Novel prussian blue nanodrug for rapid photo-thermal thrombolysis and long-term inhibition of thrombus recurrence

Abstract

This invention discloses a novel Prussian blue nanomedicine for rapid photothermal thrombolysis and long-term inhibition of thrombus recurrence. Specifically, the nanomedicine uses Prussian blue nanoparticles as a drug carrier, rutin as an antithrombotic drug that inhibits platelet aggregation, and is encapsulated with a phase change material nanomedicine prepared from a mixture of hexadecyl alcohol and oleic acid, with the surface modified by RGD peptide. Prussian blue, as an inorganic nanomaterial, inherently possesses catalase-like (CAT) activity and exhibits good light absorption in the near-infrared region. Relying on the ability of RGD peptide to specifically bind and activate platelets, the nanomedicine is first targeted and delivered to the thrombus site. Under near-infrared light irradiation, the Prussian blue nanoparticles undergo photothermal conversion, generating heat to dissolve the thrombus. Simultaneously, the thermally induced phase change triggers drug release, and the drug, in conjunction with the antioxidant effect of Prussian blue, provides a long-term inhibitory effect against thrombus recurrence. Therefore, the Prussian blue nanomedicine provided by this invention not only enables rapid photothermal thrombolysis but also provides long-term inhibition of thrombus recurrence, offering a new approach to antithrombotic treatment strategies.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to a novel Prussian blue nanomedicine for rapid photothermal thrombolysis and long-term inhibition of thrombus recurrence. Background Technology

[0002] Thrombosis refers to a small blood clot composed of insoluble fibrin, activated platelets, leukocytes, or erythrocytes. Pathological thromboembolism can lead to ischemic tissue damage and organ failure, seriously endangering human life and health. Currently, thrombolytic therapy is the preferred treatment for pathological thromboembolism, clinically mainly using fibrinolytic drugs such as urokinase (uPA) to dissolve the formed thrombus. However, due to the short circulating half-life and limited thrombus specificity of fibrinolytic drugs, the high doses required to achieve effective thrombolysis can easily lead to systemic bleeding as a side effect (T. Mei, et al. Biomaterials, 2019, 1878-5905). In addition, the insufficient thrombus permeability of fibrinolytic drugs may cause thrombus recurrence due to incomplete thrombus dissolution. These problems seriously hinder the clinical treatment of thrombotic diseases. Given the high incidence and mortality of thrombotic diseases, the rational design of new treatment strategies with high efficiency and safety has become a high priority in antithrombotic therapy.

[0003] With the rapid development of bionanotechnology, nanomedicine delivery systems have shown significant advantages in antithrombotic drug delivery, including improving the physicochemical properties of antithrombotic drugs, prolonging systemic circulation time, and reducing the risk of off-target bleeding. Furthermore, some thrombus-penetrating drug delivery strategies have attracted attention, with photothermal therapy (PTT) showing broad application prospects in thrombosis treatment. Prussian blue is a highly biosafety-compliant inorganic nanomaterial with easily tunable structure. Moreover, Prussian blue possesses strong catalase (CAT) activity, capable of catalyzing the conversion of excess hydrogen peroxide (H₂O₂) into oxygen (O₂). Additionally, Prussian blue can absorb near-infrared light in the near-infrared region and convert light energy into heat energy. These properties endow Prussian blue with application potential in the field of nanomedicine delivery. Patent ZL 202110750731.8 discloses a nano-drug delivery system, its preparation method, and its application. This system uses Prussian blue nanoparticles as drug carriers. The porous structure, high specific surface area, and pore size of Prussian blue endow the drug with a high loading capacity, effectively improving drug utilization and thus enhancing treatment efficiency. Patent ZL202210101291.8 discloses a Prussian blue nanodroplet. The Prussian blue nanodroplets prepared by this method not only achieve photothermal thrombolysis but also scavenge excess reactive oxygen species (ROS) to alleviate oxidative stress, effectively relieving the inflammatory microenvironment of thrombosis. However, these drug- and non-drug-based thrombolysis strategies only address the thromboembolism itself, neglecting the risk of thrombus recurrence. For example, incomplete thrombolysis in photothermal therapy may lead to thrombus recurrence.

[0004] To address the problem of thrombus recurrence caused by incomplete thrombolysis, combination therapy has shown significant advantages in thrombosis treatment. Studies have found that protein disulfide isomerase (PDI), expressed on the surface of platelets and endothelial cells, plays a crucial role in promoting thrombus formation. Rutin exhibits potent antiplatelet activity by inhibiting PDI-induced platelet aggregation (Lu Wang, et al. Redox Biology, 2022, 2213-2317). Based on this, this invention provides a Prussian blue nanomedicine for rapid photothermal thrombolysis and long-term inhibition of thrombus recurrence, along with its preparation method. This nanomedicine uses Prussian blue nanoparticles as a drug carrier and photothermal agent. The loaded rutin is released in a controlled manner through a thermally induced phase transition, synergistically inhibiting thrombus recurrence with the inherent antioxidant properties of Prussian blue. Compared to traditional thrombosis treatment strategies, the Prussian blue nanomedicine provided by this invention not only rapidly clears existing thrombi but also provides long-term inhibition of thrombus recurrence, offering a new approach to an integrated thrombolysis and prevention treatment strategy. Summary of the Invention

[0005] The purpose of this invention is to provide a novel Prussian blue nanomedicine for rapid photothermal thrombolysis and long-term inhibition of thrombus recurrence. Specifically, a Prussian blue nanomedicine is provided, consisting of Prussian blue loaded with the antiplatelet drug Rutin, and modified with a phase change material of hexadecyl alcohol and oleic acid, and RGD peptide. First, this Prussian blue nanomedicine exhibits excellent photothermal thrombolytic properties, rapidly clearing existing thrombi. Second, the nanomedicine possesses thermoinduced phase change properties, enabling controlled release of Rutin and effectively inhibiting platelet aggregation. Furthermore, Prussian blue alleviates oxidative stress by scavenging excess H2O2, synergistically inhibiting thrombus recurrence with the drug. In summary, the technical solution provided by this invention not only promises to achieve integrated thrombolysis and prevention in thrombus treatment but also expands the application potential of Prussian blue nanoparticles in the treatment of cardiovascular diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A novel Prussian blue nanomedicine for rapid photothermal thrombolysis and long-term inhibition of thrombus recurrence is characterized by comprising the following steps:

[0008] (1) Dissolve polyvinylpyrrolidone (PVP) powder in hydrochloric acid (HCl) solution, add potassium ferricyanide (K3[Fe(CN)6]) powder, mix and stir for 0.5-2.0 h until the solution turns yellow to obtain solution S1;

[0009] (2) Heat solution S1 until it turns dark blue to obtain solution S2; then wash and centrifuge with deionized water and anhydrous ethanol to remove the supernatant and obtain the precipitate, freeze-dry to obtain solid P1;

[0010] (3) Mix solid P1 and PVP in a mass ratio of (1-3):(2-10), dissolve in HCl, and stir for 0.5-5.0 h to obtain solution S3; then transfer it to a hydrothermal reactor and react to obtain solution S4;

[0011] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0012] (5) Dissolve rutin powder in anhydrous ethanol to obtain solution S5;

[0013] (6) Disperse solid P2 in solution S5, sonicate for 5-30 min, stir at room temperature in the dark for 12-36 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0014] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of (1-5):1, dissolve in anhydrous ethanol, mix ultrasonically for 30-90 min, remove the solvent by rotary evaporation, and dry to obtain solid P4;

[0015] (8) Mix solid P3 and solid P4 in a mass ratio of 1:(1~7), dissolve in ethanol, heat and stir, put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0016] (9) Mix 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and arginine-glycine-aspartic acid (RGD) in a mass ratio of (1-3):(1-5):(1-3), dissolve in phosphate buffer solution (PBS), and stir at room temperature for 3-12 h to obtain solution S7;

[0017] (10) Dissolve solid P5 in deionized water, add anhydrous ethanol and 3-aminopropyltriethoxysilane (APTES) in sequence, stir for 10-24 h to obtain solution S8;

[0018] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0019] (12) Mix solid P6 and solution S7 and stir for 1 to 5 hours. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0020] Preferably, in step (1), the concentration of PVP is 60.0-90.0 mg / mL, the concentration of K3[Fe(CN)6] is 2.0-6.0 mg / mL, the concentration of HCl is 0.01-0.1 mol / L, and the stirring time is 0.5-2.0 h.

[0021] Preferably, in step (2), the reaction temperature is 60-100℃ and the reaction time is 18-30h.

[0022] Preferably, in step (3), the mixing mass ratio of solid P1 to PVP is 1:(2-10), the HCl concentration is 0.1-1 mol / L, the stirring time is 0.5-5.0 h, the reaction temperature is 100-160 °C, and the reaction time is 2.5-5.0 h.

[0023] Preferably, in step (5), the concentration of Rutin is 0.25 to 4.0 mg / mL.

[0024] Preferably, in step (6), the concentration of solid P2 in the solution is 0.25–4.0 mg / mL, and the stirring time is 12–36 h.

[0025] Preferably, in step (7), the mass ratio of hexadecyl alcohol to oleic acid is (1-4):1, and the ultrasonic time is 30-60 min.

[0026] Preferably, in step (8), the mixing mass ratio of solid P3 to solid P4 is 1:(1~7), the heating temperature is 35~50℃, and the stirring time is 10~60min.

[0027] Preferably, in step (9), the mass ratio of EDC, NHS and RGD peptides is 1:1:(1-3), and the stirring time is 3-12 h;

[0028] Preferably, in step (10), the concentration of solid P5 in the solution is 0.5-2.0 mg / mL, the amount of anhydrous ethanol is calculated as 15.0-25.0 mL for every 40 mL of solution S8 prepared, the amount of APTES is calculated as a volume ratio of 1:(500-2000) to ethanol, and the stirring time is 10-24 h.

[0029] Preferably, in step (12), the concentration of solid P6 in the mixed solution is 0.5 to 3.0 mg / mL, and the stirring time is 1 to 5 h.

[0030] Compared with the prior art, the significant advantages of the present invention are:

[0031] The Prussian blue nanomedicine synthesized in this invention avoids the bleeding side effects of conventional fibrinolytic drugs. Utilizing photothermal therapy for thrombolysis, it can rapidly dissolve existing thrombi. Secondly, the thermoinduced phase transition allows for controlled drug release; the drug released after the initial thrombolytic treatment can reach deeper into the thrombus, greatly improving drug utilization and effectively inhibiting platelet aggregation. More significantly, the inherent antioxidant properties of Prussian blue effectively reduce the possibility of thrombus recurrence caused by oxidative stress. Therefore, the Prussian blue nanomedicine provided by this invention not only enables rapid photothermal thrombolysis but also provides long-term inhibition of thrombus recurrence. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the preparation process of the Prussian blue nanomedicine (T-Rutin / HMPB@PCM) of the present invention. Wherein, HMPB is Prussian blue nanoparticles, Rutin / HMPB is a Rutin-loaded nanomedicine, and Rutin / HMPB@PCM is a nanomedicine encapsulated with a phase change material.

[0033] Figure 2Morphology and particle size distribution of HMPB and T-Rutin / HMPB@PCM prepared in Example 3. (A) Transmission electron microscope image of HMPB; (B) Transmission electron microscope image of T-Rutin / HMPB@PCM; (C) Particle size distribution of HMPB; (D) Particle size distribution of T-Rutin / HMPB@PCM.

[0034] Figure 3 Photothermal performance verification of T-Rutin / HMPB@PCM prepared in Example 3. (A) Temperature rise curves of T-Rutin / HMPB@PCM after irradiation with 808nm near-infrared light at different powers for 10 min. (B) Temperature rise curves of T-Rutin / HMPB@PCM at 1.0 W / cm². 2 Photothermal conversion efficiency under high-power near-infrared light irradiation. Photothermal stability of (C)T-Rutin / HMPB@PCM under three switching cycles.

[0035] Figure 4 Transmission electron microscope images of T-Rutin / HMPB@PCM prepared in Example 3 before and after near-infrared light irradiation.

[0036] Figure 5 Cytotoxicity verification of T-Rutin / HMPB@PCM prepared in Example 3. (A) Cell viability after co-culturing with different concentrations of T-Rutin / HMPB@PCM. (B) Live / dead staining images of cells co-cultured with T-Rutin / HMPB@PCM on days 1, 3, and 7.

[0037] Figure 6 This was to verify the intracellular antioxidant properties of T-Rutin / HMPB@PCM prepared in Example 3.

[0038] Figure 7 This study validated the in vitro and in vivo thrombolytic performance of T-Rutin / HMPB@PCM prepared in Example 3. Detailed Implementation

[0039] A novel Prussian blue nanomedicine for rapid photothermal thrombolysis and long-term inhibition of thrombus recurrence is characterized by comprising the following steps:

[0040] (1) Dissolve PVP powder in HCl solution, add K3[Fe(CN)6] powder, mix and stir for 0.5-2.0 h until the solution turns yellow to obtain solution S1;

[0041] (2) Heat solution S1 until it turns dark blue to obtain solution S2; then wash and centrifuge with deionized water and anhydrous ethanol to remove the supernatant and obtain the precipitate, freeze-dry to obtain solid P1;

[0042] (3) Mix solid P1 and PVP in a mass ratio of (1-3):(2-10), dissolve in HCl, and stir for 0.5-5.0 h to obtain solution S3; then transfer it to a hydrothermal reactor and react to obtain solution S4;

[0043] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0044] (5) Dissolve Rutin powder in anhydrous ethanol to obtain solution S5;

[0045] (6) Disperse solid P2 in solution S5, sonicate for 5-30 min, stir at room temperature in the dark for 12-36 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0046] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of (1-5):1, dissolve in anhydrous ethanol, mix ultrasonically for 30-90 min, remove the solvent by rotary evaporation, and dry to obtain solid P4;

[0047] (8) Mix solid P3 and solid P4 in a mass ratio of 1:(1~7), dissolve in ethanol, heat and stir, put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0048] (9) EDC, NHS and RGD are mixed in a mass ratio of (1-3):(1-5):(1-3), then dissolved in PBS and stirred at room temperature for 3-12 hours to obtain solution S7.

[0049] (10) Dissolve solid P5 in deionized water, add anhydrous ethanol and APTES in sequence, stir for 10-24 h to obtain solution S8;

[0050] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0051] (12) Mix solid P6 and solution S7 and stir for 1 to 5 hours. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0052] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0053] Example 1

[0054] (1) Dissolve PVP powder (60 mg / mL) in HCl solution (0.01 mol / L, 40 mL), add K3[Fe(CN)6] powder (2.5 mg / mL), mix and stir for 0.5 h until the solution turns yellow to obtain solution S1;

[0055] (2) Heat solution S1 until it turns dark blue (80℃, 24h) to obtain solution S2; then wash with deionized water and anhydrous ethanol, centrifuge to remove the supernatant, obtain the precipitate, freeze dry to obtain solid P1;

[0056] (3) Mix solid P1 and PVP at a mass ratio of 1:5, dissolve in HCl, and stir for 3.0 h to obtain solution S3; then transfer it to a hydrothermal reactor (140℃, 3.0 h) and react to obtain solution S4;

[0057] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0058] (5) Dissolve Rutin powder (0.5 mg / mL) in anhydrous ethanol to obtain solution S5;

[0059] (6) Disperse solid P2 (2.5 mg / mL) in solution S5, sonicate for 30 min, stir at room temperature in the dark for 20 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0060] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of 4:1, dissolve in anhydrous ethanol, sonicate for 60 min, remove solvent by rotary evaporation, and dry to obtain solid P4.

[0061] (8) Mix solid P3 and solid P4 at a mass ratio of 1:7, dissolve in ethanol, heat and stir (40℃, 30min), put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0062] (9) EDC, NHS and RGD were mixed in a mass ratio of 1:1:3, dissolved in PBS, and stirred at room temperature for 12 hours to obtain solution S7.

[0063] (10) Dissolve solid P5 (0.5 mg / mL) in deionized water (20 mL), add anhydrous ethanol (20 mL) and APTES (40 μL) in sequence, mix and stir for 24 h to obtain solution S8;

[0064] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0065] (12) Mix solid P6 (0.5 mg / mL) and solution S7 and stir for 1 h. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0066] Example 2

[0067] (1) Dissolve PVP powder (60 mg / mL) in HCl solution (0.01 mol / L, 40 mL), add K3[Fe(CN)6] powder (2.5 mg / mL), mix and stir for 0.5 h until the solution turns yellow to obtain solution S1;

[0068] (2) Heat solution S1 until it turns dark blue (80℃, 24h) to obtain solution S2; then wash with deionized water and anhydrous ethanol, centrifuge to remove the supernatant, obtain the precipitate, freeze dry to obtain solid P1;

[0069] (3) Mix solid P1 and PVP at a mass ratio of 1:5, dissolve in HCl, and stir for 3.0 h to obtain solution S3; then transfer it to a hydrothermal reactor (140℃, 3.0 h) and react to obtain solution S4;

[0070] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0071] (5) Dissolve Rutin powder (0.5 mg / mL) in anhydrous ethanol to obtain solution S5;

[0072] (6) Disperse solid P2 (1.25 mg / mL) in solution S5, sonicate for 30 min, stir at room temperature in the dark for 20 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0073] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of 4:1, dissolve in anhydrous ethanol, sonicate for 60 min, remove solvent by rotary evaporation, and dry to obtain solid P4.

[0074] (8) Mix solid P3 and solid P4 at a mass ratio of 1:7, dissolve in ethanol, heat and stir (40℃, 30min), put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0075] (9) EDC, NHS and RGD were mixed in a mass ratio of 1:1:3, dissolved in PBS, and stirred at room temperature for 12 hours to obtain solution S7.

[0076] (10) Dissolve solid P5 (0.5 mg / mL) in deionized water (20 mL), add anhydrous ethanol (20 mL) and APTES (40 μL) in sequence, mix and stir for 24 h to obtain solution S8;

[0077] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0078] (12) Mix solid P6 (0.5 mg / mL) and solution S7 and stir for 1 h. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0079] Example 3

[0080] (1) Dissolve PVP powder (60 mg / mL) in HCl solution (0.01 mol / L, 40 mL), add K3[Fe(CN)6] powder (2.5 mg / mL), mix and stir for 0.5 h until the solution turns yellow to obtain solution S1;

[0081] (2) Heat solution S1 until it turns dark blue (80℃, 24h) to obtain solution S2; then wash with deionized water and anhydrous ethanol, centrifuge to remove the supernatant, obtain the precipitate, freeze dry to obtain solid P1;

[0082] (3) Mix solid P1 and PVP at a mass ratio of 1:5, dissolve in HCl, and stir for 3.0 h to obtain solution S3; then transfer it to a hydrothermal reactor (140℃, 3.0 h) and react to obtain solution S4;

[0083] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0084] (5) Dissolve Rutin powder (0.5 mg / mL) in anhydrous ethanol to obtain solution S5;

[0085] (6) Disperse solid P2 (1.0 mg / mL) in solution S5, sonicate for 30 min, stir at room temperature in the dark for 20 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0086] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of 4:1, dissolve in anhydrous ethanol, sonicate for 60 min, remove solvent by rotary evaporation, and dry to obtain solid P4.

[0087] (8) Mix solid P3 and solid P4 at a mass ratio of 1:7, dissolve in ethanol, heat and stir (40℃, 30min), put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0088] (9) EDC, NHS and RGD were mixed in a mass ratio of 1:1:3, dissolved in PBS, and stirred at room temperature for 12 hours to obtain solution S7.

[0089] (10) Dissolve solid P5 (0.5 mg / mL) in deionized water (20 mL), add anhydrous ethanol (20 mL) and APTES (40 μL) in sequence, mix and stir for 24 h to obtain solution S8;

[0090] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0091] (12) Mix solid P6 (0.5 mg / mL) and solution S7 and stir for 1 h. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0092] Figure 2 The images show the morphology and particle size distribution of HMPB and T-Rutin / HMPB@PCM prepared in Example 3. TEM images show that the prepared HMPB has a hollow structure and is a regular square shape. When HMPB is loaded with drugs, phase change materials, and targeting peptides, the hollow structure of T-Rutin / HMPB@PCM disappears and the edge shape becomes irregular. The particle size distribution shows that the average particle size of HMPB is 176.4 ± 35.5 nm, and the average particle size of T-Rutin / HMPB@PCM is 203.7 ± 21.6 nm. The increase in the particle size of the final product is mainly attributed to the loading of drugs and phase change materials, verifying the successful synthesis of T-Rutin / HMPB@PCM.

[0093] Figure 3 This study verifies the photothermal performance of the T-Rutin / HMPB@PCM prepared in Example 3. Figure 3 It was found that after irradiation of T-Rutin / HMPB@PCM aqueous solution (0.5 mg / mL) with near-infrared light, the solution temperature increased significantly, and the maximum temperature achievable by the T-Rutin / HMPB@PCM solution increased with increasing near-infrared light power. This photothermal rise is attributed to the Landau damping effect mediated by near-infrared light in the Prussian blue nanoparticles, which gives them excellent photothermal conversion efficiency at 1.0 W / cm². 2 Its photothermal conversion efficiency reaches 61.1% under high power irradiation. In addition, its good photostability is demonstrated by cyclically irradiating T-Rutin / HMPB@PCM with near-infrared light for three cycles.

[0094] Figure 4Transmission electron microscope images of T-Rutin / HMPB@PCM prepared in Example 3 before and after near-infrared light irradiation. Figure 4 It can be seen that liquid substances exist around the nanoparticles after near-infrared light irradiation, and the particle size increases slightly, indicating that the increase in temperature leads to solid-liquid phase transition.

[0095] Figure 5 This study validated the cytotoxicity of T-Rutin / HMPB@PCM prepared in Example 3. Cell viability analysis using the CCK8 assay showed that cells treated with different concentrations of T-Rutin / HMPB@PCM maintained high viability, all exceeding 70%, indicating good biocompatibility of T-Rutin / HMPB@PCM. Furthermore, cell live / dead staining results showed that the proliferation level of T-Rutin / HMPB@PCM-treated cells was comparable to that of untreated normal cells, further demonstrating the good biocompatibility of the synthesized nanomedicine.

[0096] Figure 6 To verify the intracellular antioxidant performance of T-Rutin / HMPB@PCM prepared in Example 3. Figure 6 It can be seen that, compared with the ROS fluorescence of cells treated with H2O2, the fluorescence level of cells treated with T-Rutin / HMPB@PCM+H2O2 was significantly reduced, and was comparable to the fluorescence level of normal cells, indicating that T-Rutin / HMPB@PCM has antioxidant properties.

[0097] Figure 7 This study validated the in vitro and in vivo thrombolytic performance of T-Rutin / HMPB@PCM prepared in Example 3. As shown in Figures A and B, the in vitro thrombolytic experiment results indicate that the thrombolytic efficiency increases with increasing near-infrared light power (1.4 W / cm²). 2 The thrombolytic efficiency reached 60%, indicating that the prepared T-Rutin / HMPB@PCM has good thrombolytic properties. Furthermore, a rat lower limb venous thrombosis model was established to verify the in vivo thrombolytic therapeutic performance of T-Rutin / HMPB@PCM. PBS and T-Rutin / HMPB@PCM were injected via the tail vein, and the blood vessels in the T-Rutin / HMPB@PCM group were irradiated with near-infrared light. Finally, the blood vessels from each group were removed, frozen, sectioned, and stained with hematoxylin and eosin (HE). As shown in Figure C, compared with the normal (Control) and PBS-treated blood vessel sections, the area of ​​the dark region in the blood vessels treated with T-Rutin / HMPB@PCM+L was significantly reduced (indicated by the white arrow), indicating a reduction in thrombus, further demonstrating the good thrombolytic properties of the prepared T-Rutin / HMPB@PCM.

[0098] Example 4

[0099] (1) Dissolve PVP powder (60 mg / mL) in HCl solution (0.01 mol / L, 40 mL), add K3[Fe(CN)6] powder (2.5 mg / mL), mix and stir for 0.5 h until the solution turns yellow to obtain solution S1;

[0100] (2) Heat solution S1 until it turns dark blue (80℃, 24h) to obtain solution S2; then wash with deionized water and anhydrous ethanol, centrifuge to remove the supernatant, obtain the precipitate, freeze dry to obtain solid P1;

[0101] (3) Mix solid P1 and PVP at a mass ratio of 1:5, dissolve in HCl, and stir for 3.0 h to obtain solution S3; then transfer it to a hydrothermal reactor (140℃, 3.0 h) and react to obtain solution S4;

[0102] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0103] (5) Dissolve Rutin powder (0.5 mg / mL) in anhydrous ethanol to obtain solution S5;

[0104] (6) Disperse solid P2 (0.63 mg / mL) in solution S5, sonicate for 30 min, stir at room temperature in the dark for 20 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0105] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of 4:1, dissolve in anhydrous ethanol, sonicate for 60 min, remove solvent by rotary evaporation, and dry to obtain solid P4.

[0106] (8) Mix solid P3 and solid P4 at a mass ratio of 1:7, dissolve in ethanol, heat and stir (40℃, 30min), put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0107] (9) EDC, NHS and RGD were mixed in a mass ratio of 1:1:3, dissolved in PBS, and stirred at room temperature for 12 hours to obtain solution S7.

[0108] (10) Dissolve solid P5 (0.5 mg / mL) in deionized water (20 mL), add anhydrous ethanol (20 mL) and APTES (40 μL) in sequence, mix and stir for 24 h to obtain solution S8;

[0109] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0110] (12) Mix solid P6 (0.5 mg / mL) and solution S7 and stir for 1 h. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0111] Example 5

[0112] (1) Dissolve PVP powder (60 mg / mL) in HCl solution (0.01 mol / L, 40 mL), add K3[Fe(CN)6] powder (2.5 mg / mL), mix and stir for 0.5 h until the solution turns yellow to obtain solution S1;

[0113] (2) Heat solution S1 until it turns dark blue (80℃, 24h) to obtain solution S2; then wash with deionized water and anhydrous ethanol, centrifuge to remove the supernatant, obtain the precipitate, freeze dry to obtain solid P1;

[0114] (3) Mix solid P1 and PVP at a mass ratio of 1:5, dissolve in HCl, and stir for 3.0 h to obtain solution S3; then transfer it to a hydrothermal reactor (140℃, 3.0 h) and react to obtain solution S4;

[0115] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0116] (5) Dissolve Rutin powder (0.5 mg / mL) in anhydrous ethanol to obtain solution S5;

[0117] (6) Disperse solid P2 (0.5 mg / mL) in solution S5, sonicate for 30 min, stir at room temperature in the dark for 20 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0118] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of 4:1, dissolve in anhydrous ethanol, sonicate for 60 min, remove solvent by rotary evaporation, and dry to obtain solid P4.

[0119] (8) Mix solid P3 and solid P4 at a mass ratio of 1:7, dissolve in ethanol, heat and stir (40℃, 30min), put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0120] (9) EDC, NHS and RGD were mixed in a mass ratio of 1:1:3, dissolved in PBS, and stirred at room temperature for 12 hours to obtain solution S7.

[0121] (10) Dissolve solid P5 (0.5 mg / mL) in deionized water (20 mL), add anhydrous ethanol (20 mL) and APTES (40 μL) in sequence, mix and stir for 24 h to obtain solution S8;

[0122] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0123] (12) Mix solid P6 (0.5 mg / mL) and solution S7 and stir for 1 h. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0124] The drug loading and in vitro thrombolytic efficiency of the Prussian blue nanomedicines provided in Examples 1-5 were compared, and the results are shown in Table 1. The results indicate that, when the concentrations of other components remain constant, the drug loading and thrombolytic efficiency of Example 3, with a drug / nanoparticle mass ratio of 0.5, were the highest, at 32.7% and 59.4%, respectively. This may be because there is an optimal ratio between the drug and nanoparticles. When the ratio is lower than this, less drug is loaded during mixing, resulting in a lower drug loading. Conversely, when the ratio is higher, the nanoparticle colloidal solution becomes unstable due to excessive drug dosage, leading to some drug not being completely encapsulated within the nanoparticles. Furthermore, the thrombolytic efficiency mainly depends on the initial photothermal therapy and subsequent antiplatelet therapy; a smaller amount of drug acting on the lesion site results in a lower thrombolytic efficiency.

[0125] Table 1. Results of drug loading and in vitro thrombolytic efficiency of Prussian blue nanomedicine.

[0126]

[0127] Comparative Example 1

[0128] (1) Dissolve PVP powder (60 mg / mL) in HCl solution (0.01 mol / L, 40 mL), add K3[Fe(CN)6] powder (4.0 mg / mL), mix and stir for 0.5 h until the solution turns yellow to obtain solution S1;

[0129] (2) Heat solution S1 until it turns dark blue (80℃, 24h) to obtain solution S2; then wash with deionized water and anhydrous ethanol, centrifuge to remove the supernatant, obtain the precipitate, freeze dry to obtain solid P1;

[0130] (3) Mix solid P1 and PVP at a mass ratio of 1:5, dissolve in HCl, and stir for 3.0 h to obtain solution S3; then transfer it to a hydrothermal reactor (140℃, 3.0 h) and react to obtain solution S4;

[0131] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0132] (5) Dissolve Rutin powder (0.5 mg / mL) in anhydrous ethanol to obtain solution S5;

[0133] (6) Disperse solid P2 (2.5 mg / mL) in solution S5, sonicate for 30 min, stir at room temperature in the dark for 20 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0134] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of 4:1, dissolve in anhydrous ethanol, sonicate for 60 min, remove solvent by rotary evaporation, and dry to obtain solid P4.

[0135] (8) Mix solid P3 and solid P4 at a mass ratio of 1:7, dissolve in ethanol, heat and stir (40℃, 30min), put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0136] (9) EDC, NHS and RGD were mixed in a mass ratio of 1:1:3, dissolved in PBS, and stirred at room temperature for 12 hours to obtain solution S7.

[0137] (10) Dissolve solid P5 (0.5 mg / mL) in deionized water (20 mL), add anhydrous ethanol (20 mL) and APTES (40 μL) in sequence, mix and stir for 24 h to obtain solution S8;

[0138] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0139] (12) Mix solid P6 (0.5 mg / mL) and solution S7 and stir for 1 h. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0140] Comparative Example 2

[0141] (1) Dissolve PVP powder (60 mg / mL) in HCl solution (0.01 mol / L, 40 mL), add K3[Fe(CN)6] powder (6.0 mg / mL), mix and stir for 0.5 h until the solution turns yellow to obtain solution S1;

[0142] (2) Heat solution S1 until it turns dark blue (80℃, 24h) to obtain solution S2; then wash with deionized water and anhydrous ethanol, centrifuge to remove the supernatant, obtain the precipitate, freeze dry to obtain solid P1;

[0143] (3) Mix solid P1 and PVP at a mass ratio of 1:5, dissolve in HCl, and stir for 3.0 h to obtain solution S3; then transfer it to a hydrothermal reactor (140℃, 3.0 h) and react to obtain solution S4;

[0144] (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2;

[0145] (5) Dissolve Rutin powder (0.5 mg / mL) in anhydrous ethanol to obtain solution S5;

[0146] (6) Disperse solid P2 (2.5 mg / mL) in solution S5, sonicate for 30 min, stir at room temperature in the dark for 20 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3;

[0147] (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of 4:1, dissolve in anhydrous ethanol, sonicate for 60 min, remove solvent by rotary evaporation, and dry to obtain solid P4.

[0148] (8) Mix solid P3 and solid P4 at a mass ratio of 1:7, dissolve in ethanol, heat and stir (40℃, 30min), put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5;

[0149] (9) EDC, NHS and RGD were mixed in a mass ratio of 1:1:3, dissolved in PBS, and stirred at room temperature for 12 hours to obtain solution S7.

[0150] (10) Dissolve solid P5 (0.5 mg / mL) in deionized water (20 mL), add anhydrous ethanol (20 mL) and APTES (40 μL) in sequence, mix and stir for 24 h to obtain solution S8;

[0151] (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6;

[0152] (12) Mix solid P6 (0.5 mg / mL) and solution S7 and stir for 1 h. Centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the Prussian blue nanomedicine.

[0153] The particle sizes of the Prussian blue nanomedicines prepared in Example 3, Comparative Example 1, and Comparative Example 2 were compared, and the results are shown in Table 2. The results indicate that, with other component concentrations remaining constant, the higher the PVP concentration, the larger the particle size of the Prussian blue nanomedicine. This is because PVP acts as a reducing agent and stabilizer; a higher PVP concentration is more conducive to the orderly self-assembly of nanoparticles. For successful drug delivery, nanoparticles must first be taken up by target cells; smaller nanoparticles are more easily taken up by epithelial cells and transported to the liver than larger nanoparticles. Therefore, the Prussian blue nanomedicine prepared in Example 3 is more conducive to metabolic circulation in vivo.

[0154] Table 2 Particle size of Prussian blue nanomedicine

[0155] Serial Number Particle size (nm) Example 3 203.7±20.5 Comparative Example 1 281.1±19.2 Comparative Example 2 317.2±35.5

[0156] The above embodiments are provided only to help understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0157] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A novel Prussian blue nanomedicine for rapid photothermal thrombolysis and long-term inhibition of thrombus recurrence, characterized in that, The nanomedicine specifically comprises Prussian blue nanoparticles as a carrier, loaded with rutin, encapsulated in a phase change material prepared by mixing hexadecyl alcohol and oleic acid, and modified with RGD peptides; the preparation method of the drug includes the following steps: (1) Dissolve polyvinylpyrrolidone powder in hydrochloric acid solution, add potassium ferricyanide powder, mix and stir for 0.5-2.0 h until the solution turns yellow to obtain solution S1; (2) Heat solution S1 until it turns dark blue to obtain solution S2; then wash and centrifuge with deionized water and anhydrous ethanol to remove the supernatant and obtain the precipitate, freeze-dry to obtain solid P1; (3) Mix solid P1 and PVP in a mass ratio of (1-3):(2-10), dissolve in HCl, and stir for 0.5-5.0 h to obtain solution S3; then transfer it to a hydrothermal reactor and react to obtain solution S4; (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2; (5) Dissolve rutin powder in anhydrous ethanol to obtain solution S5; (6) Disperse solid P2 in solution S5, sonicate for 5-30 min, stir at room temperature in the dark for 12-36 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3; (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of (1-5):1, dissolve in anhydrous ethanol, mix ultrasonically for 30-90 min, remove the solvent by rotary evaporation, and dry to obtain solid P4; (8) Mix solid P3 and solid P4 in a mass ratio of 1:(1~7), dissolve in ethanol, heat and stir, put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5; (9) Mix 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and arginine-glycine-aspartic acid RGD in a mass ratio of (1-3):(1-5):(1-3), add phosphate buffer solution to dissolve, and stir at room temperature for 3-12 hours to obtain solution S7. (10) Dissolve solid P5 in deionized water, add anhydrous ethanol and 3-aminopropyltriethoxysilane in sequence, stir for 10-24 h to obtain solution S8; (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6; (12) Mix solid P6 and solution S7 and stir for 1-5 hours, centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the product; In step (1), the concentration of polyvinylpyrrolidone in solution S1 is 60.0–90.0 mg / mL, the concentration of potassium ferricyanide powder is 2.0–6.0 mg / mL, and the concentration of hydrochloric acid is 0.01–1 mol / L.

2. A method for preparing the novel Prussian blue nanomedicine as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve polyvinylpyrrolidone powder in hydrochloric acid solution, add potassium ferricyanide powder, mix and stir for 0.5-2.0 h until the solution turns yellow to obtain solution S1; (2) Heat solution S1 until it turns dark blue to obtain solution S2; then wash and centrifuge with deionized water and anhydrous ethanol to remove the supernatant and obtain the precipitate, freeze-dry to obtain solid P1; (3) Mix solid P1 and PVP in a mass ratio of (1-3):(2-10), dissolve in HCl, and stir for 0.5-5.0 h to obtain solution S3; then transfer it to a hydrothermal reactor and react to obtain solution S4; (4) Centrifuge the solution S4 to remove the solvent, wash the precipitate with deionized water and ethanol, and freeze dry to obtain solid P2; (5) Dissolve rutin powder in anhydrous ethanol to obtain solution S5; (6) Disperse solid P2 in solution S5, sonicate for 5-30 min, stir at room temperature in the dark for 12-36 h to obtain solution S6; then remove the solvent from solution S6 by rotary evaporation, wash the precipitate with deionized water, centrifuge, and freeze dry to obtain solid P3; (7) Mix hexadecyl alcohol and oleic acid in a mass ratio of (1-5):1, dissolve in anhydrous ethanol, mix ultrasonically for 30-90 min, remove the solvent by rotary evaporation, and dry to obtain solid P4; (8) Mix solid P3 and solid P4 in a mass ratio of 1:(1~7), dissolve in ethanol, heat and stir, put in an ice bath, wash with deionized water, centrifuge, and vacuum dry to obtain solid P5; (9) Mix 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and arginine-glycine-aspartic acid RGD in a mass ratio of (1-3):(1-5):(1-3), add phosphate buffer solution to dissolve, and stir at room temperature for 3-12 hours to obtain solution S7. (10) Dissolve solid P5 in deionized water, add anhydrous ethanol and 3-aminopropyltriethoxysilane in sequence, stir for 10-24 h to obtain solution S8; (11) The solvent in solution S8 was removed by rotary evaporation, the residue was washed with deionized water and freeze-dried to obtain solid P6; (12) Mix solid P6 and solution S7 and stir for 1-5 hours, centrifuge to remove solvent, wash with PBS, and vacuum dry to obtain the product; In step (1), the concentration of polyvinylpyrrolidone in solution S1 is 60.0–90.0 mg / mL, the concentration of potassium ferricyanide powder is 2.0–6.0 mg / mL, and the concentration of hydrochloric acid is 0.01–1 mol / L.

3. The preparation method according to claim 2, characterized in that, The reaction temperature of the heating reaction described in step (2) is 60-120℃, and the reaction time is 12-36h.

4. The preparation method according to claim 2, characterized in that, The HCl concentration in step (3) is 0.1-1 mol / L, the stirring time is 0.5-5.0 h, the reaction temperature is 100-160 °C, and the reaction time is 2.5-5.0 h.

5. The preparation method according to claim 2, characterized in that, The concentration of rutin in solution S5 described in step (5) is 0.1 to 4.0 mg / mL.

6. The preparation method according to claim 2, characterized in that, The heating temperature in step (8) is 35-80℃ and the stirring time is 5-60 min.

7. The preparation method according to claim 2, characterized in that, In step (10), the concentration of solid P5 in the solution is 0.5 to 4.0 mg / mL. The amount of anhydrous ethanol used is calculated as 15.0 to 25.0 mL for every 40 mL of solution S8 prepared. The amount of 3-aminopropyltriethoxysilane mixed is calculated as a volume ratio of 1:(500 to 2000) with ethanol.

8. The preparation method according to claim 2, characterized in that, The concentration of solid P6 in the mixed solution in step (12) is 0.5 to 4.0 mg / mL, and the stirring time is 1 to 5 h.

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