to Remove Free Carboxylated Nanoparticles: Prepare 100 mL of sucrose solutions at concentrations of 60% (w / v), 45% (w / v), 30% (w / v), and 8% (w / v). Slowly layer the following gradients in an ultracentrifuge tube: 2 mL of 60% sucrose at the bottom layer, 2 mL of 45% sucrose at the middle layer, 2 mL of 30% sucrose at the top layer, and 2 mL of 8% sucrose at the top layer. Place the solution in a 4°C refrigerator for 1 hour to avoid interfacial disturbance.

[0043] Sample loading and centrifugation: Carefully add 4 mL of the coupling reaction solution to the top of the sucrose gradient. Centrifuge at 100,000 × g for 2 hours at 4°C using a swing-out rotor.

[0044] Collect the complexes and centrifuge. The high-density free nanoparticles settle to the bottom layer (60% sucrose layer), while the low-density nanoparticle-Exo complexes are distributed at the 30%-45% interface. Slowly aspirate the target layer (30%-45% interface) with a pipette and transfer to a new centrifuge tube.

[0045] Wash and concentrate the solution, dilute to 10 mL with PBS, and centrifuge at 100,000 × g for 1 hour to precipitate the complex. Discard the supernatant, resuspend the complex in 1 mL of PBS, and sterilize by filtration through a 0.22 μm filter.

[0046] Example 4: Verification of the Effect of Magnetic Targeting Therapy (Mouse Myocardial Infarction Model)

[0047] 4.1. To establish the animal model, 70 12-week-old C57BL / 6 mice weighing 20-25 g were used. The mice were housed in an SPF-protected environment with free access to food and water and a 12-hour light / dark cycle. They were fasted for 6 hours before surgery (water was not allowed). Weight was recorded. After anesthetizing the mice with an intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg), the skin and muscles were incised. The left anterior descending coronary artery (LAD) of the C57BL / 6 mice was ligated proximally to the LAD (1-2 mm below the inferior margin of the left atrial appendage) using a 7-0 Prolene suture. After chest closure, the mice were placed on a 37°C warming pad until awake, thus establishing an acute myocardial infarction model.

[0048] 4.2. Treatment Grouping: Five of the 70 mice died after modeling. The remaining 65 were divided into eight groups, each consisting of eight mice: a control group, an Exo group, and a SPION (carboxylated Fe₃O₄)-Exo group, a Preparation Example 2-Exo group, a Preparation Example 3-Exo group, a Preparation Example 4-Exo group, a Preparation Example 5-Exo group, and a Preparation Example 6-Exo group. The control group received saline injection; the Exo group received an intravenous injection of MSC exosomes (100 μg / kg); the SPION-Exo group received an intravenous injection of SPION-Exo (100 μg / kg); and the other groups received the carboxylated nanoparticle-Exo prepared in the corresponding Preparation Example (100 μg / kg). A magnetic targeting device was immediately placed on the chest of the mice (above the infarct area) and a 1.0 T magnetic field was maintained for 30 minutes.

[0049] 4.3. Results Analysis

[0050] 4.3.1 Targeting efficiency: In vivo imaging was performed on three groups of mice at 1 hour, 6 hours, 24 hours, and 48 hours after injection in step 4.2.

[0051] Anesthetize the mouse (2% isoflurane for induction, 1.5% for maintenance), shave the chest hair, apply depilatory cream, clean the skin, and place the mouse in the supine position on the imaging platform. Set the excitation wavelength to 748 nm, the emission wavelength to 780 nm, and the exposure time to 1-5 seconds (adjust based on signal intensity). Circle the cardiac region (ROI) and record the mean fluorescence intensity (MFI) and total fluorescence intensity (TFI). The field of view should cover the cardiac region.

[0052]

[0053] The results showed that there was no obvious fluorescence signal in the control group; the fluorescence signal in the Exo group reached a peak at 6 hours and then gradually decreased; the fluorescence signals in the SPION-Exo group, Preparation Example 2-Exo group, Preparation Example 3-Exo group, Preparation Example 4-Exo group, Preparation Example 5-Exo group and Preparation Example 6-Exo group reached a peak at 24 hours, and the signal duration was longer. Among them, the fluorescence signal intensity of the Preparation Example 2-Exo group in the infarct area was 3.5 times higher than that of the Exo group at 24 hours.

[0054] 4.3.2 Histological Analysis: Seven days after treatment in step 4.2, mice were sacrificed, their hearts removed, and fixed in 4% paraformaldehyde for 24 hours. Tissue samples were removed from the fixative and rinsed in PBS for 30 minutes, repeating three times.

[0055] Transfer the fixed tissue to 70% ethanol and soak for 30 minutes. Remove the tissue from 70% ethanol and transfer it to 80% ethanol and soak for 30 minutes. Remove the tissue from 80% ethanol and transfer it to 95% ethanol and soak for 30 minutes. Remove the tissue from 95% ethanol and transfer it to 100% ethanol and soak for 30 minutes. Immerse the dehydrated tissue in a 0.01 mol / L EDTA-Na solution for 8 days, then wash it with PBS solution 3 times for 2 hours each time.

[0056] The pretreated tissue fluid was perforated, and then a clearing agent was added and incubated at 37°C for 2 days.

[0057] After transparentization, the biological tissue was added with blocking solution and immersed in a shaking platform at 37°C for 12 hours. After blocking, the liquid was removed by centrifugation and the tissue was washed with PBS solution 5 times, each time for 2 hours.

[0058] The sealed biological tissue was immersed in cryopreservative solution and cooled to -80°C at a rate of 0.5°C / min. After holding for 30 minutes, it was slowly thawed to 20°C.

[0059] Add the primary antibody diluted 500 times with diluent to the ultrasonic reactor, then incubate the thawed biological tissue at 37°C in the dark under ultrasonic conditions for 15 days. After the incubation, wash it with diluent five times, each time for 10 hours.

[0060] Secondary antibody incubation: After primary antibody incubation, add secondary antibody with fluorescent dye molecules diluted 800 times with diluent into an ultrasonic reactor, incubate the biological tissue at 37°C, in the dark, and under ultrasonic conditions for 7 days, then wash with diluent 3 times, each time for 10 hours, to obtain fluorescently labeled biological tissue.

[0061] The fluorescently stained tissue was placed in a chamber and CD31 immunofluorescence staining was observed using a two-photon confocal microscope.

[0062]

[0063] The results showed that the infarct area of the Exo group decreased by 31.9% and the vascular density increased by 1.2 times; the infarct area of the SPION-Exo group decreased by 36.8% and the vascular density increased by 1.7 times; the infarct area of the Preparation 2-Exo group decreased by 55.4% and the vascular density increased by 2.4 times. The results of CD31 immunofluorescence staining were shown in Figure 2 ; It shows that the carboxylated Fe3O4:γ-Fe2O3:Co-Fe2O4 (weight ratio of 6:3:1) composition of Preparation Example 2 of the present invention exerts a synergistic effect with exosomes, promoting the release of exosome contents and vasodilation.

[0064] The above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements derived from the present invention are intended to be within the scope of protection of the claims.

Claims

1. A superparamagnetic nanoparticle-exosome complex for preparing a drug for treating myocardial infarction, characterized in that: The complex comprises exosomes, superparamagnetic iron oxide nanoparticles and a chemical coupling agent that connects the two.

2. The superparamagnetic iron oxide nanoparticles according to claim 1, characterized in that The invention relates to a superparamagnetic iron oxide nanoparticle composition comprising one of Fe3O4, γ-Fe2O3, Co-Fe2O4 or a combination thereof, preferably Fe3O4, γ-Fe2O3, Co-Fe2O4.

3. The superparamagnetic iron oxide nanoparticles according to claim 2, characterized in that The weight ratio of the composition Fe3O4:γ-Fe2O3:Co-Fe2O4 is (2-6):(3-5):(1-5), and the preferred weight ratio is 6:3:

1.

4. The chemical coupling agent according to claim 1, characterized in that Contains EDC and NHS cross-linking agent with a molar mass ratio of 5:

2.

5. A method for preparing a superparamagnetic nanoparticle-exosome complex for preparing a drug for treating myocardial infarction, comprising the following steps: STEP 1: Extract exosomes and purify them from mesenchymal stem cells through differential centrifugation and ultrafiltration purification steps; STEP 2: Modify the nanoparticles. Dissolve the superparamagnetic iron oxide nanoparticles in citric acid, sonicate, place in a constant temperature water bath, stir, cool, and centrifuge. Finally, wash to remove free citric acid to obtain; STEP 3: Constructing a complex, mixing the carboxylated superparamagnetic iron oxide nanoparticles with exosomes, adding an EDC / NHS crosslinker, and removing free carboxylated superparamagnetic iron oxide nanoparticles by sucrose density gradient centrifugation to obtain a carboxylated nanoparticle-Exo complex.

6. The preparation method according to claim 5, characterized in that The differential centrifugation was performed at 300×g, 2000×g, and 10,000×g for 6 minutes each time, and the supernatants were collected after the centrifugation. Finally, ultracentrifugation was performed at 100,000×g for 30 minutes, and the supernatant was discarded after the centrifugation to harvest the precipitate.

7. The preparation method according to claim 5, characterized in that The superparamagnetic iron oxide nanoparticles are a superparamagnetic iron oxide nanoparticle composition of Fe3O4, γ-Fe2O3, and Co-Fe2O4, with a weight ratio of 6:3:

1.

8. The preparation method according to claim 5, characterized in that The concentration of the superparamagnetic iron oxide nanoparticle composition is 1 mg / mL, the concentration of citric acid is 0.1 mol / L, and the pH is 5-6; the ultrasonic power is 100 W, and the ultrasonic time is 5 minutes; the constant temperature water bath temperature is 60±2°C, the stirring speed is 600 rpm, and the stirring time is 6 hours; the centrifugal speed is 12,000 rpm, and the centrifugation is 15 minutes.

9. The preparation method according to claim 5, characterized in that In the step of constructing the complex, the mass ratio of the carboxylated superparamagnetic iron oxide nanoparticles to the exosomes is 1:5, the molar mass ratio of the EDC / NHS crosslinker is 5:2; the sucrose density gradient is 60% (w / v), 45% (w / v), 30% (w / v), and 8% (w / v); and the centrifugation setting conditions are 4°C, 100,000×g, and centrifugation for 2 hours.

10. Use of the complex according to claim 1 in preparing a drug for treating myocardial infarction, characterized in that: It is guided to the damaged area of the heart by an external magnetic field.

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