Platelet bionic liposome based on ultrasonic response as well as preparation method and application of platelet bionic liposome

Abstract

The invention discloses a platelet bionic liposome based on ultrasonic response and a preparation method and application, and belongs to the technical field of biological medicine and nano-drug preparations, the platelet bionic liposome comprises an ultrasonic response liposome and a platelet membrane, the ultrasonic response liposome is a lipid bilayer composed of unsaturated lipid, saturated lipid and a sound-sensitive agent, an internal hydrophilic region and a double-layer hydrophobic region are formed, and the thrombolytic drug is loaded in the hydrophilic region through the hydration shell; the hydrophobic porphyrin ring of the sound-sensitive agent PPIX is driven by the hydrophobic effect to be embedded into the lipid bilayer, and the porphyrin ring of the sound-sensitive agent PPIX and the cholesterol steroid ring are subjected to pi stacking, so that the position of the sound-sensitive agent PPIX between the lipid bilayer is stabilized. The thrombus can be accurately targeted, efficient and controllable release of the medicine can be achieved at the thrombus part under ultrasonic triggering, the thrombolysis effect is remarkably enhanced, and the bleeding risk is reduced.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomedicine formulation technology, and in particular to a method for preparing and applying platelet biomimetic liposomes based on ultrasound response. Background Technology

[0002] Thrombotic diseases are among the most serious illnesses worldwide, causing the highest rates of morbidity and mortality. Thrombosis can trigger cardiovascular diseases such as ischemic stroke, myocardial infarction, and pulmonary embolism, seriously threatening human health and life. However, currently used thrombolytic drugs (such as tPA) suffer from problems such as short half-life, poor targeting, narrow therapeutic window, and high risk of systemic bleeding, which limit their efficacy and application. In recent years, the use of nanocarriers to load and deliver bioactive molecules, nanoparticles, proteins, and other substances has been considered a promising technology, offering new insights for thrombosis treatment. However, to prevent immune recognition and clearance in vivo, nanocarriers must possess excellent biocompatibility. Liposomes are a class of materials with outstanding biocompatibility; their basic structure consists of vesicles composed of a phospholipid bilayer, similar to a cell membrane, possessing a hydrophilic interior and a hydrophobic bilayer, allowing for the simultaneous encapsulation of both hydrophilic and hydrophobic drugs. For hydrophilic tPA, encapsulation within the liposome core can significantly prolong its half-life and avoid the associated bleeding risk. However, traditional liposomes lack sufficient accumulation capacity at thrombus sites and cannot achieve controlled drug release, resulting in limited therapeutic efficacy. While some nanoparticles can respond to external stimuli (such as light and heat) to precisely release drugs, the tissue penetration depth of these signals is insufficient, limiting their application scenarios. Among all external stimuli, ultrasound (US) offers superior depth penetration and focusing accuracy, making it an ideal choice for triggering spatiotemporally controlled release in clinical practice. Therefore, using ultrasound as an activation signal for liposomes can achieve higher penetration depth and precision in drug release. On the other hand, during thrombosis, platelets are activated and recruited to the thrombus, where they become a major component of thrombus formation. The membrane protein αIIbβ3 integrin on activated platelets has the function of specifically recognizing high-affinity fibrin. Reconstructing the coagulation behavior of platelets on nanoparticles can enable nanoparticles to target thrombi, while also improving the biocompatibility of the nanocarrier. Summary of the Invention

[0003] Purpose of the invention: This invention addresses the shortcomings of tPA thrombolytic drugs, such as short half-life, poor targeting ability, and high bleeding risk. It combines ultrasound-responsive controllable drug release with biomimetic targeting technology to propose an ultrasound-responsive platelet biomimetic liposome-based nanoplatform for the precise treatment of thrombosis.

[0004] Technical solution: To achieve the above objectives, the technical solution adopted by this invention is as follows: An ultrasound-responsive biomimetic platelet liposome comprises ultrasound-responsive liposomes and a platelet membrane. The ultrasound-responsive liposomes are prepared by a thin-film hydration method, comprising a lipid bilayer composed of unsaturated lipids, saturated lipids, and a sound-sensitive agent. The platelet membrane is prepared by repeated freeze-thaw cycles. The ultrasound-responsive liposomes and platelet membrane are subjected to repeated freeze-thaw treatments to obtain the ultrasound-responsive biomimetic platelet liposomes.

[0005] Preferred: Thrombolytic drugs are loaded into the hydrophilic region of ultrasound-responsive platelet biomimetic liposomes.

[0006] Another object of the present invention is to provide a method for preparing ultrasound-responsive platelet biomimetic liposomes, comprising the following steps: Step S1: Prepare ultrasound-responsive liposomes by using a thin-film hydration method with unsaturated lipids, saturated lipids, and a sound-sensitive agent.

[0007] Step S2: Platelet membranes are prepared from platelet-rich plasma using a repeated freeze-thaw process. Step S3: Ultrasonic-responsive platelet biomimetic liposomes are prepared by repeatedly freezing and thawing ultrasound-responsive liposomes and platelet membranes.

[0008] Preferably, the method for preparing ultrasound-responsive liposomes in step S1 is as follows: Step S11: Add distearylphosphatidylcholine, distearylphosphatidylglycerol, 1,2-dioleoyl-sn-glycerol-3-phosphocholine and cholesterol to the reaction vessel, and dissolve the sound-sensitive agent PPIX in a mixed solution of chloroform and methanol.

[0009] In step S12, the reaction vessel containing the mixed solution is placed on a rotary evaporator to completely evaporate the solvent. Then, it is hydrated with tissue plasminogen activator tPA and placed in an ultrasonic cleaner to form a milky white suspension.

[0010] Step S13: Centrifuge the milky white suspension to purify it and remove the supernatant to obtain ultrasound-responsive liposomes.

[0011] Preferably, the distearylphosphatidylcholine, distearylphosphatidylglycerol, 1,2-dioleoyl-sn-glycerol-3-phosphocholine and cholesterol are present in a molar ratio of 2.8-3.2:1.8-2.2:0.8-1.2:2.8-3.2.

[0012] Preferably, the concentration of the sound-sensitive agent PPIX is 0.8-1.2 wt%. The ratio of chloroform to methanol is 9:0.8-1.2. The concentration of PBS in the tissue plasminogen activator tPA is 0.25-0.3 mg / mL.

[0013] Preferably, the method for preparing platelet membranes in step S2 is as follows: Step S21: Anesthetize SD rats with isoflurane gas and collect fresh blood from the rats via the orbital vein in acidic citrate-glucose buffer.

[0014] In step S22, the collected fresh rat blood is centrifuged to separate red blood cells and white blood cells from plasma. The supernatant collected after centrifugation is used as platelet-rich plasma. Platelets are obtained by centrifugation and precipitation of platelet-rich plasma.

[0015] In step S22, the collected platelets are resuspended in a modified Tyrode buffer. The resuspended platelet suspension is subjected to repeated freeze-thaw cycles, centrifuged to collect the precipitate, and then mixed with a protease inhibitor to obtain a platelet membrane.

[0016] Preferred: Modified Tyrode buffer comprises 130-140 mM sodium chloride, 10-15 mM sodium bicarbonate, 2.8-3.0 mM potassium chloride, 0.3-0.4 mM disodium hydrogen phosphate, 0.8-1.2 mM magnesium chloride, 9-11 mM HEPES, and 1.8-2.2 µM GEL.

[0017] Preferred method: The method for preparing ultrasound-responsive platelet biomimetic liposomes in step S3 by repeated freeze-thaw cycles: Ultrasound-responsive liposomes are mixed with platelet membranes at a mass ratio of 10:0.9-1.1, allowed to stand at room temperature, and then subjected to repeated freeze-thaw cycles. Subsequently, they are extruded sequentially through polycarbonate membranes with pore sizes of 800 nm and 400 nm to obtain ultrasound-responsive platelet biomimetic liposomes.

[0018] Another object of the present invention is to provide the application of ultrasound-responsive platelet biomimetic liposomes in the preparation of drugs for treating thrombosis.

[0019] Compared with the prior art, the present invention has the following advantages: Firstly, utilizing the targeting ability of platelet membranes, and employing platelet-inspired biomimetic liposomes, a sonication agent loaded between the liposome bilayer membranes releases reactive oxygen species in response to ultrasound. The reactive oxygen species react with unsaturated lipids, causing lipid peroxidation, and the liposomes rupture to release tPA, thus achieving thrombolytic therapy. This invention can be used for the precise treatment of thrombosis, and therefore has important clinical significance and application value. Attached Figure Description

[0020] Figure 1 This is a particle size distribution chart of the platelet biomimetic liposomes in this invention.

[0021] Figure 2 This is a statistical comparison of the particle size of the platelet biomimetic liposomes before and after ultrasound in this invention.

[0022] Figure 3 The images show transmission electron microscopy (TEM) characterization of the platelet biomimetic liposomes in this invention before and after ultrasound.

[0023] Figure 4 This is a flow cytometry diagram of the platelet membrane of the platelet biomimetic liposomes in this invention.

[0024] Figure 5 The platelet membrane SDS-PAGE and WB characterization images of the platelet biomimetic liposomes in this invention are shown.

[0025] Figure 6 The image shows the DOPC oxidative Fourier transform infrared spectra of the platelet biomimetic liposomes before and after ultrasound in this invention.

[0026] Figure 7 This is an LC-MS image of DOPC before and after ultrasound in the platelet-inspired biomimetic liposomes of this invention.

[0027] Figure 8 This is a graph showing the release rate of tPA from the platelet-inspired biomimetic liposomes of this invention under the action of ultrasound.

[0028] Figure 9 This is an in vivo targeting diagram of the platelet-inspired biomimetic liposomes in this invention.

[0029] Figure 10 This is a diagram illustrating in vivo thrombolysis using platelet-inspired biomimetic liposomes, as presented in this invention.

[0030] Figure 11 This diagram illustrates the safety assessment of bleeding in a liver injury model using platelet-inspired biomimetic liposomes, as presented in this invention.

[0031] Figure 12 This is a diagram illustrating the safety assessment of tail bleeding in the platelet biomimetic liposomes of the present invention. Detailed Implementation

[0032] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0033] Example 1 This embodiment provides a platelet-inspired biomimetic liposome based on ultrasound response, such as Figure 1As shown, the invention includes ultrasound-responsive liposomes and a platelet membrane. The ultrasound-responsive liposomes consist of a lipid bilayer composed of unsaturated lipids (DOPC), saturated lipids (DSPC, DSPG, cholesterol), and a sonosensitive agent. The sonosensitive agent is embedded in the lipid bilayer and is prepared by a thin-film hydration method. The sonosensitive agent is PPIX. The hydrophobic porphyrin ring of PPIX is driven by the hydrophobic effect to embed into the lipid bilayer, and the porphyrin ring of PPIX is stacked with the cholesterol steroid ring in a π-like manner, stabilizing the position of PPIX between the lipid bilayers. The platelet membrane is prepared by repeated freeze-thaw cycles. Ultrasound-responsive liposomes and platelet membranes are subjected to repeated freeze-thaw treatments to obtain ultrasound-responsive biomimetic platelet liposomes.

[0034] Thrombolytic drugs are loaded into the hydrophilic regions of ultrasound-responsive platelet biomimetic liposomes. The thrombolytic drug is the tissue plasminogen activator tPA.

[0035] The liposomes of this invention consist of a lipid bilayer composed of unsaturated lipids (DOPC), saturated lipids (DSPC, DSPG, cholesterol), and a sonosensitive agent, forming an internal hydrophilic region and an interlayer hydrophobic region. The liposomes are prepared via a thin-film hydration method. The thrombolytic drug tPA is loaded into the hydrophilic region via a hydrated shell and prepared via the thin-film hydration method. The hydrophobic porphyrin ring of the sonosensitive agent PPIX is driven by the hydrophobic effect to embed into the lipid bilayer, and the porphyrin ring of PPIX is stacked with the cholesterol steroid ring in a π-like arrangement, stabilizing the position of PPIX between the lipid bilayers. The platelet membrane is prepared by repeated freeze-thaw cycles. Under ultrasound, the sonosensitive agent PPIX releases ROS into the phospholipid bilayer, inducing the peroxidation of unsaturated fatty acids. The polarity of the phospholipid molecules of the peroxidized unsaturated fatty acids changes, reducing membrane stability and thus releasing the thrombolytic drug. This invention simulates the peroxidation process of phospholipids on cell membranes by introducing unsaturated fatty acids and a sonosensitive agent into the construction of liposomes. Triggered by ultrasound, the sonosensitive agent releases ROS between the phospholipid bilayers, inducing the peroxidation of unsaturated fatty acids. The polarity of the phospholipid molecules of the peroxidized unsaturated fatty acids changes, reducing membrane stability and thus releasing thrombolytic drugs. By introducing the liposome into the platelet membrane, the specific binding ability of αIIbβ3 integrin on the platelet membrane to fibrin is utilized to actively target the thrombus site, achieving ultrasound response and specific recognition for thrombosis treatment.

[0036] In another embodiment, the use of ultrasound-responsive platelet biomimetic liposomes in the preparation of drugs for treating thrombosis is provided.

[0037] By leveraging the long-circulation characteristics of liposomes, the in vivo circulation time of tPA is prolonged. Under ultrasound stimulation, a sonosensitive agent loaded between the liposome bilayer releases reactive oxygen species (ROS) in response to ultrasound. These ROS react with unsaturated lipids, causing lipid peroxidation, leading to liposome rupture and the release of tPA, thus achieving thrombolytic therapy. This invention, through synergistic optimization of the platelet membrane and liposome ratio, successfully prepared liposomes with uniform particle size, high drug loading efficiency, good stability, and excellent thrombus targeting and ultrasound-responsive release. These platelet-inspired liposomes can precisely target thrombi and, under ultrasound triggering, achieve highly efficient and controllable drug release at the thrombus site, significantly enhancing the thrombolytic effect and reducing the risk of bleeding. This provides a solution to the bottleneck problems of traditional thrombolytic therapy, such as short half-life, poor targeting, and narrow therapeutic window.

[0038] Example 2 This embodiment provides a method for preparing platelet biomimetic liposomes based on ultrasound response, including the following steps: Step S1: Prepare ultrasound-responsive liposomes by using a thin-film hydration method with unsaturated lipids, saturated lipids, and a sound-sensitive agent.

[0039] Step S11: Disteazyl phosphatidylcholine, disteazyl phosphatidylglycerol, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, and cholesterol are added to a round-bottom flask, and dissolved with the sound-sensitizing agent PPIX in a mixed solution of chloroform and methanol. The molar ratio of disteazyl phosphatidylcholine, disteazyl phosphatidylglycerol, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, and cholesterol is 3:2:1:3, and the concentration of the sound-sensitizing agent PPIX is 0.1 wt%. The ratio of chloroform to methanol is 9:1.

[0040] In step S12, place the round-bottom flask containing the mixed solution on a rotary evaporator at 40 °C until the solvent is completely evaporated. Then, hydrate with tissue plasminogen activator (tPA) at a concentration of 0.25-0.3 mg / mL of PBS. Place the flask in an ultrasonic cleaner, controlling the ultrasonic power at 1 W / cm² and the frequency at 1 MHz, until a milky white suspension is formed.

[0041] Step S13: The milky white suspension is centrifuged at 12,000 rpm for 10 minutes to purify it. The supernatant is removed three times to obtain the ultrasound-responsive liposomes.

[0042] Step S2: Platelet membranes are prepared from platelet-rich plasma using a repeated freeze-thaw process. Step S21: Male SD rats were anesthetized with isoflurane gas, and fresh blood was collected from the rats via the orbital vein in acidic citrate-glucose buffer. Acidic citrate-glucose buffer (39 mM citrate, 75 mM sodium citrate, 135 mM glucose, pH=7.4).

[0043] In step S22, the collected fresh rat blood is centrifuged at 100 g for 10 minutes at 37°C to separate red blood cells and white blood cells from plasma. The supernatant collected after centrifugation is used as platelet-rich plasma. The platelet-rich plasma is then centrifuged at 1000 g for 10 minutes to precipitate platelets.

[0044] In step S22, the collected platelets were resuspended in a modified Tyrode buffer. The modified Tyrode buffer formulation is as follows: 134 mM sodium chloride, 12 mM sodium bicarbonate, 2.9 mM potassium chloride, 0.34 mM disodium hydrogen phosphate, 1 mM magnesium chloride, 10 mM HEPES, and 2 µM PGE1. The resuspended platelet suspension was subjected to repeated freeze-thaw cycles at -80°C, centrifuged 5 times (14,000 rpm, 10 minutes each time) to collect the precipitate, which was then mixed with a protease inhibitor to obtain the platelet membrane.

[0045] Step S3: Ultrasonic-responsive platelet biomimetic liposomes are prepared by repeatedly freezing and thawing ultrasound-responsive liposomes and platelet membranes.

[0046] Ultrasound-responsive liposomes were mixed with platelet membranes at a mass ratio of 10:1, allowed to stand at room temperature for 10 minutes, and then subjected to a freeze-thaw cycle of 5 times. Subsequently, the mixture was extruded 10 times through polycarbonate membranes with pore sizes of 800 nm and 400 nm, respectively, to obtain ultrasound-responsive biomimetic platelet liposomes.

[0047] Example 3 The difference between this embodiment and Example 2 is that the molar ratio of distearylphosphatidylcholine, distearylphosphatidylglycerol, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, and cholesterol is 2.8:1.8:0.8:2.8, and the concentration of the sonosensitive agent PPIX is 0.8 wt%. The ratio of chloroform to methanol is 9:0.8. The concentration of PBS in the tissue plasminogen activator tPA is 0.25 mg / mL, and the modified Tyrode buffer formulation is: 130 mM sodium chloride, 10 mM sodium bicarbonate, 2.8 mM potassium chloride, 0.3 mM disodium hydrogen phosphate, 0.8 mM magnesium chloride, 9 mM HEPES, and 1.8 µM MPGE1. The ultrasound-responsive liposomes and platelet membrane are mixed at a mass ratio of 10:0.9.

[0048] Example 4 The difference between this embodiment and Example 2 is that the molar ratio of distearylphosphatidylcholine, distearylphosphatidylglycerol, 1,2-dioleoyl-sn-glycerol-3-phosphocholine, and cholesterol is 3.2:2.2:1.2:3.2, and the concentration of the sonosensitive agent PPIX is 1.2 wt%. The ratio of chloroform to methanol is 9:1.2. The concentration of PBS in the tissue plasminogen activator tPA is 0.3 mg / mL, and the modified Tyrode buffer formulation is as follows: 140 mM sodium chloride, 15 mM sodium bicarbonate, 3.0 mM potassium chloride, 0.4 mM disodium hydrogen phosphate, 1.2 mM magnesium chloride, 11 mM HEPES, and 2.2 µM MPGE1. The ultrasound-responsive liposomes and platelet membrane are mixed at a mass ratio of 10:1.1.

[0049] Please see Figure 1 and Figure 2 As shown, the preparation and characterization of platelet biomimetic liposomes are as follows: Platelet biomimetic liposomes are subjected to ice bath sonication, with the sonication power controlled at 5%, the start-up time at 5 s, the stop-up time at 5 s, and the total time at 5 min. The samples are diluted 2 times and divided into two parts. One part is treated with ultrasound, and the other part is used for comparison and particle size statistical characterization using a laser particle size analyzer.

[0050] See results Figure 1 and Figure 2 As shown, the particle size of the platelet biomimetic liposomes is about 250 nm. Under the action of ultrasound, the platelet biomimetic liposomes oxidize and break down, and the particle size changes from 250 nm to 160 nm, showing a decreasing trend, which confirms the successful preparation of platelet biomimetic liposomes.

[0051] Please see Figure 3 As shown, the preparation and characterization of platelet-inspired liposomes were as follows: 10 µL of platelet-inspired liposomes were dropped onto a copper grid, vacuum dried for 15 minutes, and then negatively stained with 1% uranium acetate solution. All samples were observed and analyzed under a transmission electron microscope with an accelerating voltage of 100 kV.

[0052] The results are visible. Figure 3 As shown in the transmission electron microscope image, the liposomes are round in shape, and the platelet-inspired liposomes rupture under the action of ultrasound.

[0053] Please see Figure 4 and 5As shown, the degree of fusion between platelet membrane vesicles and liposomes was analyzed by flow cytometry and Western blotting. Platelet membranes were labeled with DiO, and liposomes with DiD. Samples and dyes were mixed at room temperature for 1 hour, followed by centrifugation (12,000 rpm, 10 minutes) and resuspending in PBS to remove free dye. DiD-labeled liposomes and DiO-labeled platelet membranes were mixed at a 10:1 mass ratio and subjected to repeated freeze-thaw cycles to obtain platelet-mimetic liposomes. After grouping the samples, the degree of fusion between liposomes and platelet membranes was detected by flow cytometry. The samples were divided into three groups: DiD-labeled liposomes, DiO-labeled platelets, and the two groups of platelet-mimetic liposomes prepared from the above. The proteomic profiles of platelet membranes and platelet-mimetic liposomes were analyzed by SDS-PAGE. Samples were subjected to 4-20% FuturePAGE. TM Equal amounts of protein were loaded onto the separating gel and electrophoresed using MOPS buffer. The gel was then stained with Coomassie Brilliant Blue for 30 minutes, followed by destaining in 1×TBST buffer for 2 hours until no background color remained. After SDS-PAGE gel electrophoresis, the gel was removed and subjected to Western blotting analysis. Proteins were transferred from the separating gel to a 0.45 µm PVDF membrane in loading buffer. After transfer, the PVDF membrane was blocked with 1× protein-free rapid blocking buffer at room temperature for 30 minutes. CD41 and CD61 primary antibodies were incubated with the PVDF membrane overnight, followed by washing three times with 1×TBST buffer. The membrane was then incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 2 hours. Finally, the blots were analyzed using a Tannon 5200 chemiluminescence imaging system.

[0054] The results are visible. Figure 4 and Figure 5 As shown, flow cytometry analysis revealed that most fused vesicles exhibited strong fluorescence signals from both the lipid marker DiD and the platelet membrane marker DiO, confirming successful fusion. SDS-PAGE analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that, compared to the platelet membrane, the platelet-inspired liposomes retained almost all platelet membrane proteins. Crucially, integrins llb / llla—the most abundant glycoproteins on the platelet surface—mediated high-affinity fibrin binding after activation with non-covalently coupled thromboretin (CD41) and CD61 subunits, thereby achieving thrombus targeting. Western blot experiments further confirmed the simultaneous presence of CD41 and CD61 on the surface of LPMVs, demonstrating that the platelet-inspired liposomes retained most of the thrombus-targeting function of the platelet membrane.

[0055] Please see Figure 6 and Figure 7As shown, the characterization of platelet-inspired biomimetic liposomes after oxidation was as follows: The emulsion-like liposomes before and after sonication were freeze-thawed for 24 h. Then, the liposomes were ground into a fine powder at a ratio of liposomes to potassium bromide of 1:200 and characterized using Fourier transform infrared spectroscopy. Before LC-MS testing, the liposome suspension (5 mg / mL) was sonicated (1 W / cm², 1 MHz, 10 min) for 1 hour. To inhibit oxidation, 5 mM sodium ascorbate was added to the sample. Subsequently, 1 mL of the treated liposomes was extracted with 3 mL of a methanol:chloroform (1:2, v / v) solvent mixture. The mixture was vortexed for 1 min and allowed to stand at room temperature for 10 min. After initial phase separation, the mixture was centrifuged (4℃, 3000 g, 10 min) to collect the lower organic phase. The extraction steps were then repeated. The two organic phases were combined and dried using a rotary evaporator. The treated sample was stored at -80℃. The dried sample was reconstituted with 200 μL of isopropanol / methanol (1:1, v / v). Then, 5 μL of 0.14 mg / mL LPC (12:0) internal standard was added, and the sample was centrifuged at 12000 rpm (4 °C, 10 min). The supernatant was collected after separation. Analysis was performed on an LC-MS platform.

[0056] The results are visible. Figure 6 and Figure 7 As shown, the results indicate that the structure of the DOPC component of the liposomes changed, and the platelet-inspired liposomes underwent oxidation.

[0057] Please see Figure 8 As shown, ultrasound-triggered drug release from platelet-inspired biomimetic liposomes was demonstrated. The fibrinolytic activity of tPA was tested using the chromogenic substrate S-2288. The suspension of platelet-inspired biomimetic liposomes was sonicated (1 W / cm²). -2 Centrifuge at 1 MHz for 10 minutes at different time points. Collect the supernatant and rotate it again with an equal volume of PBS. Add the supernatant and native tPA to assay buffer (0.1 M). 1 tris-HCl (pH 7.4) and S-2288 (1.0 mM) 1 The microplates were placed at 37°C. Fibrinolytic activity was calculated using absorbance per minute at 405 nm for 30 minutes.

[0058] The results are visible. Figure 8 As shown, after ultrasound treatment, approximately 60% of tPA was released from the platelet-mimetic liposomes within 1 hour, with almost no further release thereafter. In contrast, untreated platelet-mimetic liposomes released less than 10% of tPA. These results indicate that ultrasound triggers tPA release by inducing structural changes in the platelet-mimetic liposomes.

[0059] Please see Figure 9 and Figure 10 As shown, a ferric chloride-induced femoral vein thrombosis model was established in 6-8 week old male ICR mice (anesthetized with isoflurane). First, the left femoral vein of the mice was exposed with a scalpel. Then, a filter paper soaked in 10% ferric chloride (FeCl3) solution was placed on the exposed femoral vein surface for 5 minutes. After removing the filter paper, the blood vessel was flushed with sterile phosphate-buffered saline (PBS). This induced femoral vein thrombosis. Subsequently, different groups of mice were injected with 200 μL of physiological saline, natural tPA, and platelet-inspired liposomes containing an equivalent concentration of tPA (0.5 mg / kg), respectively. After 1 hour of circulation, mice in the platelet-inspired liposome group received ultrasound therapy, which involved ultrasound irradiation of the femoral vein (1 W / cm², 1 MHz, 10 min). All mice were then surgically sutured. Twelve hours later, the mice were sacrificed, and femoral vein tissue was collected for histological analysis. ImageJ software processes slice samples and determines thrombolysis efficiency by measuring the thrombus area and calculating the ratio of the thrombus area to the total area of ​​the vascular lumen.

[0060] The results are visible. Figure 9 and Figure 10 As shown, Figure 9 The fluorescence intensity in the femoral vein of mice injected with platelet-inspired liposomes was significantly enhanced, 3-5 times higher than that of other groups. In contrast, the liposomes did not show fluorescence aggregation, which proves that the platelet-inspired liposomes can effectively target thrombi in vivo, and their ability stems from the characteristics of the platelet membrane coating. Figure 10 Among all groups, mice treated with ultrasound-activated platelet-mimetic liposomes had the smallest thrombus area (approximately 23.5%). In contrast, the percentages of thrombus areas in the saline, natural tPA, and untreated platelet-mimetic liposome groups were 80.4%, 54.9%, and 67.9%, respectively.

[0061] Please see Figure 11 and Figure 12As shown, to assess the bleeding risk of platelet-based biomimetic liposomes, liver injury and tail vein bleeding models were established. Liver injury model: Male SD rats (6-8 weeks old) were anesthetized with isoflurane. After injection of saline / LPMVs / tPA via the tail vein, the abdominal cavity was opened 15 minutes later, and a 2 cm long and 1 cm wide section of liver tissue was removed from the left lateral lobe. Gauze was placed at the incision site to collect blood. Subsequently, blood was collected from the rats via the orbital cavity at different time points. The collected blood was mixed with 3.8% sodium citrate at a 9:1 ratio. One hour later, the gauze was removed and weighed; the increase in weight of the gauze was used to determine the hemorrhage mass at the liver site. The blood collected via the orbital cavity was then centrifuged at 3500 rpm, and the supernatant was collected as rat plasma. The D-dimer content in the plasma was tested to determine the changes in D-dimer levels in the rats at different time points. Finally, the rats' wounds were sutured, and their survival was observed within 12 hours. Tail vein hemorrhage model: Mice were anesthetized with isoflurane and injected via the tail vein with 200 μL of physiological saline, natural tissue plasminogen activator (tPA), or platelet-inspired liposomes containing an equivalent concentration of tPA (0.5 mg / kg). Five minutes later, a 2 cm segment of tissue was surgically removed from the distal tail. When bleeding began at the cut site, blood was immediately collected in centrifuge tubes containing 5 IU / mL heparin sodium solution until hemostasis was achieved. The optical density (OD) of the blood samples was then measured at 540 nm. 540 ), and according to OD 540 The value is used to calculate hemoglobin concentration to assess blood loss.

[0062] The results are visible. Figure 11 and Figure 12As shown, after establishing the liver injury model, D-dimer levels in all groups rapidly increased due to hemorrhage-induced coagulation activation. The D-dimer level in the tPA treatment group increased most significantly, reaching 5.5 ng / mL at 1 hour. In contrast, the D-dimer levels in the platelet-based biomimetic liposome group and the saline group were 1.19 ng / mL and 0.52 ng / mL, respectively. Furthermore, survival rates were analyzed 12 hours after treatment. The survival rate in the tPA group was only 40%, while the survival rate in the platelet-based biomimetic liposome group was as high as 80%. This indicates that platelet-based biomimetic liposomes can prevent hemorrhage complications caused by tPA leakage. In the tail vein hemorrhage model, the amount of bleeding was quantified by detecting hemoglobin levels in the blood after intravenous injection of saline, platelet-based biomimetic liposomes, and tPA, respectively. The results showed that the hemoglobin level in mice receiving platelet-based biomimetic liposomes was approximately 25% of that in the tPA group, and essentially the same as that in the saline group. Blood loss, dynamic changes in D-dimer levels, and survival rates were compared among different treatment groups to assess the bleeding risk associated with platelet-based biomimetic liposomes. Rats injected with platelet-based biomimetic liposomes experienced blood loss of approximately 8.3% of their total blood volume (TBV), comparable to the saline-injected control group (approximately 5.5%), but significantly lower than the blood loss in the tPA treatment group (approximately 20.7%).

[0063] This invention prepares platelet-inspired biomimetic liposomes via a thin-film hydration method and determines the optimal fusion ratio to be 10:1. The platelet-inspired biomimetic liposomes exhibit uniform size, with a particle size of approximately 250 nm. Utilizing the targeting capability of the platelet membrane, the biomimetic liposomes, under the influence of ultrasound, release reactive oxygen species (ROS) via a sonication agent loaded between the liposome bilayer. The ROS react with unsaturated lipids, causing lipid peroxidation, leading to liposome rupture and the release of tPA, thus achieving thrombolytic therapy. This invention utilizes the long-circulation characteristics of liposomes to prolong the in vivo circulation time of thrombolytic drugs, and leverages the targeting ability of platelets to precisely target thrombi. Under the action of ultrasound, the acoustic sensitizer PPIX loaded between the liposome bilayer membranes responds to the ultrasound and releases reactive oxygen species. The reactive oxygen species react with unsaturated lipids, causing lipid peroxidation, and the liposomes rupture to release tPA, thereby achieving thrombolytic therapy.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A platelet-inspired biomimetic liposome based on ultrasound response, characterized in that, The invention includes ultrasound-responsive liposomes and platelet membranes. The ultrasound-responsive liposomes consist of a lipid bilayer composed of unsaturated lipids, saturated lipids, and a sonosensitive agent, with the sonosensitive agent embedded in the lipid bilayer. The ultrasound-responsive liposomes and platelet membranes are subjected to repeated freeze-thaw cycles to obtain ultrasound-responsive biomimetic platelet liposomes. Thrombolytic drugs are loaded into the hydrophilic regions of the ultrasound-responsive biomimetic platelet liposomes.

2. The ultrasound-responsive platelet biomimetic liposome according to claim 1, characterized in that: The sonosensitive agent is PPIX. The hydrophobic porphyrin ring of PPIX is driven by the hydrophobic effect to embed into the lipid bilayer, and the porphyrin ring of PPIX is stacked with the cholesterol steroid ring in a π-like manner, which stabilizes the position of PPIX between the lipid bilayer.

3. The ultrasound-responsive platelet biomimetic liposome according to claim 2, characterized in that: The ultrasound-responsive liposomes were prepared by thin-film hydration; the platelet membranes were prepared by repeated freeze-thaw cycles.

4. A method for preparing ultrasound-responsive platelet biomimetic liposomes as described in claim 1, characterized in that, Includes the following steps: Step S1: Prepare ultrasound-responsive liposomes by using a thin-film hydration method with unsaturated lipids, saturated lipids, and a sound-sensing agent; Step S2: Platelet membranes are prepared from platelet-rich plasma using a repeated freeze-thaw process; Step S3: Ultrasonic-responsive platelet biomimetic liposomes are prepared by repeatedly freezing and thawing ultrasound-responsive liposomes and platelet membranes.

5. The preparation method according to claim 4, characterized in that: The method for preparing ultrasound-responsive liposomes in step S1 is as follows: Step S11: Add distearylphosphatidylcholine, distearylphosphatidylglycerol, 1,2-dioleoyl-sn-glycerol-3-phosphocholine and cholesterol to the reaction vessel, and dissolve them with the sound-sensitive agent PPIX in a mixed solution of chloroform and methanol. Step S12: Place the reaction vessel containing the mixed solution on a rotary evaporator to completely evaporate the solvent; then hydrate it with tissue plasminogen activator tPA, place it in an ultrasonic cleaner, and sonicate it to form a milky white suspension. Step S13: Centrifuge the milky white suspension to purify it and remove the supernatant to obtain ultrasound-responsive liposomes.

6. The preparation method according to claim 5, characterized in that: The distearylphosphatidylcholine, distearylphosphatidylglycerol, 1,2-dioleoyl-sn-glycerol-3-phosphocholine and cholesterol are present in a molar ratio of 2.8-3.2:1.8-2.2:0.8-1.2:2.8-3.2; the concentration of the sound-sensitive agent PPIX is 0.8-1.2 wt%; the ratio of chloroform to methanol is 9:0.8-1.2; and the concentration of PBS in the tissue plasminogen activator tPA is 0.25-0.3 mg / mL.

7. The preparation method according to claim 6, characterized in that: The method for preparing platelet membranes in step S2 is as follows: Step S21: Anesthetize SD rats with isoflurane gas and collect fresh blood from the rats through the orbital vein in acidic citrate-glucose buffer. Step S22: The collected fresh rat blood is centrifuged to separate red blood cells and white blood cells from plasma. The supernatant collected after centrifugation is used as platelet-rich plasma. Platelets are obtained by centrifugation and precipitation of platelet-rich plasma. In step S22, the collected platelets are resuspended in modified Tyrode buffer; the resuspended platelet suspension is subjected to repeated freeze-thaw cycles, centrifuged to collect the precipitate, and then mixed with a protease inhibitor to obtain a platelet membrane.

8. The preparation method according to claim 7, characterized in that: The modified Tyrode buffer contains 130-140 mM sodium chloride, 10-15 mM sodium bicarbonate, 2.8-3.0 mM potassium chloride, 0.3-0.4 mM disodium hydrogen phosphate, 0.8-1.2 mM magnesium chloride, 9-11 mM HEPES, and 1.8-2.2 µM MPGE1.

9. The preparation method according to claim 8, characterized in that: The method for preparing ultrasound-responsive platelet biomimetic liposomes by repeated freeze-thaw cycles in step S3 is as follows: ultrasound-responsive liposomes are mixed with platelet membranes at a mass ratio of 10:0.9-1.1, allowed to stand at room temperature, and then subjected to repeated freeze-thaw cycles; subsequently, they are extruded through polycarbonate membranes with pore sizes of 800 nm and 400 nm to obtain ultrasound-responsive platelet biomimetic liposomes.

10. The use of the ultrasound-responsive platelet biomimetic liposome as described in claim 1 in the preparation of a drug for treating thrombosis.

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