Platelet membrane coated curcumin nano material as well as preparation method and application thereof

Abstract

The invention provides a platelet membrane coated curcumin nano material and a preparation method and application thereof.The preparation method includes the steps that polylactic acid-polyglycolate powder is added into dichloromethane to be dissolved, then perfluoropentane and curcumin powder are added to be fully mixed, polyvinyl alcohol and isopropyl alcohol are added, under the ice bath condition, magnetic stirring is conducted for a reaction, and the platelet membrane coated curcumin nano material is obtained; and finally, fusing the basic nano material with the platelet membrane, so as to obtain the platelet membrane coated nano material P-Cur-PFP coated PC. Curcumin is directly released to a vascular lesion part, so that the microenvironment of damaged blood vessels can be improved, and PLG has the advantages of good biocompatibility and high drug release controllable speed; by utilizing LIFU guidance, the perfluoropentane can be subjected to phase change, microbubbles are generated, and venous thrombosis is dissolved; meanwhile, by utilizing a technology of coating a nano material with a platelet membrane, not only is the targeting property of the nano material enhanced, but also the stability and the biocompatibility in a living body are improved; the preparation method is simple and convenient to popularize and apply.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of targeted drugs, and in particular to a platelet membrane-wrapped curcumin nanomaterial and a preparation method and application thereof. Background Art

[0002] Venous thromboembolism (VTE) is a general term for deep vein thrombosis (DVT) and pulmonary embolism (PE). DVT refers to a venous reflux disorder caused by abnormal coagulation of blood in the deep veins. It often occurs in the lower limbs, and rarely in the mesenteric veins, upper limb veins, jugular veins or intracranial venous systems. If the thrombus breaks off and blocks the pulmonary artery, it will cause PE. The risk of DVT and PE in pregnant women and the mortality rate caused by VTE are significantly higher than those in the normal population. Venous thromboembolism can lead to miscarriage, intrauterine growth restriction of the fetus, gestational hypertension, placental abruption and other conditions, posing a serious threat to the lives of mothers and children.

[0003] In recent years, the incidence of VTE during pregnancy and puerperium has increased significantly, with the number of older pregnant women, obese women, and pregnancy complications or comorbidities increasing. Screening for high-risk factors for VTE and conducting early prevention can effectively reduce its incidence.

[0004] However, current treatment options for venous thrombosis during pregnancy have some limitations, including the following:

[0005] 1. Clinical drugs have side effects on the fetus: Most clinical drugs may have side effects on the fetus when circulating in the body of a pregnant woman, such as miscarriage, deformity, bleeding and other serious consequences.

[0006] 2. Insufficient drug targeting: Traditional drug treatment cannot achieve effective targeting, which may cause widespread distribution of drugs, leading to unnecessary side effects or affecting efficacy.

[0007] 3. Drug release is difficult to control: The release rate of most drugs in the body is difficult to control, which may cause fluctuations in drug concentration and affect efficacy.

[0008] 4. Systemic side effects: Some drugs in clinical practice need to be used for a long time, which may cause serious systemic side effects, such as bleeding, neurodevelopmental disorders, etc.

[0009] 5. Diagnostic limitations: Traditional imaging technology has limitations in diagnosing venous thrombosis in pregnant women. For example, in some cases, the reliability of diagnosis is low and more accurate diagnostic technology is needed. Summary of the invention

[0010] The purpose of the present invention is to provide a method for preparing a platelet membrane-wrapped curcumin nanomaterial, its products and applications, in view of the problems of high side effects, insufficient drug targeting and poor drug release control in the existing therapeutic drugs for venous thrombosis during pregnancy. Curcumin is directly released to the site of vascular lesions, which can improve the microenvironment of damaged blood vessels. PLG has the advantages of good biocompatibility and fast controllable drug release. Using LIFU guidance, perfluoropentane can undergo phase change, generate microbubbles, and dissolve venous thrombus. At the same time, the present application uses the technology of platelet membrane-wrapped nanomaterials, which not only enhances the targeting of nanomaterials, but also improves the stability and biocompatibility in vivo. The preparation method is simple and easy to promote and apply.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] A method for preparing a platelet membrane-encapsulated curcumin nanomaterial comprises the following steps:

[0013] Step 1, adding polylactic acid-polyglycolic acid ester powder into dichloromethane and dissolving by ultrasonication;

[0014] Step 2, adding perfluoropentane and curcumin powder to the mixed solution obtained in step 1, respectively, and mixing them thoroughly by ultrasonication;

[0015] Step 3, adding polyvinyl alcohol and isopropanol to the mixed solution obtained in step 2, and mixing by ultrasonication;

[0016] Step 4: Under ice bath conditions, the mixed solution obtained in step 3 is magnetically stirred, and then centrifuged in a low-temperature centrifuge to obtain the basic nanomaterial Cur-PFP@PC;

[0017] Step 5: Mix the basic nanomaterial Cur-PFP@PC obtained in step 4 with the platelet membrane, and perform ultrasonic treatment to obtain the platelet membrane-coated nanomaterial P-Cur-PFP@PC.

[0018] The present invention provides a method for preparing a platelet membrane-wrapped curcumin nanomaterial, firstly adding polylactic acid-polyglycolate powder into dichloromethane to dissolve, then adding perfluoropentane and curcumin powder to fully mix, then adding polyvinyl alcohol and isopropanol, and reacting under ice bath conditions with magnetic stirring to obtain a basic nanomaterial Cur-PFP@PC, and finally fusing the basic nanomaterial with the platelet membrane to obtain a platelet membrane-wrapped nanomaterial P-Cur-PFP@PC. The prepared basic nanomaterial Cur-PFP@PC uses PLGA as a basic material, and curcumin (Cur) and perfluoropentane (PFP) as core components. After use, curcumin is directly released into the vascular lesion site, reacts with hydrogen peroxide (H2O2) in the thrombus formation area to produce oxygen (O2), and inhibits pro-inflammatory mediators, cytokines and inflammatory signaling pathways, which can improve the microenvironment of damaged blood vessels, further prevent the formation of thrombi, and improve the efficacy of thrombus treatment. PLGA (polylactic acid-polyglycolate) is a high molecular weight nanomaterial with the advantages of biodegradability, good biocompatibility, structural modifiability, and controllable drug release speed. In addition, the composite material provided by the present invention is added with a new contrast agent perfluoropentane. After the targeted drug is used, low-intensity focused ultrasound (LIFU) is used to guide the perfluoropentane to undergo a phase change, undergo a transition from liquid to gas, and generate microbubbles, which use the cavitation effect to dissolve venous thrombi, showing good diagnostic and therapeutic effects. At the same time, the present application uses the technology of platelet membrane coating nanomaterials, which not only enhances the targeting of nanomaterials, but also improves their stability and biocompatibility in vivo. The preparation method is simple and easy to promote and apply.

[0019] Furthermore, lactic acid-glycolic acid is a constituent material of the outer membrane of the nanoparticles, and is used in step 1 to form an oil-in-water primary emulsion.

[0020] Furthermore, in step 3, the addition of polyvinyl alcohol and isopropyl alcohol is used for sphering, thereby stabilizing the structure of the nanoparticles.

[0021] Furthermore, in step 4, the basic nanomaterial Cur-PFP@PC is in a liquid state, and centrifugation is performed to remove impurities that have not formed into balls during the manufacturing process.

[0022] Furthermore, in the mixed solution obtained in step 1, the mass concentration of polylactic acid-polyglycolate is 20 to 30 mg / mL.

[0023] Furthermore, in step 2, 180 to 300 microliters of perfluoropentane are added for every 10 mg of curcumin powder.

[0024] Furthermore, in step 3, the mass concentration of polyvinyl alcohol is 3-5%; the mass concentration of isopropanol is 1-3%.

[0025] Furthermore, the added volume ratio of polyvinyl alcohol to isopropyl alcohol is 0.5-1:1.

[0026] Furthermore, in step 5, the platelet membrane is extracted from platelet-rich plasma by freeze-thaw method.

[0027] Furthermore, in step 5, 5 to 15 ml of the basic nanomaterial Cur-PFP@PC is added to 1 mL of the platelet membrane solution. Studies have found that the addition ratio of platelet membrane to basic nanomaterial Cur-PFP@PC is a key factor affecting the performance of the final material. Too large or too small a ratio will result in a decrease in performance. Preferably, 10 to 15 ml of the basic nanomaterial Cur-PFP@PC is added to 1 mL of the platelet membrane solution.

[0028] Another purpose of the present invention is to protect the platelet membrane-wrapped curcumin nanomaterial product obtained by the above preparation method.

[0029] The material product is prepared by the preparation method of platelet membrane-encapsulated curcumin nanomaterial as mentioned above.

[0030] The platelet membrane-wrapped curcumin nanomaterial provided by the present invention can be used for targeted thrombolytic therapy, achieve precise and controlled drug release, and repair damaged blood vessels, thereby avoiding adverse effects associated with traditional long-term medication and having good safety.

[0031] The platelet membrane-encapsulated curcumin nanomaterial prepared in this application has the following advantages:

[0032] 1. Enhanced targeting and selectivity: Through the coating of platelet membranes, P-Cur-PFP@PC nanomaterials can mimic the natural behavior of platelets and achieve targeting of diseased venous thrombi. This targeting is much better than the existing non-specific drug delivery system, reducing potential damage to healthy tissues and improving efficacy.

[0033] 2. Improve treatment efficiency: Using low-intensity focused ultrasound (LIFU) to activate the phase change effect caused by PFP can generate microbubbles at the target site, thereby enhancing the thrombolytic effect, while improving the diagnostic accuracy of the treatment site through enhanced ultrasound signals. This mechanism can effectively enhance the thrombolytic efficiency, and it is achieved in a non-invasive way.

[0034] 3. Reduce systemic side effects: Due to the targeting and controlled release capabilities of nanomaterials, the drugs mainly act on the lesion area, reducing systemic drug exposure. Compared with traditional intravenous drugs, it can significantly reduce systemic side effects and improve the safety of treatment.

[0035] 4. Excellent biocompatibility and safety: The platelet membrane coverage strategy provides good biocompatibility and reduces the body's immune response to nanomaterials. Curcumin (Cur) itself, as a natural extract, has good antioxidant and anti-inflammatory effects, further improving the biosafety of nanomaterials.

[0036] 5. Friendly to pregnant women and fetuses: The present invention takes into account the special physiological state of pregnant women, reduces the potential risks of drugs to the fetus through targeted and local treatment, and provides a relatively safe strategy for the treatment of venous thrombosis during pregnancy.

[0037] 6. Comprehensively improve diagnostic capabilities: The nanomaterial can provide clearer signals in ultrasound imaging, providing more accurate diagnostic capabilities for those situations that are difficult to diagnose with traditional imaging technology (for example: pregnant women due to obesity or blood clots in the deep veins).

[0038] In general, this invention provides a safer, more effective and targeted treatment option compared to the existing technology, and significantly improves the safety, efficiency, targeting and diagnostic capabilities of the treatment of venous thrombosis during pregnancy.

[0039] The use of the above-mentioned material products in the preparation of drugs for preventing and / or treating venous thrombosis.

[0040] Furthermore, after medication, low-intensity focused ultrasound technology is used to guide the treatment area.

[0041] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0042] 1. The present invention provides a method for preparing a platelet membrane-wrapped curcumin nanomaterial, firstly adding polylactic acid-polyglycolate powder to dichloromethane to dissolve, then adding perfluoropentane and curcumin powder to fully mix, then adding polyvinyl alcohol and isopropanol, and reacting under ice bath conditions with magnetic stirring to obtain a basic nanomaterial Cur-PFP@PC, and finally fusing the basic nanomaterial with the platelet membrane to obtain a platelet membrane-coated nanomaterial P-Cur-PFP@PC. The prepared basic nanomaterial Cur-PFP@PC uses PLGA as a basic material, and curcumin (Cur) and perfluoropentane (PFP) as core components. After use, curcumin is directly released to the vascular lesion site, reacts with hydrogen peroxide (H2O2) in the thrombus formation area to produce oxygen (O2), and inhibits pro-inflammatory mediators, cytokines and inflammatory signaling pathways, which can improve the microenvironment of damaged blood vessels, further prevent the formation of thrombi, and improve the efficacy of thrombus treatment. PLGA (polylactic acid-polyglycolate) is a high molecular weight nanomaterial with the advantages of biodegradability, good biocompatibility, structural modifiability, and controllable drug release speed. In addition, the composite material provided by the present invention is added with a new contrast agent perfluoropentane. After the targeted drug is used, low-intensity focused ultrasound (LIFU) is used to guide the perfluoropentane to undergo a phase change, undergo a transition from liquid to gas, and generate microbubbles, which use the cavitation effect to dissolve venous thrombi, showing good diagnostic and therapeutic effects. At the same time, the present application uses the technology of platelet membrane coating nanomaterials, which not only enhances the targeting of nanomaterials, but also improves their stability and biocompatibility in vivo. The preparation method is simple and easy to promote and apply.

[0043] 2. The platelet membrane-encapsulated curcumin nanomaterial provided by the present invention can be used for targeted thrombolytic therapy, achieve precise and controlled drug release, and repair damaged blood vessels, thereby avoiding the adverse effects associated with traditional long-term medication and having good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Diagram of the synthesis process of P-Cur-PFP@PC nanodroplets and schematic diagram of P-Cur-PFP@PC-mediated specific therapy.

[0045] Figure 2 Diagram of the design process and characterization of nanodroplets; Figure 2 A in the figure is the TEM image of Cur-PFP@PC and P-Cur-PFP@PC; Figure 2 Figure B is the size diagram of Cur-PFP@PC and P-Cur-PFP@PC; Figure 2 C in the figure is the zeta potential diagram of Cur-PFP@PC and P-Cur-PFP@PC; Figure 2D in the figure is the Western blot analysis of CD41 and GAPDH in platelets, Cur-PFP@PC and P-Cur-PFP@PC; Figure 2 E in the figure is the SDS-PAGE gel electrophoresis of platelets, Cur-PFP@PC and P-Cur-PFP@PC; Figure 2 F in the figure is the stability analysis diagram of P-Cur-PFP@PC in PBS and FBS; Figure 2 G in the figure is the metabolic rate analysis chart of pregnant mice by fluorescence microscopy, ****P<0.0001 (n=3).

[0046] Figure 3 The phase transition of nanomaterials in vitro and the US images in vivo; Figure 3 A in the figure is the optical microscope image of P-Cur-PFP@PC after LIFU irradiation at different time points, scale bar = 50 μm; Figure 3 B in the figure is the ultrasound image of P-Cur-PFP@PC at different time points under in vitro LIFU irradiation (B model and CEUS model); Figure 3 C in the figure is the ultrasound images of saline, Cur, Cur-PFP@PC and P-Cur-PFP@PC before and after LIFU irradiation in vivo (b model and ultrasound contrast model), (n=3).

[0047] Figure 4 This is a graph showing the safety test results of nanomaterials; Figure 4 A in the figure is the effect of Cur-PFP@PC and P-Cur-PFP@PC on HUVEC cells at specified drug concentrations; Figure 4 BC in the figure is the hemolysis rate diagram of different concentrations of Cur-PFP@PC and P-Cur-PFP@PC; Figure 4 D in the figure is the platelet aggregation rate diagram of saline, Cur-PFP@PC and P-Cur-PFP@PC; Figure 4 Figure E shows the H&E staining test results of the heart, liver, spleen, lung, and kidney of ICR rats after different treatments, scale bar = 100 μm, (n = 4).

[0048] Figure 5 Provides a map for targeted testing of nanomaterials in vivo and in vitro; Figure 5 A in the figure is the result of the targeting test of Cur-PFP@PC and P-Cur-PFP@PC on activated platelets analyzed by confocal microscopy. The cell nucleus was stained with DIPA (blue), Cur-PFP@PC and P-Cur-PFP@PC were labeled with Dil (red), platelets were stained with DIPA and labeled with Calcein-AM (green), scale bar: 10 μm; Figure 5B in the figure is the flow cytometry detection results of Cur-PFP@PC and P-Cur-PFP@PC at different time points, ****p<0.0001; Figure 5 C in the figure is the result of frozen section analysis of the targeted thrombus of Cur-PFP@PC and P-Cur-PFP@PC in vivo. The cell nucleus was stained with DIPA (blue), Cur-PFP@PC and P-Cur-PFP@PC were labeled with Dil (red), and the thrombus was labeled with CD42 (green). Scale bar: 500 μm (n=3).

[0049] Figure 6 The results of in vivo and in vitro thrombolysis tests are shown in Figure 2. Figure 6 A in the figure shows the appearance of thrombus after incubation with PBS, Cur, Cur-PFP@PC, P-Cur PFP@PC, Cur-PFP@PC+LIFU, and P-Cur-PFP@PC+LIFU (7 W cm-2 LIFU irradiation) at different time points; Figure 6 B in the figure is the test graph of thrombus weight in each group, and the values ​​are mean ± sd, ****P < 0.0001; Figure 6 C in the figure is the SEM analysis of the fibrin network in each group, scale bar: 10 μm; Figure 6 D in the figure is the ultrasound image of saline, Cur, Cur-PFP@PC, and P-Cur-PFP@PC before and after in vivo LIFU irradiation (b model) thrombolytic effect diagram; Figure 6 E in the figure is the H&E staining test picture of the posterior cavity after different treatments (n=4).

[0050] Figure 7 It is a test diagram for the removal of ROS in vivo and in vitro; Figure 7 AC in the figure is the analysis diagram of scavenging effect of PBS and Cur on ROS; Figure 7 D in the figure is a fluorescence microscopy analysis of the clearance of free oxygen in cells, scale bar: 200 μm; Figure 7 E in the figure is the quantitative analysis of intracellular ROS clearance rate, ****P<0.0001; Figure 7 F in the figure is the Detectionkit analysis chart of ROS clearance rate in vivo, ****P<0.0001.

[0051] Figure 8 : Expression analysis of inflammatory factors after treatment and cell repair; among them, Figure 8 AC in the figure are the results of Elisa kit analysis of IL-1, IL-6, and TNF-α content in each group of cells, ****P<0.0001; Figure 8D in the figure shows the wound healing experiment images and healing rate test results of PBS, Cur, Cur-PFP@PC, and P-Cur-PFP@PC after incubation, respectively. The last two groups were added with LIFU (7W cm-2LIFU irradiation) for 24h. Scale bar: 20μm; Figure 8 E in the figure is a quantitative analysis chart of the wound healing rate (100%) of each group.

[0052] Fig. 9 : Live safety test result diagram; Fig. 9 AF in the figure are the biochemical function test diagrams of leukocytes, erythrocytes, platelets and liver of ICR mice after treatment; Fig. 9 G in the figure is the test results of fetal body and placenta weight of each group, the values ​​are mean ± sd, ****P < 0.0001; Fig. 9 H in the figure is the H&E staining of the heart, liver, spleen, lung and atrium of the offspring mice after different treatments (n=4). DETAILED DESCRIPTION

[0053] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0054] 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 the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0055] Platelet membrane extracted by freeze-thaw method, specific operation:

[0056] 1. Transfer the purchased SD rat platelet-rich plasma into a sterile 1.5 ml EP tube and centrifuge at 3000 rpm for 5 minutes;

[0057] 2. After centrifugation, discard the supernatant, wash twice, store in PBS solution containing protease inhibitors, and place in a 4°C refrigerator for later use;

[0058] 3. Place the platelet solution in a -80°C ultra-low temperature refrigerator for 30 minutes, then transfer it to room temperature to slowly melt. Repeat this step 5 times to obtain the platelet membrane.

[0059] Example 1

[0060] 1. Material Preparation

[0061] Preparation of curcumin and perfluoropentane based nanomaterials Cur-PFP@PC

[0062] 50 mg of PLGA (polylactic acid-polyglycolic acid) powder was weighed and dissolved in 2 mL of dichloromethane by ultrasonication.

[0063] Then, 200 μL of perfluoropentane (PFP) and 10 mg of curcumin powder were added and thoroughly mixed by ultrasonication.

[0064] 8 mL of 4% polyvinyl alcohol (PVA) and 10 mL of 2% isopropanol were added and sonicated again for 2 minutes.

[0065] The mixture was magnetically stirred in an ice bath at 4°C for 3 h and then centrifuged in a low-temperature centrifuge at 4°C for 5 min to obtain the basic nanomaterial Cur-PFP@PC.

[0066] Preparation of platelet membrane-coated nanomaterial P-Cur-PFP@PC

[0067] 10 ml of Cur-PFP@PC was taken and mixed with 1 ml of extracted platelet membrane, and ultrasonically treated with a sonicator at a power of 45 W to obtain the platelet membrane-coated nanomaterial P-Cur-PFP@PC.

[0068] Curcumin (Cur) is the main component found in turmeric rhizomes, which is known for its anti-inflammatory and antioxidant properties. Curcumin can directly neutralize free radicals (ROS and RNS) and maintain the activities of multiple antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH), playing a vital role in regulating oxidative stress in various diseases. However, suboptimal sample preparation and analytical methods often hinder the accurate evaluation of bioactivity and its clinical efficacy.

[0069] PLGA (polylactic acid-polyglycolic acid) is a high molecular weight nanomaterial approved by the US Food and Drug Administration (FDA). It has biodegradability, good biocompatibility, structural modifiability, and fast controllable drug release, making it widely used in the field of drug delivery. However, it is difficult to control intracellular delivery to target organelles. This application designs biomimetic platelet membrane-loaded nanomaterials to simulate nanoscale platelets with strong innate targeting capabilities. Polylactic acid-glycolic acid) (PLGA) loaded with curcumin (Cur) and perfluoropentane (PFP) was prepared and wrapped by platelet membranes. Then, we enhanced the delivery of venous thrombosis treatment drugs by ultrasound (US) and LIFU stimulation of the phase change of PFP.

[0070] PFP is a new type of ultrasound contrast agent that undergoes liquid-gas conversion and produces microbubbles that are stimulated by external factors. This leads to acoustic droplet vaporization (ADV), which uses the cavitation effect to dissolve venous thrombi and has important diagnostic value and therapeutic effects ( Figure 1). Early diagnosis of thrombosis is limited by inherent deficiencies of traditional imaging techniques in clinical applications. The reliability of the US diagnosis is reduced in some cases, such as obese pregnant women, significant limb swelling, small thrombus volume, and deep vascular location. The nanomaterials designed in the present invention undergo a phase and produce highly echogenic microbubbles, which further enhance the ultrasound signal and improve the accuracy and sensitivity of venous thrombosis diagnosis. A multifunctional molecular imaging treatment method with comprehensive advantages is established, which has the biocompatibility and targeting of early diagnosis, precise treatment, and efficacy monitoring of pregnancy-related venous thrombosis. In addition, this therapy is a targeted drug delivery system that relies on the nanomaterials designed by us. In the living state, specific molecular changes at the organ, cell, and even subcellular levels can be detected to guide disease treatment at the molecular level.

[0071] like Figure 1 As shown, the vascular endothelium has several important functions, including hemostasis. During thrombosis and thrombolysis, endothelial damage occurs, leading to platelet activation and excessive production of reactive oxygen species (ROS), exacerbating oxidative stress. This further promotes platelet activation and aggregation, triggers inflammatory factors, worsens the condition of damaged blood vessels, and increases the risk of recurrence of venous thrombosis. In order to improve the microenvironment of damaged blood vessels, the nanomaterials of the present application release curcumin directly into the site of vascular lesions to remove reactive oxygen. Curcumin reacts with hydrogen peroxide (H2O2) in the thrombosis area to produce oxygen (O2), while inhibiting proinflammatory mediators, cytokines and inflammatory signaling pathways. This improvement in the vascular microenvironment further prevents the formation of thrombi and improves the efficacy of thrombosis treatment.

[0072] 2.0 Results and Discussion

[0073] 2.1 Synthesis and characterization of P-Cur-PFP@PC nanomaterials

[0074] We synthesized Cur-PFP@PC by emulsion solvent evaporation method and combined platelet membrane vesicles (PMVs) with Cur-PFP@PC to prepare platelet membrane-coated nanomaterial P-CurPFP@PC.

[0075] The morphology of the prepared P-Cur-PFP@PC was obtained by TEM (Hitachi H-7600, Japan), and the structure of the platelet membrane was detected by SEM (scanning electron microscope). Western Blot (WB) and Coomassie blue staining confirmed the platelet membrane extraction. The zeta potential and average particle size of Cur-PFP@PC and P-CUR-PFP@PC were determined by a dynamic light scattering instrument (DSL, Brookhaven Instruments ZEN3600, USA). Transmission electron microscopy (TEM) showed the spherical nature of the prepared nanomaterials, and P-Cur-PFP@PC showed an obvious core-shell structure compared with Cur-PFP@PC ( Figure 2 A in Figure 1). Dynamic light scattering (DLS) showed that the hydrodynamic diameter of Cur-PFP@PC was 234.3±8.63nm, and that of P-Cur-PFP@PC was 267±2.61nm. The particle size increased after PMV coating (P<0.0001) ( Figure 2 (B). Zeta potential changed from -5.86±0.97mv to -17.95±1.98mv (P<0.0001). Figure 2 C), indicating successful outer membrane coating.

[0076] In order to confirm that the outer membrane is a platelet membrane, we verified the presence of functional proteins on the nanomaterials wrapped on the membrane. The results showed that the protein band of P-Cur-PFP@PC was consistent with the PLT protein band, indicating that P-Cur-PFP@PC inherited the protein components of the platelet membrane, while PLGA did not contain PLT protein ( Figure 2 D in the figure). At the same time, SDS-PAGE gel electrophoresis confirmed the retention of platelet membrane proteins on P-Cur-PFP@PC nanomaterials. It was found that although P-Cur-PFP@PC showed a weaker protein band, it had the same protein composition as the platelet membrane, while Cur-PFP@PC did not contain protein components ( Figure 2 E).

[0077] This result further confirmed that the outer transparent membrane of P-Cur-PFP@PC nanomaterial is a platelet membrane and contains platelet protein components.

[0078] To evaluate a drug delivery system, it must have a suitable particle size, good stability, drug loading capacity, and a long in vivo circulation period. P-Cur-PFP@PC was placed in PBS and fetal bovine serum for 7 days, and the particle size remained at around 250nm, with no significant change, and excellent stability ( Figure 2F in the figure). Finally, the encapsulation efficiency and drug loading rate of curcumin were determined. The encapsulation efficiency was 70.87% ± 0.150, and the drug loading rate was 8.50% ± 0.021. The metabolic rates of Cur-PFP@PC and P-Cur-PFP@PC in pregnant mice were compared. P-Cur-PFP@PC had a longer circulation than Cur-PFP@PC. Starting from 6 hours, the metabolic rates of the two nanomaterials were significantly different ( Figure 2 G in.

[0079] In summary, bionic platelet membrane-loaded curcumin nanomaterials (P-Cur-PFP@PC) were successfully prepared, which showed good stability, drug loading capacity and good circulation ability.

[0080] 2.2 Phase change characteristics and imaging evaluation of P-CUR-PFP@PC nanomaterials

[0081] In order to further observe the ADV effect of PFP, the PFP was observed under an optical microscope ( Figure 3 A in the figure). With the increase of LIFU irradiation time, the volume began to increase significantly at 5 minutes and reached the maximum value at 10 minutes. After 15 minutes, it gradually shrank and cracked. This transition shows that LIFU has excellent performance in the phase transition of nanomaterials. In addition, we observed the ultrasonic signal of P-Cur-PFP@PC in both the B model and the CEUS model ( Figure 3 B in the figure). The number of microbubbles gradually increased, and the ultrasound signal gradually strengthened. After 10 minutes, the signal reached a peak, providing the clearest image. Based on this, it is assumed that when P-Cur-PFP@PC effectively targets the thrombus and accumulates, LIFU irradiation induces a phase change and releases microbubbles. It then causes an increase in the ultrasound signal at the thrombus site, making up for the disadvantage that traditional imaging may be affected by external conditions. We injected mice with venous thrombosis with saline, Cur, Cur-PFP@PC, and P-Cur-PFP@PC. After LIFU irradiation, the results showed that the ultrasound signal in the P-Cur-PFP@PC group was significantly enhanced, while the ultrasound signals in the other groups were weak ( Figure 3 in; C).

[0082] 2.3 Safety evaluation of nanomaterials

[0083] To evaluate cell viability, HUVECs were incubated with different concentrations of P-Cur-PFP@PC and Cur-PFP@PC nanomaterials for 12 h. The cell viability was close to 100% ( Figure 4 A), indicating that these nanomaterials are not harmful to normal cells, even at high concentrations.

[0084] In addition, an in vitro hemolysis rate test was performed. 2% of red blood cells were collected and suspended in PBS. Different concentrations of P-Cur-PFP@PC and Cur-PFP@PC nanomaterials (0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL) were co-incubated for 4 hours. The hemolysis rate was measured using an enzyme-linked immunosorbent assay. The results are shown in Figure 2. Figure 4 B in Figure 4 As shown in C. The hemolysis rates at different concentrations remained within the normal range, indicating that these nanomaterials have good biocompatibility and no hemolysis in vitro.

[0085] After incubating mouse whole blood with P-Cur-PFP@PC and Cur-PFP@PC nanomaterials, platelet aggregation was detected ( Figure 4 D). Both nanomaterials did not activate platelets, thereby reducing the risk of thrombosis, compared with a saline control group.

[0086] To confirm the safety of the nanomaterials in vivo, mice were intravenously injected with P-Cur-PFP@PC and Cur-PFP@PC nanomaterials at a dose three times the therapeutic dose, and the control group was injected with saline. After 7 days of treatment, there were no structural changes in the HE-stained organs of the nanomaterial-treated group compared with the control group, indicating that these nanomaterials played a safe role in vivo ( Figure 4 E).

[0087] 2.4 Evaluation of targeting ability of nanomaterials

[0088] In vitro, HUVECs were incubated with dil-labeled P-Cur-PFP@PC (Dil-P-Cur-PFP@PC) and Cur-PFP@PC (Dil-Cur-PFP@PC). Under a confocal fiber microscope, Dil-P-Cur-PFP@PC with red fluorescence showed excellent co-localization with platelets, and was significantly better than Dil-Cur-PFP@PC, indicating that the platelet membrane on the surface of P-Cur-PFP@PC has excellent targeting ability ( Figure 5 A).

[0089] To detect the phagocytic effect of HUVECs on Dil-P-Cur-PFP@PC and Dil-Cur-PFP@PC, 2×10 5 HUVECs cells were seeded in each well of a 6-well plate and cultured for 24 hours. Dil-P-Cur-PFP@PC and Dil-Cur-PFP@PC nanomaterials were added, and PBS was used as a control. The cells were incubated for 1, 2, 4, and 6 hours, and flow cytometry analysis was performed after digestion. With the extension of incubation time, the phagocytic ability of cells increased, reaching a peak at 4 hours, and then gradually decreased. In addition, the ability of cells to phagocytose Dil-P-Cur-PFP@PC was significantly better than that of Dil-Cur-PFP@PC( Figure 5 These results are consistent with the confocal microscopy results, indicating that the Dil-P-Cur-PFP@PC nanomaterial has superior targeting ability.

[0090] The targeting of these nanomaterials was evaluated in ICR mice at gestational day 13.5, and FeCl3 induced endothelial cell damage in the inferior vena cava and thrombus formation (thrombus group). Subsequently, Dil-P-Cur-PFP@PC and Dil-Cur-PFP@PC were injected into the tail vein. Five minutes later, frozen venous thrombus sections were stained with CD42. The targeted nanomaterials (red) showed good colocalization with fibrin (green) within the thrombus, while the Dil-Cur-PFP@PC group showed obvious colocalization, while the Dil-P-Cur-PFP@PC group ( Figure 5 C). These findings demonstrate the effective targeting of thrombi by targeted nanomaterials and suggest the potential for targeted thrombolysis.

[0091] 2.5 Evaluation of thrombolysis with nanomaterials

[0092] Mice and cells: ICR mice (6 weeks old) were purchased from Chongqing Enbi Biotechnology Co., Ltd.

[0093] All mice were housed under grade IVC conditions in the animal facility of Chongqing Medical University, and all animal experiments strictly adhered to the compliance standards of the Institutional Animal Care and Use Committee. All animal handling procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese Association for Laboratory Animal Science and followed the guidelines of the Animal Welfare Act. This animal study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (approval number: 2020-790). HUVECs cells were provided by the Maternal-Fetal Laboratory of the First Affiliated Hospital of Chongqing Medical University, and the parameters of the hypoxic culture chamber were 37°C, 1% O2, 5% CO2, and 94% N2.

[0094] Phase change properties and in vitro release of nanomaterials: 2 ml of P-Cur-PFP@PC was added to the gelatin model and released at 7 W / cm -2 The acoustic power is irradiated with LIFU.

[0095] Ultrasound images were collected at different time intervals after irradiation, including B-ultrasound and ultrasound contrast imaging, modes (n=3).

[0096] Venous blood was collected from rats and blood clots were formed at room temperature. Six experimental groups were set up: PBS group, Cur group, Cur-PFP@PC group, P-Cur-PFP@PC group, Cur-PFP@PC+LIFU group, and P-Cur-PFP@PC+LIFU group. The blood clot weight was measured every 5 minutes, and laser confocal scanning microscopy was performed after 60 minutes. Figure 6 A and Figure 6 As shown in B, the weight of the blood clot was reduced in the LIFU irradiation group compared with the non-LIFU group. In the LIFU group, the thrombolytic effect of P-Cur-PFP@PC+LIFU was better than that of Cur-PFP@PC+LIFU. In the non-LIFU group, the blood clot weight did not change significantly, and the results were consistent by measurement. There was no statistically significant difference between the non-LIFU groups, and the difference between the LIFU groups was statistically significant (P<0.0001). In addition, confocal scanning electron microscopy observed that the fibrin network in the LIFU group was significantly loose ( Figure 6 C).

[0097] The thrombolytic ability of pregnant ICR mice was evaluated, and an inferior vena cava thrombosis model was successfully established. The mice were injected with 200uL of normal saline, Cur (5mg / ml), 200uL of Dil-Cur-PFP@PC (5mg / ml), and 200uL of Dil-P-Cur-PFP@PC (5mg / ml) through the tail vein. The mice were then irradiated with LIFU for 10 minutes, and the thrombus volume of each group was detected using the B model. The thrombus volume of the control group and the Cur group gradually increased over time, the thrombus volume of the Dil-Cur-PFP@PC group was slightly reduced compared with before LIFU radiotherapy, and the thrombus size of the Dil-P-Cur-PFP@PC group was significantly reduced ( Figure 6 D in the figure). Comparing the HE staining of thrombi in each group, the thrombolytic effect of the non-LIFU group was worse than that of the LIFU group, while the thrombolytic effect of the Dil-P-Cur-PFP@PC+LIFU group was the best ( Figure 6 E), which is consistent with the observation results of the ultrasound imaging system.

[0098] 2.6 Evaluation of the effect of nanomaterials in removing reactive oxygen species

[0099] To verify the role of Cur in scavenging ROS in vitro, we used PBS as a control group to evaluate the ability of Cur to scavenge superoxide radicals, singlet oxygen, and hydroxyl radicals.

[0100] like Figure 7 A in Figure 7B and Figure 7 As shown in C, Cur has a significant ROS scavenging effect compared with PBS. As mentioned above, we divided HUVECs cells into different groups for incubation, detected intracellular ROS, and observed the fluorescence signal under a fluorescence microscope ( Figure 7 D in the figure). Compared with the non-LIFU group, the LIFU group had a stronger ROS scavenging ability in the cells. Quantitative analysis by fluorescence microplate reader showed that compared with the non-LIFU group, the LIFU group had a significant ROS scavenging effect ( Figure 7 E). There was no statistically significant difference between P-Cur-PFP@PC+LIFU and Cur-PFP@PC+LIFU. It is worth noting that despite the lack of strong targeting in the Cur-PFP@PC group, all nanomaterials were dissolved in the cell culture medium and were confined by the space of the six-well plate. Under LIFU irradiation, a phase transition occurred, achieving Acoustic Droplet Vaporization (ADV), allowing Cur to enter the cell to eliminate ROS. In contrast, due to the less solubility of free Cur, its effective entry into the cytoplasm to eliminate ROS is limited. This further verifies the hydrophobicity and instability that directly affect its clinical trials, and the prepared nanomaterials successfully solve these problems.

[0101] In addition, the ROS scavenging ability of the nanomaterials in successfully simulated ICR pregnant mice was evaluated. Starting from day 13.5 of pregnancy, normal saline, Cur, Cur-PFP@PC, and P-Cur-PFP@PC (5 mg / ml, 200 μL) were injected into the tail vein every day. The latter two groups received 5 minutes of LIFU radiotherapy at the thrombus site. After 4 days of treatment, thrombi were extracted from the mice that died suddenly, and tissue ROS was detected in some thrombi using a detection kit. The results showed that the P-Cur-PFP@PC+LIFU group showed the most prominent ROS scavenging effect ( Figure 7 F), indicating that the Cur loaded by the nanomaterial can successfully reach the thrombus site, be targeted and released, and effectively remove oxygen free radicals.

[0102] 2.7 Detection of the inhibition of inflammatory cell infiltration by nanomaterials

[0103] In order to detect the inhibitory effect on inflammatory cell infiltration, we placed HUVECs cells in a hypoxic incubator for 24 hours to induce endothelial cell damage. The cells were divided into 6 groups as before, and PBS was used as the control group. After incubation for 24 hours, the cell supernatant was collected, and the expression levels of inflammatory factors and IL-1, IL-6, and TNF-α in each group were detected by ELISA kits ( Figure 8 A in Figure 8 B in Figure 8C in the figure). The expression level in the control group was the highest, while the expression level in the P-Cur-PFP@PC+LIFU group was the lowest, followed by the Cur-PFP@PC+LIFU group. The differences among the groups were statistically significant (P<0.0001).

[0104] 2.8 Evaluation of HUVECs Migration Promotion

[0105] HUVECs cells were cultured under hypoxic conditions for 24 hours, and scratch tests were performed using a 200 μL pipette tip. The cells were divided into 6 groups, and the migration of cells was observed under an inverted microscope for 24 hours according to the pre-treatment conditions ( Figure 8 D in Figure 8 E). The P-Cur-PFP@PC+LIFU group showed a dominant migration effect, promoting the repair of damaged endothelial cells and promoting the healing of damaged blood vessels.

[0106] 2.9 In vivo safety evaluation after treatment

[0107] After thrombosis of the inferior vena cava in pregnant ICR mice, 3 times the dose of P-Cur-PFP@PC and Cur-PFP@PC nanomaterials were given to pregnant ICR mice starting from day 8.5 of pregnancy. The control group was pregnant mice without thrombosis and injected with normal saline. The LIFU group received 5 minutes of LIFU radiotherapy every day until day 16.5 of pregnancy. Eye blood samples were collected for hematology, biochemistry and coagulation function tests, and the results of each group were within the normal range ( Fig. 9 A in Fig. 9 B in Fig. 9 C in Fig. 9 D in Fig. 9 E in Fig. 9 F).

[0108] Record fetal body weight and placenta weight ( Fig. 9 G), the weight gain of fetal mice in the P-Cur-PFP@PC+LIFU group was significantly greater than that in other groups, and the difference between the groups was statistically significant (P<0.0001). However, there was no difference in the placental weight measurement results. We believe that it may be that the basic weight of the placenta is light, and its changes are expressed at the molecular level. We need to continue to discuss. In addition, no abnormalities were found in the HE staining of fetal organs fed to 4 weeks ( Fig. 9 In summary, our nanomaterials not only have therapeutic effects on thrombosis but also have high safety and no adverse effects on pregnant mice and their offspring.

[0109] Example 2

[0110] The main factors for venous thrombosis during pregnancy are elevated estrogen levels, elevated coagulation status, advanced maternal age, cesarean section, prolonged bed rest, reduced blood flow, multiple pregnancy, uterine compression, inferior vena cava reflux obstruction, preeclampsia, thrombin release, and increased coagulation activity. Currently, there are no noninvasive, safe, and reliable clinical trials available for this condition. In addition, traditional imaging methods have limitations in diagnosing venous thrombosis in pregnant women.

[0111] The present application provides a new method for nanomaterial encapsulation involving platelet membranes. This allows nanomaterials to directly mimic the characteristics of source cells and replicate highly complex biological functions on their surface. As a result, they exhibit long blood circulation time, good biocompatibility, and high-precision targeting, thereby solving the problem of insufficient binding and targeting capabilities of previous molecular probes in the context of venous thrombosis. Once the thrombus is specifically captured by the nanomaterials wrapped by the platelet membrane and exposed to LIFU irradiation, the cavitation effect is used to destroy the internal structure of the thrombus, increasing its sensitivity to dissolution, thereby achieving deep dissolution of the thrombus. In addition, LIFU is thrombus-specific, allowing local thrombus treatment without the need for systemic anticoagulation therapy, thereby reducing long-term side effects and drug treatment.

[0112] The microbubbles generated in this process enhance ultrasound imaging signals, promote visual thrombolysis, ensure precise positioning, control drug release, improve the damaged vascular microenvironment, repair damaged blood vessels, and reduce the risk of thrombosis recurrence. This provides a promising insight pathway for further exploring the integration of hormonal changes, immune responses, and thrombosis-mediated signaling in pregnant women.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing platelet membrane-encapsulated curcumin nanomaterials, characterized in that: The following steps are involved: Step 1, adding lactic acid-glycolic acid powder into dichloromethane and dissolving by ultrasonication; Step 2, adding perfluoropentane and curcumin powder to the mixed solution obtained in step 1, respectively, and mixing them thoroughly by ultrasonication; Step 3, adding polyvinyl alcohol and isopropanol to the mixed solution obtained in step 2, and mixing by ultrasonication; Step 4: Under ice bath conditions, the mixed solution obtained in step 3 is subjected to magnetic stirring, and then centrifuged in a low-temperature centrifuge to obtain the basic nanomaterial Cur-PFP@PC; Step 5: Mix the basic nanomaterial Cur-PFP@PC obtained in step 4 with the platelet membrane, and perform ultrasonic treatment to obtain the platelet membrane-coated nanomaterial P-Cur-PFP@PC.

2. The method for preparing the platelet membrane-encapsulated curcumin nanomaterial according to claim 1, characterized in that: In the mixed solution obtained in step 1, the mass concentration of polylactic acid-polyglycolate is 20-30 mg / mL.

3. The method for preparing the platelet membrane-encapsulated curcumin nanomaterial according to claim 2, characterized in that: In the step 2, 180 to 300 microliters of perfluoropentane are added for every 10 mg of curcumin powder.

4. The method for preparing the platelet membrane-encapsulated curcumin nanomaterial according to claim 3, characterized in that: In the step 3, the mass concentration of polyvinyl alcohol is 3-5%; the mass concentration of isopropanol is 1-3%.

5. The method for preparing the platelet membrane-encapsulated curcumin nanomaterial according to claim 4, characterized in that: The added volume ratio of polyvinyl alcohol and isopropyl alcohol is 0.5-1:

1.

6. The method for preparing the platelet membrane-encapsulated curcumin nanomaterial according to claim 1, characterized in that: In step 5, the platelet membrane is extracted from platelet-rich plasma by freeze-thaw method.

7. The method for preparing the platelet membrane-encapsulated curcumin nanomaterial according to claim 6, characterized in that: In the step 5, 5 to 15 ml of the basic nanomaterial Cur-PFP@PC is added to 1 mL of the platelet membrane solution.

8. A material product prepared by the method for preparing platelet membrane-encapsulated curcumin nanomaterial according to any one of claims 1 to 7.

9. Use of the material product as claimed in claim 8 in the preparation of drugs for preventing and / or treating venous thrombosis.

10. The use according to claim 9, after administration of the medicine, low intensity focused ultrasound technology is used to guide the treatment area.

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