Ultrasound-responsive phase change nanoparticles, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses an ultrasonic responsive phase change nanoparticle as well as a preparation method and application thereof. The ultrasonic responsive phase change nanoparticle comprises a spherical shell, the spherical shell is a thrombus targeting peptide modified chitosan oligosaccharide-eicosapentaenoic acid compound, and the spherical shell encapsulates perfluorohexane. The preparation method comprises preparation of eicosapentaenoic acid-chitosan oligosaccharide-thrombus targeting peptide and encapsulation of perfluorohexane under ultrasonic oscillation of eicosapentaenoic acid-chitosan oligosaccharide-thrombus targeting peptide, and finally the ultrasonic responsive phase change nanoparticle is obtained. The ultrasonic responsive phase change nanoparticle can realize intravenous thrombolysis and healing of venous ulcers simultaneously.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an ultrasonically responsive phase change nanoparticle, its preparation method, and its application. Background Technology

[0002] Old thrombosis causes narrowing or blockage of the venous lumen, resulting in long-term and continuous venous hypertension. At the junction of the diseased blood vessels, the congested tissue is hypoxic and metabolic products accumulate, leading to the formation of lower extremity venous ulcers, the end point of the disease.

[0003] Currently available thrombolytic drugs (such as urokinase) cannot completely dissolve old thrombi that have already formed. Even interventional surgery can only recanalize occluded segments of central veins such as the iliac and femoral veins, making it difficult to completely improve local blood supply and also unable to simultaneously promote ulcer healing. Summary of the Invention

[0004] The present invention aims to provide an ultrasound-responsive phase change nanoparticle, its preparation method and application, which can simultaneously achieve intravenous thrombolysis and venous ulcer healing.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an ultrasonically responsive phase change nanoparticle comprising a spherical shell, wherein the spherical shell is a chitosan oligosaccharide-eicosapentaenoic acid compound modified with a thrombus-targeting peptide, and perfluorohexane is encapsulated within the spherical shell.

[0006] To achieve the above objectives, the present invention also provides another technical solution: a method for preparing ultrasonically responsive phase change nanoparticles, comprising the following steps: S1, take chitosan oligosaccharide, eicosapentaenoic acid, and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate, and dissolve them separately in formamide to obtain three solutions. Mix the three solutions with triethylamine and react to obtain a reaction solution. S2, 6-(maleimide)hexanoic acid succinimide ester is dissolved in formamide and added to the reaction solution in S1 to react and obtain eicosapentaenoic acid-chitosan oligosaccharide-maleimide intermediate; then the thrombosis-targeting peptide is dissolved in formamide and coupled with the intermediate to obtain eicosapentaenoic acid-chitosan oligosaccharide-thrombosis-targeting peptide. S3, the eicosapentaenoic acid-chitosan oligosaccharide-thrombosis-targeting peptide in S2 is dissolved in deionized water, and perfluorohexane is added under ultrasonic vibration to obtain ultrasonically responsive phase change nanoparticles.

[0007] The reagents or raw materials involved in this application have the following abbreviations, and the abbreviations used in the following descriptions refer to the corresponding reagents or raw materials: Chitosan oligosaccharide, abbreviated as COS; Eicosapentaenoic acid, abbreviated as EPA; Perfluorohexane, abbreviated as PFH; Benzotriazol-1-yl-oxytripyrrolidinyl phosphate hexafluorophosphate, abbreviated as PyBop; 6-(maleimide)hexanoic acid succinimide ester, abbreviated as NHS-6-MAL; Maleimide group, abbreviated as MAL; The ultrasonically responsive phase change nanoparticles of this application are referred to as PFH@ECC.

[0008] Reports indicate that persistent inflammation and the accumulation of reactive oxygen species (ROS) are not only core characteristics of thrombosis but also key drivers in the development and progression of venous ulcers. During thrombosis, a large number of inflammatory cells are recruited, triggering an inflammatory cascade. The resulting ROS exacerbate endothelial damage, consolidating the pathological process of thrombosis. Simultaneously, due to persistent distal venous return obstruction, venous ulcers remain in a state of chronic inflammation-repair imbalance, hindering epithelial repair and making long-term healing difficult. Studies have mentioned that related bioactive substances produced after thrombosis (such as platelet-derived growth factor and extracellular vesicles) may continuously maintain local inflammatory responses and ROS production, thereby promoting the development of chronic ulcers. Therefore, the inventors hypothesize that improving the inflammatory state and ROS imbalance in the disease microenvironment after thrombolysis may be a potential strategy to promote ulcer healing.

[0009] However, in the existing technology, the aforementioned thrombolysis and the improvement of the disease microenvironment after thrombolysis can only be achieved by using drugs alone, and the effect is not good.

[0010] During their experiments, the inventors accidentally linked chitosan oligosaccharides and eicosapentaenoic acid (EPA). Chitosan oligosaccharides are hydrophilic, while EPA is hydrophobic; their combination could potentially serve as a carrier for nanoparticles. EPA possesses strong anti-inflammatory properties. If the chitosan oligosaccharide-EPA combination can indeed act as a carrier, and can also encapsulate thrombolytic drugs, the inventors hypothesize that it could potentially achieve both thrombolysis and anti-inflammation simultaneously, thus enabling thrombolysis and ulcer healing.

[0011] Therefore, the inventors conducted experiments based on this idea and successfully synthesized the expected nanoparticles, namely the ultrasound-responsive phase change nanoparticles of this application, using the method described in this application. Furthermore, experimental verification showed that these ultrasound-responsive phase change nanoparticles have thrombolytic and ulcer-healing effects.

[0012] In fact, chitosan oligosaccharide is a depolymerization product of chitosan, with a very low degree of polymerization and is highly soluble in water. Therefore, relying solely on chitosan oligosaccharide is insufficient for drug encapsulation. For example, encapsulating eicosapentaenoic acid (EPA), which has anti-inflammatory properties, is impossible. Furthermore, perfluorohexane exhibits phase transition capabilities, expanding with even slight temperature changes. Low-polymerization-degree chitosan oligosaccharide is very thin and unstable, making direct encapsulation of EPA with chitosan oligosaccharide even more challenging. However, linking EPA with chitosan oligosaccharide increases the stability of the chitosan-EPA combination in aqueous solution and also increases its thickness. These changes in properties make chitosan oligosaccharide-EPA a viable nanoparticle carrier.

[0013] Linking chitosan oligosaccharide with other anti-inflammatory unsaturated fatty acids is also difficult due to the steric hindrance in the synthesis of unsaturated fatty acids. Even replacing chitosan oligosaccharide with other hydrophobic substances, such as low-molecular-weight heparin, makes it difficult to link with unsaturated fatty acids, including eicosapentaenoic acid (EPA). Only by operating chitosan oligosaccharide and EPA according to the method of this application can the linking be achieved, and the resulting product exhibits good stability, serving not only as a nanoparticle carrier but also directly exerting anti-inflammatory effects.

[0014] In this application, a chitosan oligosaccharide-eicosapentaenoic acid compound was prepared as a carrier and modified with a thrombus-targeting peptide. Finally, perfluorohexane was encapsulated under ultrasonic vibration. The thrombus-targeting peptide guided PFH@ECC NPs to target the thrombus site. Under low-intensity focused ultrasound (LIFU), the perfluorohexane core underwent a liquid-gas phase transition, driving the nanoparticles to expand until they ruptured. The resulting mechanical force destroyed blood cells and the thrombus matrix, creating fissures and micropores, thereby initiating sonomechanical thrombolysis. Simultaneously, through the carrier-drug hybridization mechanism, chitosan oligosaccharide exerted an antioxidant effect, and eicosapentaenoic acid exerted an anti-inflammatory effect, jointly promoting ulcer healing.

[0015] Furthermore, the mass ratio of chitosan oligosaccharide to eicosapentaenoic acid is (1:1) to (1:3).

[0016] By controlling the mass ratio of the two within this range, the modification rate of eicosapentaenoic acid on chitosan oligosaccharide can be guaranteed. If the modification rate is low, that is, if there is less eicosapentaenoic acid attached to chitosan oligosaccharide, it will be difficult to use it as a carrier to encapsulate perfluorohexane.

[0017] The mass ratio of chitosan oligosaccharide to eicosapentaenoic acid is 120:202.

[0018] Furthermore, in S1, the solution of benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate is first mixed with the solution of eicosapentaenoic acid and reacted, and then the solution of chitosan oligosaccharide and triethylamine are added and mixed and reacted.

[0019] Benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate, or PyBop, is used here as a condensation activator. PyBop is first reacted with eicosapentaenoic acid (EPA) to activate the carboxyl group of EPA, promoting the formation of amide bonds and facilitating its linkage with chitosan oligosaccharides. Triethylamine acts as a basic acid-binding agent to ensure the system remains alkaline and neutralizes the acids generated during the reaction. This method allows for the maximum possible modification of chitosan oligosaccharides with eicosapentaenoic acid.

[0020] Furthermore, the reaction temperatures in S1 and S2 are both room temperature.

[0021] Furthermore, the molar ratio of benzotriazol-1-yl-oxytripyrrolidinyl phosphorus hexafluorophosphate to eicosapentaenoic acid is 2:1.

[0022] Controlling the temperature to room temperature and the molar ratio of benzotriazol-1-yl-oxytripyrrolidine phosphorus hexafluorophosphate to eicosapentaenoic acid (EPA) are both beneficial for modifying the chitosan oligosaccharide with as much EPA as possible, so as to ensure that the final chitosan oligosaccharide-EPA compound can be used as a nanocarrier.

[0023] In summary, the ultrasonically responsive phase change nanoparticles prepared in this application possess both thrombolytic and ulcer-healing-promoting effects. Based on this, this application also proposes the following technical solutions: Option 1: Application of ultrasound-responsive phase change nanoparticles in the preparation of drugs for treating venous thrombosis.

[0024] Option 2: Application of an ultrasound-responsive phase change nanoparticle in the preparation of a drug for treating venous ulcers.

[0025] Option 3: Application of an ultrasound-responsive phase change nanoparticle in the preparation of a drug for simultaneously treating venous thrombosis and venous ulcers. Attached Figure Description

[0026] Figure 1 The image shows the hydrogen nuclear magnetic resonance spectrum of the EPA-COS-CREKA nanoshell in this embodiment of the invention.

[0027] Figure 2 These are representative physical photos of ECC and PFH@ECC.

[0028] Figure 3 Transmission electron microscopy (TEM) images of ECC and PFH@ECC.

[0029] Figure 4 Plots of polydispersity index (PDI) for ECC and PFH@ECC (n = 5).

[0030] Figure 5 This is a graph showing the DLS particle size variation of PFH@ECC over one week.

[0031] Figure 6 The graph shows the cytotoxicity of different concentrations of PFH@ECC on HUVECs (n=5).

[0032] Figure 7 The graph shows the cytotoxicity of RS1 cells at different concentrations of PFH@ECC (n=5).

[0033] Figure 8 Image showing the live and dead cell staining results of HUVECs after PFH@ECC treatment (n=3).

[0034] Figure 9 Image showing the live and dead cell staining results of RS1 after PFH@ECC treatment (n=3).

[0035] Figure 10 This is an experimental diagram showing the scratch healing of HUVECs after H2O2-induced oxidative damage, treated with different concentrations of PFH@ECC.

[0036] Figure 11 This is an experimental diagram of scratch healing of RS1 after H2O2-induced oxidative damage, treated with different concentrations of PFH@ECC.

[0037] Figure 12 This is a quantitative analysis of the migration rate of HUVECs treated with different concentrations of PFH@ECC under ROS stimulation conditions (n ​​= 3).

[0038] Figure 13 This is a quantitative analysis graph of RS1 migration rate after treatment with different concentrations of PFH@ECC under ROS stimulation conditions (n=3).

[0039] Figure 14 DCFH-DA fluorescence imaging of ROS levels in HUVECs after PFH@ECC treatment.

[0040] Figure 15 DCFH-DA fluorescence imaging of ROS levels in RS1 after PFH@ECC treatment.

[0041] Figure 16 This is a quantitative analysis of ROS levels in HUVECs after treatment with different concentrations of PFH@ECC (n = 3).

[0042] Figure 17 This is a quantitative analysis chart of ROS levels in RS1 after treatment with different concentrations of PFH@ECC (n = 3).

[0043] Figure 18 The image shows the results of quantitative analysis of ROS levels in HUVECs by flow cytometry after DCFH-DA staining.

[0044] Figure 19 The image shows the results of quantitative analysis of ROS levels in RS1 cells by flow cytometry after DCFH-DA staining.

[0045] Figure 20 Representative B-mode ultrasound and contrast-enhanced ultrasound (CEUS) images of ECC and PFH@ECC at different time points after low-intensity focused ultrasound (LIFU) irradiation.

[0046] Figure 21 Optical microscope image of the PFH@ECC phase transition process.

[0047] Figure 22 Thermographic images of PBS and PFH@ECC after low-intensity focused ultrasound (LIFU) irradiation.

[0048] Figure 23 The target enrichment of PFH@EC and PFH@ECC in fibrin clots and whole blood clots (n = 3).

[0049] Figure 24 Photographs showing the dissolution of chronic thrombi over time after different treatments.

[0050] Figure 25 The thrombolysis rate of thrombi after different treatments varies over time (n = 3); the upper X-axis represents the incubation time of the control group, uPA group, and PFH@ECC group, and the lower X-axis represents the LIFU irradiation time of the ECC+LIFU, PFH@EC+LIFU, and PFH@ECC+LIFU groups.

[0051] Figure 26 Scanning electron microscopy (SEM) images showing changes in the surface morphology of thrombi after different treatments.

[0052] Figure 27 Fluorescence microscopy images of fibrinogen (red) distribution in whole blood clots after different treatments; yellow arrows indicate areas of clot dissolution.

[0053] Figure 28 Ex vivo fluorescence imaging of Cy7-PFH@EC and Cy7-PFH@ECC thrombus accumulation at the IVC (inferior vena cava) thrombus site (n = 3).

[0054] Figure 29 Quantitative fluorescence analysis of Cy7-PFH@EC and Cy7-PFH@ECC accumulation at the IVC (inferior vena cava) thrombus site (n = 3).

[0055] Figure 30 Ultrasound images of IVC recanalization after different thrombolytic therapies.

[0056] Figure 31 A quantitative analysis of IVC vessel thrombolysis rates after different thrombolytic therapies (n=3).

[0057] Figure 32 H&E staining images of thrombus sections after different thrombolytic therapies.

[0058] Figure 33 Immunofluorescence staining images of TNF-α and IL-10 in thrombus tissue after different thrombolytic therapies.

[0059] Figure 34 The endothelial integrity of inferior vena cava sections after different treatments, as shown by H&E staining.

[0060] Figure 35 Representative wound photographs at specified time points after different treatments.

[0061] Figure 36 This is a graph representing a quantitative assessment of wound healing rates after different treatments.

[0062] Figure 37 H&E staining images of wound tissue after different treatments and thrombolytic therapies.

[0063] Figure 38 Masson trichrome staining images of wound tissue after different treatments (n=3), from left to right: G1 group, G2 group, G3 group, G4 group, G5 group, and G6 group.

[0064] Figure 39 This is a quantitative analysis diagram of collagen deposition after different treatments.

[0065] Figure 40 Immunofluorescence staining images of TNF-α and IL-10 in wound tissue after different thrombolytic therapies.

[0066] Figure 41 H&E staining images of major organs (including heart, liver, spleen, lung and kidney) of SD rats after different treatments.

[0067] Figure 42 To evaluate the hematological and serum biochemical safety of SD rats after different treatments, including complete blood parameters (AE) and liver and kidney function indicators (FK) (n=5).

[0068] Figure 43 Volcano plot of differentially expressed genes (DEGs) between the PFH@ECC group, the normal group, and the control group.

[0069] Figure 44This is a GO enrichment analysis diagram of differentially expressed genes in wound tissue of the PFH@ECC group compared with the control group.

[0070] Figure 45 This is a graph showing the enrichment of differentially expressed genes in the KEGG pathway in the wound tissue of the PFH@ECC group compared to the control group. Detailed Implementation

[0071] The following detailed description provides further details on specific implementation methods.

[0072] This embodiment provides an ultrasound-responsive phase change nanoparticle, comprising a spherical shell, wherein the spherical shell is a chitosan oligosaccharide-eicosapentaenoic acid compound modified with a thrombus-targeting peptide, and perfluorohexane is encapsulated within the spherical shell. The mass ratio of chitosan oligosaccharide to eicosapentaenoic acid ranges from (1:1) to (1:3), and in this embodiment, it is specifically 120:202.

[0073] The preparation method of the above-mentioned ultrasonically responsive phase change nanoparticles includes the following steps in sequence: S1, weigh 120 mg of chitosan oligosaccharide and dissolve it in 2 mL of formamide to obtain a chitosan oligosaccharide solution; weigh 202 mg of eicosapentaenoic acid, i.e., 0.67 mmol (1.0 eq.) of eicosapentaenoic acid, and dissolve it in 0.5 mL of formamide to obtain an eicosapentaenoic acid solution; weigh 1.34 mmol (2.0 eq.) of benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate, i.e., PyBop, and dissolve it in 0.5 mL of formamide to obtain a PyBop solution.

[0074] First, eicosapentaenoic acid solution and PyBop solution were mixed and reacted at room temperature (25°C) for 4 h; then chitosan oligosaccharide solution and 2.01 mmol (3.0 eq.) triethylamine were added and mixed and reacted at room temperature for 6 h to obtain the first reaction solution.

[0075] In reaction S2, 0.67 mmol (1.0 eq.) of 6-(maleimide)hexanoic acid succinimide ester, i.e., 0.67 mmol (1.0 eq.) NHS-6-MAL, was dissolved in 0.2 mL of formamide to obtain an NHS-6-MAL solution. This NHS-6-MAL solution was then added to the first reaction solution in reaction S1, and the mixture was reacted at room temperature for 2 h to obtain the second reaction solution. The second reaction solution was added to 40 mL of acetone for precipitation, and PyBop was removed by washing three times with acetone to obtain eicosapentaenoic acid-chitosan oligosaccharide-maleimide.

[0076] Thrombosis-targeting peptides were dissolved in formamide. In this example, 0.603 mmol (0.9 eq.) of CREKA peptide was dissolved in 0.2 mL of formamide to obtain a CREKA peptide solution. The CREKA peptide solution was added to the eicosapentaenoic acid-chitosan oligosaccharide-MAL mixture described above and reacted at room temperature for 3 h to obtain the third reaction solution. The third reaction solution was transferred to 40 mL of icy diethyl ether for precipitation to obtain eicosapentaenoic acid-chitosan oligosaccharide-CREKA peptide, i.e., EPA-COS-CREKA.

[0077] S3: Weigh 10 mg of EPA-COS-CREKA from S2, dissolve it in 4 mL of deionized water, place it in an ice-water bath, and sonicate it for 6 min using an ultrasonic oscillator (Sonics & Material Inc., USA) at 50 W. While sonicating, add 200 μL of perfluorohexane. Finally, centrifuge at 11000 rpm for 10 min to obtain the finished ultrasonically responsive phase change nanoparticles, namely PFH@ECC.

[0078] The resulting PFH@ECC needs to be stored at a low temperature of 4°C for a long period of time before further use.

[0079] The following experiment demonstrates the successful preparation and effectiveness of the ultrasonically responsive phase change nanoparticles in this embodiment.

[0080] PFH, fibrinogen, and thrombin used in this example were purchased from Sigma Aldrich (St. Louis, USA). CREKA and Cy7 were purchased from MedChemexpress (New Jersey, USA). Chitosan oligosaccharide and docosapentaenoic acid were purchased from Xi'an Ruixi Biotechnology Co., Ltd. (Xi'an, China). Urokinase, reactive oxygen species assay kit, and Beyo3D™ calcein / PI cell viability and cytotoxicity assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China).

[0081] Experiment 1: Successful preparation and characterization of PFH@ECC 1. The EPA-COS-CREKA proton NMR spectrum obtained in S2 is as follows: Figure 1 As shown, the characteristic signals of EPA and CREKA after being connected to the COS backbone are clearly visible, indicating that EPA-COS-CREKA was successfully synthesized.

[0082] 2. As a comparison, a control group ECC was also prepared. The preparation method of the ECC was generally the same as that of PFH@ECC, except that in S3, no additional 200uL of perfluorohexane was added during the ultrasonic treatment. Figure 2The comparison of the two images after ultrasound shows that the color of the PFH@ECC group has turned milky white, indicating that PFH has been successfully encapsulated in the ECC nanoshell.

[0083] 3. Transmission electron microscopy (TEM) images of PFH@ECC and ECC, such as Figure 3 As shown, the two images at the top represent ECC, and the two images at the bottom represent PFH@ECC. From Figure 3 It can be seen that the diameter of PFH@ECC is larger than that of ECC without PFH, but both formulations exhibit a uniform and stable spherical morphology. Dynamic light scattering (DLS) further measured the average particle sizes of ECC and PFH@ECC to be 128.8 ± 25.93 nm and 257.4 ± 18.43 nm, respectively, which is consistent with the TEM results.

[0084] 4. The results of the polydispersity index determination are shown in [the table below]. Figure 4 As can be seen, PFH@ECC exhibits better dispersion than ECC.

[0085] 5. After incubating PFH@ECC in pure water or PBS for one week, the average particle size of PFH@ECC is as follows: Figure 5 As shown, the average particle size increase is less than 20%, demonstrating the good colloidal stability of this nanoplatform.

[0086] Experiment 2: Cellular protective effects of PFH@ECC against oxidative stress and inflammation Human umbilical vein endothelial cells (HUVECs) are an important component of the blood vessel wall, while rat skin fibroblasts (RS1) are present throughout the entire skin wound healing process, and their normal function directly determines the quality, speed, and aesthetics of healing.

[0087] 1. In this experiment, HUVECs and RS1 cells in the logarithmic growth phase were seeded in 96-well plates and cultured for 24 hours. Subsequently, they were co-incubated with different concentrations of PFH@ECC for 24 hours. The in vitro biosafety of PFH@ECC was assessed using the Cell Counting Kit-8 (CCK-8). The results are as follows: Figure 6 and Figure 7 As shown in the figure, after 24 hours of co-incubation with different concentrations of PFH@ECC, both HUVECs and RS1 cells maintained high viability. OD was measured using a microplate reader. 450 Cell viability is calculated using the following formula: Cell viability (%) = (Average OD of experimental groups) 450 -Mean OD of the blank control group 450 ) / (mean OD of the control group 450 -Mean OD of the blank control group 450()×100%. Among them, healthy HUVECs and RS1 showed the highest viability after co-incubation with 1 mg / mL PFH@ECC, reaching 112% and 104% respectively, demonstrating a certain ability to promote cell proliferation and migration.

[0088] In addition, the cells co-incubated with PFH@ECC (1 mg / mL) were subjected to live / dead cell staining, and the staining results of HUVECs and RS1 cells were observed under a microscope, as shown below. Figure 8 and Figure 9 As shown, a large number of surviving adherent cells can still be seen, and there was no large-scale cell death.

[0089] The above results confirm that PFH@ECC has excellent biocompatibility.

[0090] 2. The protective effect of PFH@ECC on cells under oxidative stress was evaluated by scratch assay.

[0091] HUVECs and RS1 cells in logarithmic growth phase were seeded in 6-well plates and cultured for 24 hours to form a monolayer. Wounds were created on each well of the monolayer using a 200 μL pipette tip, and the wound areas were marked. After washing with PBS to remove detached cells, HUVECs and RS1 cells were exposed to H2O2 to induce oxidative stress, followed by treatment with different concentrations of PFH@ECC. The progress of wound closure was monitored at 0, 24, and 48 hours to determine whether the nanoparticles could restore the migration and proliferation functions of damaged cells. The results are shown below. Figure 10 and 11 As shown in the figure, PFH@ECC treatment significantly promoted wound closure in both cell types. By 48 hours, cell migration rate was significantly increased, as indicated by... Figure 12 and Figure 13 As shown, HUVECs reached 65.72 ± 3.41% and RS1 reached 95.37 ± 1.74%, effectively reversing the inhibitory effect of oxidative stress.

[0092] To further verify the antioxidant capacity of PFH@ECC, HUVECs and RS1 cells were seeded in confocal culture dishes and cultured for 24 hours. After incubation, cells were treated individually with H2O2 (300 μM) or co-incubated with different concentrations of PFH@ECC for 4 hours, with normal cells serving as a blank control. Subsequently, cells were incubated with DCFH-DA (10 μM) for 30 minutes, and intracellular fluorescence intensity was observed using a laser scanning confocal microscope to quantitatively detect intracellular reactive oxygen species (ROS) levels. The results are shown in the figure below. Figure 14 , 15As shown, PFH@ECC treatment significantly reduced intracellular ROS production, and the fluorescence intensity was negatively correlated with the PFH@ECC concentration. Quantitative analysis of fluorescence intensity further supported this result, as shown in the figures below. Figure 16 , 17 As shown. Its potential mechanism may be that the amino and hydroxyl groups in chitosan oligosaccharides can neutralize free radicals and effectively block the propagation of the oxidation chain reaction.

[0093] Furthermore, this experiment used flow cytometry to analyze intracellular ROS expression, and the results are as follows: Figure 18 , 19 As shown, compared with the control group, the expression of ROS in cells was significantly reduced after PFH@ECC treatment, which is consistent with the fluorescence imaging results.

[0094] The above results indicate that PFH@ECC still possesses strong antioxidant activity in the cellular environment, effectively maintaining redox homeostasis and protecting cells from oxidative damage.

[0095] Experiment 3: Thrombolytic effect of PFH@ECC 1. Phase transition experiment of PFH@ECC under low-intensity focused ultrasound stimulation To investigate the phase transition effect of PFH@ECC under low-intensity focused ultrasound stimulation, a photoacoustic imaging system (VIVO2100; FUJIFILM VisualSonics, Canada) and an optical imaging system (Leica Mateo FL RUO, Germany) were used for detection. A 3% agarose gel was prepared to simulate tissue, and ECC and PFH@ECC were incorporated into it, respectively. The mixture was then irradiated with low-intensity focused ultrasound (3 W cm⁻²) for 10 minutes. B-mode ultrasound and contrast-enhanced ultrasound (CEUS) images were acquired at different time points (0, 2, 4, 6, 8, and 10 minutes). The results are shown below. Figure 10 As shown, before LIFU irradiation, PFH@ECC exhibited weak echo signals in both B-mode ultrasound and contrast-enhanced ultrasound (CEUS) imaging. After LIFU irradiation, the echo intensity of the PFH@ECC group gradually increased, reaching a peak at 6 minutes, and then gradually decreased. This may be due to the rupture of the nanodroplets mediated by phase transition caused by prolonged ultrasound treatment. In contrast, the ECC group did not show significant signal changes.

[0096] To more intuitively observe the phase transition process of nanoparticles, the morphological changes of PFH@ECC during low-intensity focused ultrasound irradiation were also monitored using optical microscopy. The results are as follows: Figure 21 As shown, it can be seen that with the passage of ultrasonic irradiation time, PFH@ECC expands significantly from the nanoscale to the microscale, with a diameter approaching 20 μm.

[0097] To investigate whether ultrasound irradiation causes a significant temperature increase, PFH@ECC (2.5 mg mL⁻¹) was placed in test tubes and subjected to either low-intensity focused irradiation for 6 minutes or no treatment. Temperature was measured at different time points (0, 1, 2, 3, 4, 5, and 6 minutes) using an infrared thermometer, and quantitative analysis was performed using the thermal imaging software AnalyzIR. The results are as follows: Figure 22 As shown, during the entire 6-minute ultrasonic irradiation process, the temperature of the PFH@ECC suspension showed only a negligible increase, remaining well below 36°C, which is the reported phase transition threshold for PFH. These results rule out spontaneous phase transition breakup caused by ultrasonic heating, indicating that the observed phase transition was primarily driven by acoustic effects triggered by LIFU.

[0098] 2. In vitro ultrasound thrombolysis experiment of PFH@ECC on chronic thrombosis ①In vitro targeting experiments of PFH@ECC Fibrinogen (10 mg mL⁻¹) and thrombin (10 units mL⁻¹) were thoroughly mixed and incubated at 37 °C until the solution completely solidified to form a stable gel, which was then used for subsequent experiments. Similarly, fresh whole blood from SD rats was mixed with an appropriate amount of calcium chloride solution (10 mM) and incubated at 37 °C to promote the formation and maturation of blood clots, thereby generating chronic thrombosis. The resulting fibrin gels and blood clots were randomly divided into two groups (n = 3 per group): the Cy7-PFH@EC group (2.5 mg mL⁻¹) and the Cy7-PFH@ECC group (2.5 mg mL⁻¹). In the Cy7-PFH@EC group, Cy7 was coupled to the shell of the control group PFH@EC (without CREKA peptide); in the Cy7-PFH@ECC group, Cy7 was coupled to the shell of PFH@ECC. Each group was co-incubated with the corresponding formulation at 37 °C. Samples were washed with physiological saline at specified time points (0, 15, 30, and 60 minutes) and analyzed using a multimodal in vivo animal imaging system (AniView100 Pro, Guangzhou Bolaite Biotechnology Co., Ltd., China). Results are as follows: Figure 23 As shown, after incubation for 60 minutes with fibrin clots or intact thrombus clots under light-protected conditions, the Cy7-PFH@EC group exhibited only weak fluorescence signals in both models. In contrast, the Cy7-PFH@ECC group showed significantly enhanced and specific fluorescence signals in fibrin clots, while the signal in intact thrombus clots was relatively weak. This difference suggests that the enhanced enrichment is mediated by CREKA, which preferentially targets the fibrin network in the complex thrombus microenvironment, rather than other cellular components.

[0099] ②In vitro thrombolysis experiment of PFH@ECC Blood from SD rats was used to prepare cylindrical thrombi 1 cm long, which were then incubated at 37°C for 72 hours to form chronic thrombi for subsequent thrombolysis studies. The thrombi were divided into six groups and treated differently: G1: PBS; G2: urokinase (uPA); G3: PFH@ECC; G4: ECC+LIFU; G5: PFH@ECC+LIFU; G6: PFH@ECC+LIFU. The thrombi in each group were weighed and photographed at different time points after treatment. The photographic results are shown below. Figure 24 As shown. The concentration of nanoparticles used in this experiment was 2.5 mg mL⁻¹, and the LIFU intensity was 3 W cm⁻². The thrombolysis rate was calculated using the following formula: Thrombolysis rate (%) = [(Initial thrombus weight - Post-treatment thrombus weight) / Initial thrombus weight] × 100%. The thrombolysis rate results are shown below. Figure 25 As shown.

[0100] from Figure 24 , Figure 25 It can be seen that after 6 minutes of LIFU irradiation, PFH@EC mainly undergoes cavitation on the thrombus surface, with limited penetration into the thrombus matrix. In contrast, CREKA-functionalized PFH@ECC can re-anchor to the newly exposed fibrin interface, penetrating deeper into the thrombus matrix and disrupting the internal structure of the thrombus, thus exhibiting significant mechano-mediated thrombolytic activity. The thrombolytic effect of PFH@ECC combined with LIFU is comparable to that of 6 hours of urokinase treatment. Furthermore, no significant changes in clot size or morphology were observed in either the PFH@ECC group without LIFU irradiation or the ECC+LIFU group, indicating that PFH encapsulation is crucial for achieving LIFU-responsive thrombolysis.

[0101] To further observe the surface morphology of thrombi after different treatments, a scanning electron microscope (HITACHIS 4800, Hitachi High Technology Co., Ltd., Japan) was used to observe the changes in the surface structure of the treated thrombi. The results are as follows: Figure 26 As shown, in the control group, erythrocytes, platelets, and fibrin were tightly embedded within the clot matrix. In contrast, PFH@ECC combined with LIFU treatment significantly induced the dissociation and collapse of blood cells and fibrin, accompanied by the disruption of the fibrin network.

[0102] To further observe the in vitro thrombolytic effect, the treated thrombi were stained with antifibrinogen antibodies and then observed under a microscope. The results were consistent. Figure 27 As shown, the fibrin scaffold underwent extensive degradation after PFH@ECC combined with LIFU treatment.

[0103] 3. In vivo ultrasound thrombolysis experiment of PFH@ECC on chronic thrombosis ①In vivo targeting experiments of PFH@ECC Six SD rats were used. On day 1, the inferior vena cava was dissected to fully expose the vessels. FeCl3 was then added to the inferior vena cava to form a stable thrombus. The abdominal wound was then re-sutured. On day 28, after the formation of an old thrombus, the rats were randomly divided into two groups of three. The rats were injected via tail vein with CY7-labeled PFH@EC and PFH@ECC, respectively. Four hours later, the inferior vena cava containing the thrombus was harvested for in vitro fluorescence imaging. The results are as follows: Figure 28 As shown, a significant fluorescent signal appeared at the thrombus site in rats injected with Cy7-PFH@ECC. Quantitative analysis confirmed that, as Figure 29 As shown, the cumulative amount of Cy7-PFH@ECC at the thrombus site was significantly higher than that of non-targeted Cy7-PFH@EC, verifying its in vivo targeting effect, which is consistent with the results of in vitro targeting experiments.

[0104] ②In vivo thrombolysis experiment of PFH@ECC To investigate the in vivo thrombolytic effect of PFH@ECC on chronic thrombosis, a chronic thrombosis model of the inferior vena cava (IVC) was established in SD rats. Eighteen rats (8-12 weeks old) were divided into six groups of three: G1: normal group; G2: control group; G3: PFH@ECC group; G4: ECC + LIFU group; G5: PFH@ECC + LIFU group; and G6: PFH@ECC + LIFU group. Except for the normal group, all other groups underwent induction of IVC thrombosis. The specific method for thrombosis formation was as follows: rats were anesthetized, and the IVC was exposed by incision along the midline of the abdomen. A 2×2 mm filter paper soaked in 10% FeCl3 solution was placed on the exposed IVC for 10 minutes to induce thrombosis. After thrombosis formation on day 1, the rats were continued to be fed until a stable chronic thrombus was formed. Treatment was initiated on day 29 and continued for 5 consecutive days. The control group received normal saline injections, while the treatment group received 2.5 mg / mL of the designated preparation, with LIFU irradiation administered or withholding depending on the situation. On day 35, the volume of residual thrombus in the inferior vena cava was assessed by ultrasound imaging, and the results were as follows: Figure 30 As shown.

[0105] As can be seen, the thrombus in the inferior vena cava of rats treated with PFH@ECC+LIFU almost completely dissolved, approaching the state of normal tissue. This significant thrombolytic effect is attributed to CREKA-mediated thrombus targeting and the strong mechanical force generated by the LIFU-triggered PFH phase transition. In contrast, the treatment effect was extremely low without LIFU irradiation or PFH loading. Similarly, the thrombolytic performance of unmodified PFH@ECC was also reduced due to the lack of active targeting capability.

[0106] The cross-sectional area of ​​the thrombus was measured using ImageJ software, and the thrombolysis rate was calculated using the following formula: Thrombolysis rate (%) = [(Initial thrombus cross-sectional area - Post-treatment thrombus cross-sectional area / Initial thrombus cross-sectional area] × 100%. The results are as follows: Figure 31 As shown, the thrombolysis rates of the PFH@ECC (−1.49 ± 5.24%), ECC + LIFU (5.86 ± 8.72%), and PFH@EC + LIFU (32.90 ± 10.29%) groups were significantly lower than those of the PFH@ECC + LIFU group, which reached 59.12 ± 11.81%. (Equation: 0.1181)

[0107] The rats were then euthanized, the inferior vena cava was isolated and photographed, and thrombus samples were collected for H&E staining and immunofluorescence staining. The results were as follows: Figure 32 and Figure 33 As shown in the figure, PFH@ECC + LIFU treatment has an enhanced thrombolytic effect. After treatment with PFH@ECC matrix, TNF-α and DHE fluorescence signals are significantly reduced, while IL-10 fluorescence signals are increased, indicating that PFH@ECC has dual anti-inflammatory and antioxidant effects in vivo.

[0108] In addition, to assess the safety of LIFU irradiation, the inferior vena cava (IVC) of SD rats was surgically exposed after administration of the appropriate formulation for local LIFU treatment. Endothelial staining results showed no detectable endothelial damage or disruption of vascular integrity under 3 W cm⁻² LIFU irradiation, thus confirming the biosafety of this treatment parameter. See details below. Figure 34 As shown.

[0109] 4. Experiment on PFH@ECC promoting the healing of lower extremity ulcers A full-thickness resection wound (1 cm in diameter) was created in the lateral iliac vessel region of the hind limb of 8- to 10-week-old SD rats. The rats were randomly divided into six groups: G1: Normal tissue group (external iliac artery only, treated with saline); G2: Saline group (thrombotic wound model only, treated with saline); G3: PFH@ECC group (thrombotic wound model, treated with PFH@ECC); G4: ECC+LIFU group (thrombotic wound model, treated with ECC+LIFU); G5: PFH@EC+LIFU group (thrombotic wound model, treated with PFH@EC+LIFU); G6: PFH@ECC+LIFU group (thrombotic wound model, treated with PFH@ECC+LIFU). The concentration of the treatment in groups G3-G6 (4 treatment groups) was 2.5 mg / mL. -1The dosage was 1 mL for all doses, and the LIFU intensity was 3 W / cm². -2 The irradiation time was 10 minutes. Treatment was continued for 6 days. On the 7th day, the mice were euthanized and leg skin was collected. Wound images were recorded daily. Results are as follows: Figure 35 As shown. ImageJ software was used to analyze the wound area, and the healing rate was calculated using the following formula: Healing rate (%) = [(Initial wound area - Post-treatment / Initial wound area] × 100%. The results are as follows. Figure 36 As shown.

[0110] As can be seen, compared with the Saline group, both the Normal tissue group and the PFH@ECC+LIFU group showed significant wound closure after 5 days of treatment. Notably, the wound closure rate of the PFH@ECC+LIFU group reached 81.83% on day 5, while the wound in the Saline group remained largely unhealed. By day 7, a large amount of new hair growth was observed around the wounds in both the Normal tissue group and the PFH@ECC+LIFU group, accompanied by significantly improved wound repair. This enhanced healing response may stem from PFH@ECC-mediated microenvironment remodeling, characterized by highly efficient thrombolysis and reduction of local inflammation and oxidative stress.

[0111] This experiment also involved H&E staining of skin tissue sections from SD rats to assess tissue regeneration during wound healing. The results are as follows: Figure 37 As shown in the figure, H&E staining revealed persistent and significant inflammatory infiltration in the control group. In contrast, PFH@ECC combined with LIFU treatment significantly reduced the inflammatory response, and its tissue structure was closer to that of normal tissue. Notably, PFH@ECC + LIFU treatment promoted the formation of more epithelial structures and hair follicles, and the tissue maturity was also higher than in other groups.

[0112] During the remodeling phase, adequate collagen deposition and remodeling are crucial for improving the tensile strength of new skin and accelerating wound healing. However, excessive accumulation of reactive oxygen species (ROS) can impair collagen fiber synthesis. Therefore, this experiment further assessed collagen deposition on day 7 using histological analysis and Masson's trichrome staining, with results as follows: Figure 38 and Figure 39 As shown in the figure, compared with other groups, the PFH@ECC +LIFU group showed significantly enhanced collagen deposition, indicating that new skin tissue had begun to form.

[0113] Immunofluorescence staining further showed that inflammation was relieved after PFH@ECC combined with LIFU treatment, such as Figure 40 As shown.

[0114] The above results indicate that PFH@ECC combined with LIFU accelerates wound healing by reducing inflammation, promoting collagen deposition, and facilitating the formation of mature epithelial structures.

[0115] 5. Transcriptome analysis of venous ulcer skin in SD rats To systematically elucidate the molecular mechanism of nanoplatform-mediated therapy, this experiment collected wound tissue from SD rats treated with PFH@ECC + LIFU for RNA sequencing (RNA-seq). By comparing data from multiple groups—the PFH@ECC + LIFU group (i.e., skin ulceration treated with PFH@ECC + LIFU), the control group (control group, i.e., skin ulceration without treatment), and the normal group (normal skin group)—differentially expressed genes and their functional characteristics were systematically identified. The results are as follows: Figure 43 As shown, PFH@ECC + LIFU treatment significantly altered the gene expression profile compared to the control group. Differential expression analysis between the PFH@ECC + LIFU group and the control group revealed extensive gene regulatory abnormalities, indicating that this treatment profoundly remodeled the transcriptional landscape of the ulcer microenvironment.

[0116] To further elucidate the biological functions of differentially expressed genes (DEGs), GO enrichment analysis was performed on differentially expressed genes between the PFH@ECC + LIFU group and the control group. The results are as follows: Figure 44 As shown, these genes are significantly enriched in multiple biological processes closely related to skin repair, including epidermal development, tissue development, keratinocyte differentiation, and epithelial cell differentiation. Furthermore, processes related to cell structure and tissue remodeling, such as intermediate filament organization and cytoskeleton remodeling, are also significantly enriched. These results suggest that anti-inflammatory and antioxidant therapies may promote epidermal regeneration and tissue remodeling, thereby accelerating the healing of venous ulcer wounds.

[0117] KEGG pathway analysis further revealed that these differentially expressed genes were mainly enriched in pathways regulating inflammation and tissue repair, particularly arachidonic acid metabolism, the Wnt signaling pathway, cytokine-cytokine receptor interactions, and tight junction pathways, such as... Figure 45 As shown in the figure, the significant enrichment of arachidonic acid metabolism in these pathways indicates that lipid inflammatory mediators play an important role in ulcer repair, while the Wnt signaling pathway is closely related to tissue regeneration and cell proliferation. Taken together, these results suggest that PFH@ECC + LIFU treatment not only modulates the inflammatory response but may also accelerate wound repair by promoting regeneration-related signaling pathways.

[0118] 6. Biosafety evaluation experiment SD rats were randomly divided into five groups (n=5 per group): saline group, PFH@ECC group, ECC+LIFU group, PFH@EC+LIFU group, and PFH@ECC+LIFU group. After intravenous injection of saline or the corresponding nanomedicine preparation (2.5 mg / mL, 1 mL) via the tail vein, the groups receiving LIFU irradiation underwent ultrasonic irradiation. Twenty-four hours after circulation, H&E staining was performed on major organs, including the heart, liver, spleen, lungs, and kidneys. The results are shown below. Figure 41 As shown in the figure, no obvious morphological abnormalities or tissue damage were observed in the nanoparticle treatment group compared with the saline group. Whole blood and plasma were collected from SD rats for routine blood tests and liver and kidney function tests. The results are as follows: Figure 42 As shown, routine blood parameters and plasma biochemical indicators did not show significant abnormalities among the groups, indicating that PFH@ECC has good in vivo biocompatibility under the tested treatment conditions.

[0119] In summary, the ultrasound-responsive phase-change nanoparticles provided in this application not only effectively achieve mechanical thrombolysis of refractory chronic thrombosis but also simultaneously regulate the anti-inflammatory and antioxidant microenvironment. By combining vascular recanalization with tissue regeneration, a paradigm shift has been achieved from simply "clearing blood vessels" to "achieving tissue repair through systemic network intervention," providing a promising alternative to traditional intervention methods, especially suitable for microcirculatory disorders that are difficult to access with traditional surgery. This strategy, by achieving targeted, controllable, and minimally invasive treatment, provides a novel theoretical framework and a translatable clinical pathway for managing thrombotic complications and promoting chronic wound healing.

[0120] For those skilled in the art, without departing from the concept of the technical solution of this invention, several modifications and improvements can be made, and these should also be considered within the scope of protection of this invention. These will not affect the effectiveness of the implementation of this patent or the practicality of the patent.

Claims

1. An ultrasonically responsive phase change nanoparticle, characterized in that: It includes a spherical shell, which is a chitosan oligosaccharide-eicosapentaenoic acid compound modified with thrombus-targeting peptides, and perfluorohexane is encapsulated inside the spherical shell.

2. The ultrasonically responsive phase change nanoparticle according to claim 1, characterized in that: The mass ratio of chitosan oligosaccharide to eicosapentaenoic acid is (1:1) to (1:3).

3. The ultrasonically responsive phase change nanoparticle according to claim 2, characterized in that: The mass ratio of chitosan oligosaccharide to eicosapentaenoic acid is 120:

202.

4. A method for preparing ultrasonically responsive phase change nanoparticles according to any one of claims 1-3, characterized in that: The steps are as follows: S1, take chitosan oligosaccharide, eicosapentaenoic acid, and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate, and dissolve them separately in formamide to obtain three solutions. Mix the three solutions with triethylamine and react to obtain a reaction solution. S2, 6-(maleimide)hexanoic acid succinimide ester is dissolved in formamide and added to the reaction solution in S1 to react and obtain eicosapentaenoic acid-chitosan oligosaccharide-maleimide intermediate; then the thrombosis-targeting peptide is dissolved in formamide and coupled with the intermediate to obtain eicosapentaenoic acid-chitosan oligosaccharide-thrombosis-targeting peptide. S3, the eicosapentaenoic acid-chitosan oligosaccharide-thrombosis-targeting peptide in S2 is dissolved in deionized water, and perfluorohexane is added under ultrasonic vibration to obtain ultrasonically responsive phase change nanoparticles.

5. The method for preparing ultrasonically responsive phase change nanoparticles according to claim 4, characterized in that: In S1, a solution of benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate is first mixed with a solution of eicosapentaenoic acid and reacted. Then, a solution of chitosan oligosaccharide and triethylamine are added and mixed and reacted.

6. The method for preparing ultrasonically responsive phase change nanoparticles according to claim 4, characterized in that: The reactions in S1 and S2 were both at room temperature.

7. The method for preparing ultrasonically responsive phase change nanoparticles according to claim 5, characterized in that: The molar ratio of benzotriazol-1-yl-oxytripyrrolidinyl phosphorus hexafluorophosphate to eicosapentaenoic acid is 2:

1.

8. The use of an ultrasound-responsive phase change nanoparticle according to any one of claims 1-3 in the preparation of a drug for treating venous thrombosis.

9. The use of an ultrasound-responsive phase change nanoparticle according to any one of claims 1-3 in the preparation of a drug for treating venous ulcers.

10. The use of an ultrasound-responsive phase change nanoparticle according to any one of claims 1-3 in the preparation of a drug for simultaneously treating venous thrombosis and venous ulcers.

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