Ferroptosis inhibitor-loaded bionic nano-carrier and application thereof

Abstract

The invention discloses a biomimetic nano-carrier loaded with a ferroptosis inhibitor and application of the biomimetic nano-carrier. A bacterial vesicle and a platelet membrane are subjected to extrusion to obtain a hybrid vesicle, a ferroptosis inhibitor Fer-1 is loaded in the hybrid vesicle, maleimide activated urokinase and DSPE-PEG2000-CREKA are connected to the outside of the hybrid vesicle in a chemical coupling and physical insertion mode, and the final preparation is obtained. The nano-carrier reaches a thrombus part in a multi-covalent targeting manner and releases a thrombolytic drug urokinase and an anti-inflammatory drug Fer-1 at a fixed point, so that the effects of precisely dissolving thrombus and improving a thrombus microenvironment are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a biomimetic nanocarrier loaded with ferroptosis inhibitors and its applications. Background Art

[0002] At present, cardiovascular diseases have gradually become an important factor threatening human life and health, and cardiovascular thromboembolic diseases have the problems of acute onset and short treatment windows, and have become serious public health problems. Thromboembolic diseases can be divided into arterial thrombosis, venous thrombosis, and microthrombosis. Among them, arterial thrombosis formed under high shear conditions is mainly composed of platelet aggregates bound together by fibrin chains; compared with venous thrombosis, arterial thrombosis under high blood flow shear force conditions causes greater harm. However, there is currently little clinical research on arterial thrombosis. Therefore, developing an effective antithrombotic therapy for arterial thrombosis is the top priority in solving cardiovascular problems at present. Clinically, thrombosis is managed through two complementary strategies: prevention and treatment. Since thrombus treatment mainly targets two processes, platelet activation and the coagulation cascade system, antithrombotic drugs mainly consist of drugs such as antiplatelet aggregation drugs, anticoagulant drugs, and drugs that activate plasmin for thrombolysis.

[0003] To enhance drug retention at the site of action and minimize adverse reactions to off-target tissues and improve the therapeutic effect of existing drugs, it is necessary to select and develop appropriate delivery systems to achieve targeted and controlled drug release. However, nanocarriers face problems such as poor targeting ability, short in vivo circulation time, and cell barrier penetrability. Based on the characteristics of rich platelets, inflammatory cells, damaged endothelial cells, and thrombin in arterial thrombosis and the inflammatory microenvironment, designing targeted carriers targeting the coagulation cascade system, platelets, or inflammatory cells and optimizing the design of drug carriers are still major challenges to ensure safe, sustainable, and precise drug delivery in vivo. As a new type of biomimetic nanodelivery carrier, platelets have a unique inner membrane system and a powerful intracellular transport ability. Drugs can be loaded on the surface or internalized into platelets. After the drug-loaded platelets reach the damaged or inflamed site, platelets secrete nanoscale vesicles and internal granules to achieve precise recognition and site-specific release of the target site, achieving the effects of reducing drug toxicity and reducing drug loss. Therefore, using platelets as the main carrier material and delivering thrombolytic drugs and anti-inflammatory drugs to the thrombus site by various drug-loading methods is the top priority for achieving efficient thrombolysis and improving the thrombus microenvironment.

[0004] Ferrostatin-1 (Fer-1) is an effective ferroptosis-specific inhibitor that can prevent the accumulation of lipid ROS, inhibit lipid peroxidation, and reduce labile iron in cells. Recent studies have shown that Fer-1 can downregulate the expression of prostaglandin endoperoxide synthase 2, upregulate the expression of GPX4 and nuclear factor E2-related factor 2 (Nrf2) proteins, and protect cells by inhibiting oxidative stress and reducing ROS production. However, as a promising pharmacological molecule, Fer-1 faces inherent stability and hydrophobicity problems, which limit its application in vivo.

[0005] Based on this, the present invention designs and prepares hybrid vesicles using platelet membranes and bacterial vesicles as biomimetic nanocarriers. Then, the ferroptosis inhibitor Fer-1 is encapsulated inside them, and the thrombolytic drug urokinase and the fibrin-targeting peptide CREKA are conjugated outside them, delivering the thrombolytic drug to the thrombus site to achieve the effect of highly efficient and precise thrombolysis. Summary of the Invention

[0006] One of the objectives of the present invention is to provide a biomimetic nanocarrier loaded with a ferroptosis inhibitor, which is prepared by the following steps:

[0007] Step 1: Lactobacillus plantarum WCFS1 is expanded in culture and then bacterial vesicles are prepared by ultrafiltration concentration-ultracentrifugation method;

[0008] Step 2: Platelets are separated and platelet membranes are extracted by the repeated freeze-thaw method;

[0009] Step 3: The platelet membranes are mixed with phospholipids, and platelet membrane vesicles are obtained after sonication. Then, the bacterial vesicles are mixed and extruded to obtain hybrid vesicles. The hybrid vesicles are mixed with the ferroptosis inhibitor Fer-1 to obtain drug-loaded hybrid vesicles;

[0010] Step 4: Urokinase is conjugated to the surface of the drug-loaded hybrid vesicles through Sulfo-SMCC;

[0011] Step 5: The drug-loaded hybrid vesicles conjugated with urokinase are incubated with DSPE-PEG 2000 -CREKA to obtain the biomimetic nanocarrier loaded with a ferroptosis inhibitor.

[0012] Furthermore, the mass ratio of the phospholipid DSPE-PEG 2000 to platelet membrane protein is 1:1 - 4:1, preferably 4:1.

[0013] Furthermore, the mass ratio of the bacterial vesicle protein to the platelet membrane protein is 1:20 - 1:5, preferably 1:10.

[0014] Further, the mass ratio of the ferroptosis inhibitor Fer-1 to the platelet membrane protein is 1:5 - 1:20, preferably 1:5.

[0015] Further, the mass ratio of urokinase to the platelet membrane protein is 1:1.

[0016] Further, the DSPE-PEG 2000 -CREKA and the platelet membrane protein have a mass ratio of 1:4.

[0017] In one embodiment of the present invention, the preparation process is as follows: According to the mass ratio of M DSPE-PEG2000 :M PM = 4:1, the platelet membrane suspension is uniformly mixed with powdered DSPE-PEG2000, and placed under a probe sonicator for probe sonication for 5 min to obtain platelet membrane vesicles (PMV); after standing overnight at 4°C, according to the mass ratio of M Levs :M PM = 1:10, the mixture of Levs and PMV is placed in a 37°C horizontal shaker and shaken at a speed of 100 rpm for 30 min, and then passed through 400 nm and 200 nm carbonate membranes 7 times each to obtain hybrid vesicles (PLevs); according to the mass ratio of M Fer-1 :M PM = 1:5, the powdered solid Fer-1 is uniformly mixed with PLevs, and also placed in a 37°C constant temperature horizontal shaker and shaken at a speed of 100 rpm for 2 h, and centrifuged and washed twice to obtain the final drug-loaded hybrid vesicles F@PLevs.

[0018] The second object of the present invention is to provide the application of the above-mentioned biomimetic nanocarrier loaded with a ferroptosis inhibitor in the preparation of an anti-thrombotic drug.

[0019] Based on the extrusion method of bacterial vesicles (Levs) and platelet membranes (PM) to obtain hybrid vesicles (PLevs), Fer-1 is loaded inside, and maleimide-activated urokinase and DSPE-PEG2000-CREKA are connected in a chemically conjugated and physically inserted manner outside to obtain the final preparation F@PLevs-C&U. This nanocarrier will reach the thrombus site in a multiple covalent targeting manner and release the thrombolytic drug urokinase and the anti-inflammatory drug Fer-1 at a fixed point, achieving the effects of precise thrombolysis and improving the thrombus microenvironment.

[0020] The present invention designs and prepares a bacterial-platelet hybrid vesicle system for the targeted delivery of thrombolytic drugs and the improvement of the thrombus microenvironment, providing new ideas for the treatment of thromboembolic diseases. Description of the Drawings

[0021] Figure 1Transmission electron microscopy image of bacterial vesicles Levs.

[0022] Figure 2 For platelet membranes with different DSPE-PEG 2000 Particle size potential of platelet membrane vesicles with different dosage ratios.

[0023] Figure 3 Particle size potential of platelet membrane vesicles with different probe sonication times.

[0024] Figure 4 Particle size potential of drug-loaded hybrid vesicles with different dosage ratios of bacterial vesicles Levs and platelet membranes.

[0025] Figure 5 Drug loading and encapsulation efficiency of drug-loaded hybrid vesicles with different amounts of Fer-1 added.

[0026] Figure 6 Transmission electron microscopy images of PMV (A), PLevs (B), F@PLevs (C), and F@PLevs-C&U (D).

[0027] Figure 7 Particle size potential of F@PLevs-C&U after being placed for 7 days.

[0028] Figure 8 In vitro release curves of Fer-1, F@PLevs, and F@PLevs-C.

[0029] Figure 9 In vitro thrombolysis results of F@PLevs-C&U. Detailed implementation manners

[0030] The preferred implementation manners of the present invention will be described in detail below in conjunction with the embodiments. It should be understood that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0031] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0032] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Example 1

[0034] I. Extraction of bacterial vesicles Levs

[0035] The Lactobacillus plantarum WCFS1 strain stored at -80°C was evenly spread on an MRS agar plate by continuous streaking, and left standing at 37°C for 24 h, and spherical convex colonies were visible. Single colonies with clear edges and far from other colonies were selected. A pipette was used to pick up the single colonies and placed them in 20 mL of broth, and then repeatedly pipetted evenly. After that, they were placed in a shaker at 37°C and cultured at a rotation speed of 100 rpm for 18 h. When the bacterial OD600 reached 1.0, the next amplification was carried out. The strain seed liquid was transferred into 200 mL of fresh MRS broth medium according to a volume fraction of 0.1%. After mixing evenly, the culture solution was taken out and the absorbance value at 600 nm was measured under an ultraviolet spectrophotometer, which was the OD600 value at 0 h. Subsequently, the bacterial solution was placed in a horizontal shaker at 37°C and cultured at a rotation speed of 100 rpm. Samples were taken at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, and 48 h to measure the OD600 value.

[0036] When the bacteria to be expanded entered the logarithmic growth phase (OD600 = 1.0), the supernatant was taken for gradient centrifugation. That is, it was centrifuged at a rotation speed of 12000g for 30 min to remove most of the bacterial precipitate, and then filtered through a 0.45 μm filter membrane to obtain the primary filtrate. The primary filtrate was centrifuged again at a rotation speed of 12000g for 20 min and filtered through a 0.22 μm filter membrane to obtain the re-filtrate. For further concentration, the re-filtrate was placed in a 100 kDa ultrafiltration centrifugal tube and ultrafiltration concentrated multiple times at a frequency of 3500 rpm for 30 min, so that 1 L of the bacterial solution was concentrated to 20 mL. The concentrated solution was placed in an ultra-high-speed centrifuge for high-speed centrifugation to obtain vesicle precipitate (4°C, 180000g, 120 min). The enriched bacterial vesicles (Levs) were dispersed and resuspended with a mixture of PBS and protease inhibitor, and then placed at -80°C for standby.

[0037] The particle size and potential of the vesicles were measured by a nanoparticle size potentiometer. The hydrated particle size of Levs was 114.1 ± 3.8 nm, and the PDI was 0.276 ± 0.117; the zeta potential was -5.42 ± 1.53 mv, indicating that the particle size of the vesicles was relatively uniform and the surface was negatively charged.

[0038] As Figure 1 shown, under a transmission electron microscope, the Levs after negative staining with phosphotungstic acid showed an oval double-membrane vesicle structure with a size between 50 - 200 nm.

[0039] II. Isolation and preparation of platelet membranes

[0040] 1. Isolation of platelets (PLT)

[0041] Fresh arterial blood was collected from the abdominal aorta of ICR mice and stored in an anticoagulant tube containing potassium citrate (1:9). Platelets were purified by gradient centrifugation and centrifuged at room temperature (200g, 10 min) to obtain platelet-rich plasma (PRP). To prevent platelet activation, PBS buffer containing 1 mmol / L EDTA and 2 mmol / L PGE1 was added to the PRP, and centrifuged again (900g, 20 min). The lower precipitate was taken and resuspended in pre-cooled Cocktail buffer (0.1M PBS, 2 mM EDTA, 10-6M PGE1), and the above suspension was stored at -80°C.

[0042] 2. Extraction process of platelet membrane (PM)

[0043] The platelet membrane was extracted by the method of repeated freezing and thawing. Specifically: the platelet suspension frozen at -80°C was thawed in a 37°C water bath and then refrozen in an -80°C refrigerator for 2 h, repeated 5 times, and centrifuged (10000g, 1 h). After washing three times with PBS solution, the precipitate was collected and resuspended in Cocktail buffer. The platelet membrane was suspended in the prepared freeze-dried reference solution (9.5 mmol / L HEPES, 142.5 mmol / L NaCl, 4.8 mmol / KCl, 1 mmol / L MgCl2), with 20% w / w trehalose as a saccharide protectant. The sample was pre-frozen at -80°C for 4 h and then placed in a freeze dryer for freeze-drying with a temperature control program of -60°C, -40°C, -20°C, -10°C, 0°C, 5°C, 10°C, 25°C for storage.

[0044] III. Preparation of Fer-1-loaded hybrid vesicles (F@PLevs)

[0045] 1. Preparation process

[0046] The freeze-dried platelet membrane (PM) and bacterial vesicles (Levs) were resuspended separately with PBS buffer, and the protein content of the platelet membrane (M PM ) and the protein content of Levs (M Levs ) were detected by the BCA method.

[0047] The platelet membrane suspension was mixed with DSPE-PEG 2000Mix evenly and place under a probe sonicator for probe sonication to obtain platelet membrane vesicles (PMV). After standing overnight at 4 °C, place the mixture of Levs and PMV in a horizontal shaker at 37 °C and shake at a speed of 100 rpm for 30 min. Then, pass through a 400 nm and a 200 nm carbonate membrane 7 times each to obtain hybrid vesicles (PLevs). Mix the powdered solid Fer-1 evenly with PLevs, also place it in a constant temperature horizontal shaker at 37 °C and shake at a speed of 100 rpm for 2 h, and centrifuge and wash twice to obtain the final drug-loaded hybrid vesicles F@PLevs.

[0048] 2. M DSPE-PEG2000 :M PM

[0049] Mix the platelet membrane suspension with phospholipid DSPE-PEG 2000 in different ratios evenly (M DSPE-PEG2000 :M PM = 0, 1,

[0050] 2, 3, 4), and perform probe sonication under a probe sonicator for 5 min. Taking the particle size potential as the investigation index, investigate the influence of the mass ratio on the particle size and potential of PMV.

[0051] As Figure 2 shown, with the addition of phospholipid, the potential of PMV shows a continuous decreasing trend, while the particle size shows a trend of decreasing first, then increasing, and then decreasing again. In the vesicles with mass ratios of 1:1, 2:1, and 3:1, the particle size is uneven and PDI > 0.3. In the vesicles with a mass ratio of 4:1, the particle size of PMV is relatively uniform and PDI is 0.158 ± 0.047.

[0052] 3. Probe sonication time

[0053] Place the mixed solution under a probe sonicator, ensure that the probe sonication power remains unchanged (on for 1 s, off for 2 s, power 30%), and only change the probe sonication time (5 min, 6 min, 7 min, 8 min, 9 min). Taking the particle size and PDI as the investigation indexes, explore the influence of the probe sonication time on the particle size distribution of PMV.

[0054] As Figure 3 shown, at 7 min, the particle size of PMV is the smallest, which is 155.17 ± 3.79 nm. After that, with the increase of the probe time, the particle size reaches 467.54 ± 64.26 nm at 9 min. Therefore, 7 min is selected as the final sonication time.

[0055] 4. M Levs :M PM

[0056] F@PLevs was prepared by ultrasonic cavitation-extrusion method. Based on the membrane protein concentration, the prescribed amounts of PMV and Levs were shaken in a horizontal shaker at 37 °C and 100 rpm for 30 min according to the mass ratio of membrane protein of 5:1, 10:1, 15:1, and 20:1. Then, the mixture was extruded through a 400 nm and a 200 nm carbonate membrane 7 times in sequence to obtain the F@PLevs solution.

[0057] As Figure 4 shown, with the increase of the Levs membrane component, the particle size of PLevs increased first and then decreased. When the mass ratio of PMV to Levs membrane protein was 10:1 and 20:1, the particle sizes were 231.6 ± 14.2 nm and 263.4 ± 6.3 nm, respectively, and the PDI were 0.195 ± 0.021 and 0.116 ± 0.011. When the mass ratio of PMV to Levs membrane protein was 10:1, the particle size was 290.6 ± 7.0 nm and the PDI was 0.124 ± 0.010. Therefore, the hybrid vesicles with a mass ratio of membrane protein of 10:1 were used for subsequent experiments.

[0058] 5. M Fer-1 : M PM

[0059] Only the addition amount of powdered Fer-1 was changed, i.e., M Fer-1 : M PM = 1:10, 1:5, 1:3, 1:2, and other experimental conditions were the same. The effects of the addition amount of Fer-1 on the drug loading and encapsulation efficiency of F@PLevs were investigated.

[0060] The determination processes of the encapsulation efficiency and drug loading are as follows:

[0061] The prepared F@PLevs was centrifuged at 4 °C (3500 rpm, 15 min) to remove free Fer-1. After collecting the supernatant, PBS buffer was added and centrifuged again at a centrifugal force of 1800000 g for 1.5 h. The obtained white precipitate was F@PLevs. After extraction with dichloromethane and drying with nitrogen, it was dissolved in the mobile phase, filtered through a 0.22 μm microporous membrane, and analyzed by HPLC injection. The peak area was recorded, and the encapsulation efficiency (EE%) and drug loading (LE%) could be calculated by the following formulas:

[0062] EE(%) = W0 / W1 × 100%

[0063] LE(%) = W0 / W × 100%.

[0064] Among them, the content of Fer-1 encapsulated by F@PLevs was calculated as W0, the total drug dosage was W1, and the total mass of the materials in F@PLevs (only calculating the mass of membrane protein and phospholipid) was W.

[0065] The liquid chromatography conditions for Fer-1 are as follows: chromatographic column: RP-C18 column ( C18, 5 μm, 250 mm × 4.6 mm, Welch); mobile phase: acetonitrile - formic acid (0.05%) (50:50); detection wavelength: 254 nm; flow rate: 1 mL / min; column temperature: 35 °C; injection volume: 20 μL.

[0066] As Figure 5 shown, based on the calculation formula of the encapsulation efficiency, which is the ratio of the Fer-1 concentration in F@PLevs to the total drug dosage, it can be found that as the Fer-1 dosage increases, the encapsulation efficiency shows a gradually decreasing trend. At the same time, the drug loading further reflects the ratio of Fer-1 in the total carrier components in this vesicle system. When M Fer-1 : M PM = 1:5, the drug loading of F@PLevs reaches the highest at 2.25% ± 0.31%. When M Fer-1 : M PM = 1:3 and 1:2, the drug loadings (LE) of the obtained PLevs are 2.16% and 2.14% respectively, and there is no significant difference among the three groups (P > 0.05). Therefore, when M Fer-1 : M PM = 1:5, the proportion of Fer-1 in the total carrier components reaches the maximum, and the drug loading reaches the highest level, which is the final drug dosage.

[0067] Finally, the optimal formulation parameters for preparing F@PLevs are as follows: the mass ratios of phospholipid (DSPE-PEG2000), bacterial vesicles (Levs) protein, and ferroptosis inhibitor (Fer-1) to platelet membrane protein are 4:1, 1:10, and 1:5 respectively. The probe sonication time is 7 min, and the drug addition method is the powder direct mixing method. On this basis, hybrid vesicles (F@PLevs) with an encapsulation efficiency of 55.17% are obtained by the sonication-extrusion-incubation method.

[0068] IV. Preparation of F@PLevs-U and F@PLevs-C&U

[0069] 1. Preparation of F@PLevs-U

[0070] The amino groups on the surface of urokinase are coupled with Sulfo-SMCC to form a preliminary conjugate, and then form the final complex F@PLevs-U with the thiol groups on the surface of the hybrid vesicles modified by Traut's reagent.

[0071] Dissolve 1 mg of urokinase (UPA) in physiological saline (0.4 M HEPES), adjust the osmotic pressure to 550 mmol / L, and ensure that the pH in the system is 7.4. According to the molar ratio of UPA:Sulfo-SMCC = 10:1, add Sulfo-SMCC dropwise to the UPA buffer solution. After adding 0.02% Tween 80 to the reaction system, remove the oxygen in the solution and introduce nitrogen for protection. Then, make the reaction continue at 4 °C with a rotation speed of 200 rpm for 3 h to obtain the UPA-SMCC conjugate. Place the product in a 10 kDa ultrafiltration tube and centrifuge at a centrifugal force of 4000 g for 30 min to remove the unreacted Sulfo-SMCC. Mix freshly prepared F@PLevs with 1.0 mg / mL of 2-iminothiolane hydrochloride (Traut's reagent), stir at a rotation speed of 200 rpm at 4 °C for 1 h to induce the activation of amino groups on the surface of F@PLevs into thiol groups. After washing with PBS, at 4 °C, mix the activated F@PLevs with UPA-SMCC in proportion and continue to stir at a rotation speed of 200 rpm for 3 h to obtain the drug-loaded hybrid vesicles conjugated with UPA (F@PLevs-U).

[0072] Based on the results of the determination of thiol group content by the DTNB method and the determination of urokinase content by the enzyme-linked immunosorbent assay, the mass ratio of urokinase to platelet membrane protein was determined to be 1:1, and the complex F@PLevs-U with a reaction efficiency of 55.8% could be obtained.

[0073] 2. Preparation of F@PLevs-C&U

[0074] Take freshly prepared F@PLevs-U and mix it according to the mass ratio of DSPE-PEG 2000 -CREKA:F@PLevs-U (calculated based on the content of platelet membrane protein) = 1:4. After incubating at a constant temperature with shaking at 37 °C for 30 min, centrifuge the physical mixture at 180,000 g for 2 h, discard the supernatant, and redissolve the precipitate with PBS to obtain CREKA-modified F@PLevs-U (F@PLevs-C&U).

[0075] Meanwhile, take freshly prepared F@PLevs and prepare CREKA-modified F@PLevs (F@PLevs-C) according to the above method.

[0076] The above preparations are characterized and detected below.

[0077] 1. Determination of particle size and potential

[0078] Take freshly prepared PMV, PLevs, F@PLevs, F@PLevs-U, F@PLevs-C&U solutions and dilute them with water to a membrane protein concentration of 0.1 mg / ml, and then measure the hydrated particle size and potential.

[0079] The particle size of PMV was 114.2 ± 17.98 nm, the PDI was 0.470 ± 0.013, and the zeta potential was -12.3 ± 0.916 mV.

[0080] The particle size of PLevs was 129.8 ± 0.416 nm, the PDI was 0.231 ± 0.010, and the zeta potential was -10.4 ± 4.74 mV. The particle size of the preparation was appropriate and the distribution was uniform, indicating that the ultrasonic-extrusion method could successfully retain the larger vesicles of F@PMV and retain the relatively uniform hybrid vesicles, improving the stability of the vesicles.

[0081] The particle size of F@PLevs was 135.2 ± 4.513 nm, the PDI was 0.465 ± 0.091, and the zeta potential was -13.4 ± 0.713 mV. Compared with PLevs, the zeta potential of F@PLevs was enhanced, indicating an increase in the stability of the self-assembled complex.

[0082] The particle size of F@PLevs-U was 168.3 ± 9.014 nm, the PDI was 0.340 ± 0.086, and the zeta potential was -12.8 ± 0.987 mV, indicating that after the amino groups on the surface of the hybrid vesicles were successfully converted to thiol groups and UPA was linked through Sulfo-SMCC, it was manifested as a change in the vesicle potential and an increase in particle size.

[0083] The particle size of F@PLevs-C&U was 180.6 ± 7.242 nm, the PDI was 0.392 ± 0.020, and the zeta potential was -14.8 ± 2.36 mV, indicating that with the addition of CREKA, the absolute value of the zeta potential on the surface of the vesicles increased slightly.

[0084] 2. Transmission electron microscopy analysis

[0085] Characterize the morphologies of the prepared PMV, PLevs, F@PLevs, F@PLevs-C&U by TEM, and the results are as Figure 6As shown. PMV, PLevs, F@PLevs, and F@PLevs-C&U all presented a bilayer vesicle structure with a spherical shape under a transmission electron microscope. The particle size was about 200 nm. The results of TEM were basically consistent with those measured by a particle size analyzer, indicating that the prepared PMV, F@PLevs, F@PLevs-U, and F@PLevs-C&U had appropriate particle sizes and good morphologies. By comparing and observing PMV and PLevs, it was found that under the condition of a membrane protein concentration ratio of 10:1, a membrane structure with a width of about 10 - 20 nm was visible on the outside of the nanocarrier, which was consistent with the cell membrane thickness, indicating the successful construction of the hybrid membrane vesicle. At the same time, Figure 6 In C, no obvious insoluble particles were visible outside F@PLevs, indicating that most insoluble particles were removed by centrifugation during the preparation process. By comparing F@PLevs and F@PLevs-C&U, it was obvious that the thickness of the outer membrane vesicle of the bilayer membrane vesicle increased.

[0086] 3. Investigation of storage stability

[0087] Take freshly prepared F@PLevs-C&U and store it in a 4°C refrigerator for one week to observe its storage stability. Samples were taken at 0, 1, 3, 5, and 7 days respectively, and the particle size and potential were continuously measured according to the aforementioned method to investigate its storage stability within seven days.

[0088] After testing within 7 days, the appearance of the nano - preparation was clear and transparent, without obvious black particle accumulation, and the nano - particle size and potential analyzer detected as Figure 7 , its particle size remained in the range of 250 - 320 nm, and the potential remained at about - 18 mV, without obvious fluctuations, indicating that the negatively charged nanoparticles were in good condition in the solution, and F@PLevs-C&U had good stability under this condition.

[0089] 4. In vitro drug release study

[0090] Take 1.0 mL of each of free Fer - 1, F@PLevs, and F@Plevs - C solutions. Among them, the free Fer - 1 powder was dialyzed in the form of a suspension, placed in a dialysis bag with a molecular weight cut - off of 25 kDa, and 0.1% SDS in a phosphate buffer solution with pH = 7.4 was used as the release medium. The total volume of the release medium was 20 mL. Ensure that the conditions in the thermostatic horizontal shaker were 37°C and 100 rpm. Sampling needles were used to take out the external release liquid at 1, 2, 4, 6, 8, 12, 24, 36 h, and 48 h respectively, and an equal amount of release medium was replenished. The samples taken at different time points were freeze - dried by a freeze - dryer and analyzed by HPLC to determine the content of Fer - 1 in the samples, and the cumulative release rate at different time points was calculated to plot the in vitro drug release curve.

[0091] The results of the release of free Fer-1, F@PLevs and F@PLevs-C in the release medium at pH 7.4 for 48 h are as follows Figure 8 As shown, free Fer-1 showed a characteristic of rapid release, and the cumulative release rates at 24 h and 48 h reached 68.20% ± 6.15% and 89.50% ± 0.71% respectively. It was speculated that the free drug could be almost completely released within 48 h. Both the F@PLevs group and the F@PLevs-C group had a sustained-release effect, and the release amounts at 2 h were 2.50% ± 0.16% and 2.71% ± 0.27% respectively, indicating that there was no burst release phenomenon during the drug release process. Compared with the free Fer-1 group, the cumulative release rates of Fer-1 in the F@PLevs group and the F@PLevs-C group at 24 h were 16.75% ± 2.61% and 18.82% ± 4.64% respectively, indicating that the drug was slowly and continuously released inside the nanon preparation within the first 24 h, and there was a significant difference in the cumulative drug release amount compared with the free drug group (**p≤0.01). When the release time reached 48 h, the cumulative release rates of Fer-1 in the two nanon preparations were 27.23% ± 1.42% and 32.67% ± 3.31% respectively (p>0.05). The above results not only showed that the hybrid vesicles were stable under the condition of pH 7.4 and there was no obvious drug leakage phenomenon, but also showed a sustained-release effect in this release medium, providing a basis for realizing long-circulating targeted delivery in vivo, and the modification of CREKA did not have a great impact on the drug release characteristics of the hybrid vesicles.

[0092] 5. Study on in vitro thrombolytic ability

[0093] After mixing the arterial plasma in SD rats with calcium chloride solution (20 mM) and thrombin solution (10 μM) evenly, 200 μL of the mixture was placed in a transparent 96-well plate. After incubation at a constant temperature and static standing at low temperature, the thrombus mass showed a uniform block structure without obvious cracks and breakages on the surface. On the premise of ensuring the integrity of the thrombus mass structure, the thrombus masses were weighed separately and placed in a 12-well plate for in vitro thrombolytic experiments to investigate the in vitro thrombolytic ability of F@PLevs-C&U. The thrombus masses were randomly divided into five groups with 3 replicates. On the premise of ensuring that the UPA concentration in each preparation was 40 μg / mL, PBS, urokinase-type plasminogen activator (UPA), F@PLevs-C, F@PLevs-U, and F@PLevs-C&U were added to the thrombus masses respectively. The dissolution of the thrombus masses was investigated by observing the weight and appearance of the thrombus masses at 0, 1, 2, 3, and 4 h under static state at 37 °C, and the thrombolytic efficiency was calculated by the mass of the thrombus masses before the start of thrombolysis in each group and the weights of the thrombus masses at different time points.

[0094] Use uniform-sized blood clots weighing about 0.04 - 0.06 mg, and incubate the free drug UPA, the drug-free preparation F@PLevs-C, and the preparation groups F@PLevs-C&U and F@PLevs-U containing thrombolytic drugs with the blood clots for 4 h. The results are as Figure 9 shown. It can be seen from the results that the weights of the blood clots in the PBS group and the F@PLevs-C group changed slightly at 1 h, decreasing from 0.0448 ± 0.01 and 0.0537 ± 0.005 to 0.0407 ± 0.008 and 0.0433 ± 0.004 respectively. It is speculated that this is due to slight loosening of the blood clots caused by physical operations, resulting in slight dissolution. Within 2 - 4 h, the weight of the blood clots in the F@PLevs-C group continued to decrease and the thrombolysis rate gradually increased to 26.86% ± 3.02%. There was no significant difference compared with the PBS group, indicating that F@PLevs-C does not contain fibrinolytic components, which verifies the safety of the preparation from the side. By analyzing the groups containing the thrombolytic drug UPA, when the concentration of UPA was 40 μg / mL, the volume of the blood clots in the free UPA group gradually decreased, and the thrombolysis rates at 1 h, 2 h, 3 h, and 4 h reached 49.29% ± 1.53%, 61.96% ± 2.26%, 72.08% ± 2.91%, and 78.95% ± 3.53% respectively, indicating that the thrombolytic drug UPA can achieve rapid in vitro dissolution of blood clots and still maintain fibrinolytic activity within 4 h. Therefore, compared with the free drug UPA group, the thrombolysis rate of the F@PLevs-U group was lower in the first 3 h, and the thrombolysis rate only reached 54.08% at 3 h, indicating that the release rate of the UPA drug coupled to the surface of the hybrid vesicles is relatively slow. The reason is speculated to be that urokinase is tightly connected through the addition reaction of maleimide and thiol, resulting in slow release of UPA in the buffer. However, the blood clots in the F@PLevs-C&U group showed excellent dissolution performance, and the thrombolysis rate reached 75.70% ± 3.11% at 4 h. And at 3 h, obvious splitting of individual blood clots could be observed, that is, one blood clot broke into two smaller blood clots. This phenomenon is consistent with the research basis that urokinase can enter the interior of the blood clot to cause surface dissolution and internal collapse of the blood clot, as well as the research conclusion that the fibrin-targeting peptide CREKA increases the penetration and adhesion of the nanoplatform in the blood clot. Therefore, F@PLevs-C&U has good application prospects and thrombolysis performance as a targeted delivery carrier for thrombolytic drugs to dissolve blood clots at the thrombus site.

Claims

1. A biomimetic nanocarrier loaded with ferroptosis inhibitors, characterized in that, It is prepared by the following steps: Step 1: After the Lactobacillus plantarum WCFS1 is expanded and cultured, bacterial vesicles are prepared by ultrafiltration concentration-ultracentrifugation method; Step 2: Platelets are separated, and platelet membranes are extracted by the repeated freeze-thaw method; Step 3: The platelet membranes are mixed with phospholipids, and platelet membrane vesicles are obtained after ultrasonic treatment. Then, the bacterial vesicles are mixed and extruded to obtain hybrid vesicles. The hybrid vesicles are mixed with the ferroptosis inhibitor Fer-1 to obtain drug-loaded hybrid vesicles; Step 4: Urokinase is conjugated to the surface of the drug-loaded hybrid vesicles through Sulfo-SMCC; Step 5: Incubate the drug-loaded hybrid vesicles conjugated with urokinase with DSPE-PEG 2000 -CREKA to obtain the biomimetic nanocarrier loaded with ferroptosis inhibitor.

2. The biomimetic nanocarrier according to claim 1, characterized in that, The phospholipid DSPE-PEG 2000 has a mass ratio with platelet membrane protein of 1:1 - 4:

1.

3. The bionic nanocarrier according to claim 1, wherein The mass ratio of the bacterial vesicle protein to the platelet membrane protein is 1:20 - 1:

5.

4. The biomimetic nanocarrier according to claim 1, characterized in that, The mass ratio of the ferroptosis inhibitor Fer-1 to the platelet membrane protein is 1:5 - 1:

20.

5. The biomimetic nanocarrier according to claim 1, characterized in that, The mass ratio of urokinase to the platelet membrane protein is 1:

1.

6. The biomimetic nanocarrier according to claim 1, wherein The DSPE-PEG 2000 -CREKA to platelet membrane protein mass ratio is 1:

4.

7. Use of the biomimetic nanocarrier loaded with ferroptosis inhibitor according to any one of claims 1 - 6 in the preparation of antithrombotic drugs.

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