Blood cell membrane camouflage bacteria-driven micro-robot drug delivery system, preparation method and application

Abstract

This invention discloses a blood cell membrane-masked bacterial-driven microrobot drug delivery system, belonging to the field of microrobot technology. Addressing the low targeted delivery efficiency of existing technologies, this system utilizes engineered probiotic EcN as a self-driven motor, with its surface modified with platelet-neutrophil hybrid membrane nanovesicles encapsulating the thrombolytic drug rt-PA. This system leverages the autonomous movement and natural rod-like structure of the probiotics to achieve active targeting and deep penetration of thrombi. Through the synergistic effect of the platelet-neutrophil dual membranes, it accurately identifies thrombi and triggers drug release in response to PLA2 within the thrombus microenvironment. Experiments show that this system significantly prolongs the drug half-life (approximately 177 times that of free drug), effectively reduces the risk of bleeding, and simultaneously achieves rapid thrombolysis and inhibits secondary thrombus formation. This system provides a new strategy for the efficient and safe treatment of thrombotic diseases.

Description

Technical Field

[0001] This invention belongs to the field of microrobot technology, specifically relating to a blood cell membrane disguised as a bacterial-driven microrobot drug delivery system, its manufacturing method, and its application. Background Technology

[0002] Thrombotic diseases (such as myocardial infarction and stroke) are among the leading causes of death and disability worldwide. Currently, thrombolytic drugs are the primary treatment in clinical practice, with recombinant tissue plasminogen activator (rt-PA) being the most commonly used. However, traditional thrombolytic drug therapy has the following main technical limitations: (1) Systemic administration of free drugs: Thrombolytic drugs such as rt-PA are directly injected intravenously, and the drugs reach the thrombus site through blood circulation to exert thrombolytic effects.

[0003] (2) Cell membrane-masked nanocarrier drug delivery system: Thrombolytic drugs are encapsulated in natural cell membranes such as platelet membranes and erythrocyte membranes to prepare nanoscale vesicles or nanoparticles. The natural targeting properties of cell membranes enhance the accumulation of drugs at the thrombus site. For example, nanocarriers encapsulated in platelet membranes can bind to activated platelets at the thrombus site through receptors on the platelet membrane surface, achieving targeted delivery.

[0004] (3) Artificial synthetic micromotor system: Develop micromotors with autonomous movement capabilities, such as tubular, rod-shaped or spherical micromotors, driven by chemical fuels (such as H2O2) or external fields (magnetic field, sound field) to carry drugs to the thrombus site and improve drug delivery efficiency.

[0005] The above technical solution has the following disadvantages: (1) Short half-life of free drug and high risk of bleeding: The biological half-life of free rt-PA is extremely short (only about 4 minutes), requiring frequent administration of large doses, which significantly increases the risk of serious bleeding complications such as intracranial hemorrhage.

[0006] (2) Limited efficiency of passive targeted delivery: Traditional cell membrane camouflaged nanocarriers mainly rely on chemokine gradients, hemodynamics and passive diffusion mechanisms to accumulate in the lesion site. This is a passive targeting strategy, which has limited delivery efficiency and timeliness, making it difficult to achieve rapid and efficient thrombus targeted delivery.

[0007] (3) Insufficient thrombus penetration ability: Traditional nanocarriers (such as liposomes, polymer nanoparticles, and inorganic nanoparticles) lack self-propulsion ability and mainly rely on passive diffusion or hemodynamic transport, making it difficult to penetrate deep into the thrombus, resulting in insufficient drug accumulation in the core of the thrombus and unsatisfactory thrombolytic effect.

[0008] (4) Poor biocompatibility of artificial micromotors: Most existing synthetic micromotors rely on toxic fuels (such as H2O2) or external field drive, which have biocompatibility problems and limit their autonomous navigation ability in complex vascular networks; and although artificially synthesized non-spherical carriers can improve circulation, they cannot generate continuous autonomous movement, and surface functionalization often damages the activity of target ligands.

[0009] (5) Limited single-target effect: Targeting strategies that use only cell membrane camouflage (such as only platelet membrane or only neutrophil membrane) have limited targeting ability and cannot make full use of the pathological mechanisms involving multiple cells in the process of thrombosis to achieve efficient targeting.

[0010] (6) Biosafety issues of probiotic carriers: When probiotics are used directly as drug carriers for intravenous administration, the probiotics may multiply in the body, causing inflammatory reactions and liver and kidney toxicity, posing a biosafety risk. Summary of the Invention

[0011] To address one or more of the above-mentioned deficiencies or improvement needs of the existing technology, the present invention provides a blood cell membrane-masked bacterial-driven microrobot drug delivery system, including... Engineered probiotics, and drug-loaded blood cell membrane hybrid nanovesicles modified on the surface of the engineered probiotics; The drug-loaded blood cell membrane hybrid nanovesicles include hybrid membrane vesicles of platelet membrane and neutrophil membrane, and thrombolytic drugs loaded in the hybrid membrane vesicles of platelet membrane and neutrophil membrane.

[0012] As a further improvement of the present invention, the engineered probiotic is Escherichia coli Nissle1917.

[0013] As a further improvement of the present invention, the thrombolytic drug is a recombinant tissue plasminogen activator.

[0014] As a further improvement of the present invention, the drug-loaded blood cell membrane hybrid nanovesicles are modified on the surface of the engineered probiotics via biotin-streptavidin coupling.

[0015] As a further improvement of the present invention, the particle size of the drug-loaded blood cell membrane hybrid nanovesicles is 100-110 nm.

[0016] The present invention also provides a method for preparing the above-mentioned blood cell membrane camouflaged bacterial-driven microrobot drug delivery system, characterized by comprising the following steps: S1. Neutrophil membranes and platelet membranes were extracted separately. S2. Mix the neutrophil membrane and platelet membrane to prepare platelet-neutrophil hybrid membrane vesicles; S3. Encapsulate the thrombolytic drug inside the platelet-neutrophil hybrid membrane vesicle to obtain drug-loaded blood cell membrane hybrid nanovesicles; S4. Modify the drug-loaded hybridization membrane nanovesicles onto the surface of engineered probiotics.

[0017] As a further improvement of the present invention, the preparation of the platelet-neutrophil hybrid membrane vesicles includes: Step 1, resuspending equal amounts of neutrophil precipitate and platelet precipitate in PBS and repeatedly freezing and thawing them; Step 2: Centrifuge the lysed suspension and collect the supernatant and precipitate separately. Resuspend the precipitate in PBS and centrifuge to obtain a mixed cell membrane vesicle precipitate. Step 3: Resuspend the mixed cell membrane vesicle pellet in PBS containing protease inhibitors, centrifuge and wash to obtain purified platelet-neutrophil hybrid membrane vesicles.

[0018] As a further improvement of the present invention, the preparation of the drug-loaded hybrid membrane nanovesicles includes: mixing the platelet-neutrophil hybrid membrane nanovesicles with a thrombolytic drug at a mass ratio of 10:1, ultrasonicating, and then homogenizing by extrusion through a 200nm polycarbonate porous membrane.

[0019] As a further improvement of the present invention, the modification specifically includes: biotinylation modification of the drug-loaded hybrid membrane nanovesicles, biotinylation modification and streptavidin modification of the engineered probiotics in sequence, and then incubation and combination of the biotinylated drug-loaded hybrid membrane nanovesicles and the engineered probiotics that have been biotinylated and streptavidin modified in sequence.

[0020] The present invention also provides the application of the above-described delivery system in a medicament for treating thrombotic diseases.

[0021] In summary, the beneficial effects of the above-described technical solutions conceived by this invention compared with the prior art include: (1) Significantly prolongs the drug half-life and improves bioavailability In vivo pharmacokinetic studies showed that the biological half-life of free rt-PA is only about 0.07 hours, while the biological half-life of EcN-PN / rt-PA is significantly prolonged to 13.52 hours, with a mean residence time of 19.51 hours, approximately 177 times that of free rt-PA. This significant prolongation of the drug half-life reduces the frequency of dosing and the systemic dosage, while achieving a sustained thrombolytic effect and effectively inhibiting secondary thrombosis.

[0022] (2) Autonomous movement capability, significantly improving the efficiency of targeted delivery. This invention utilizes the natural flagellar structure of EcN probiotics as a bio-motor, generating autonomous movement through flagellar waving. Experimental results show that EcN-PN / rt-PA retains most of the movement capabilities of EcN, exhibiting a typical "run-tumble" zigzag movement trajectory. Compared with traditional passively diffused nanocarriers, autonomous movement enables the microrobots to actively migrate towards the thrombus site, significantly improving the timeliness and efficiency of drug delivery. In vitro Transwell experiments confirmed that EcN-PN / rt-PA's targeting ability for platelets and neutrophils is significantly higher than that of unmodified EcN, and the migration amount in the 37℃ active movement group is significantly higher than that in the 0℃ static inhibition group, demonstrating that the targeting effect mainly comes from the synergistic effect of active movement and specific recognition.

[0023] (3) Natural rod-shaped structure, which enhances the depth of thrombus penetration. This invention utilizes the natural rod-shaped morphology of EcN probiotics (approximately 2-3 μm in length), which exhibits superior thrombus penetration ability compared to traditional spherical carriers. The non-spherical structure generates a torsional-rotational complex motion during circulation, significantly enhancing its interaction with thrombi on the blood vessel wall while simultaneously reducing macrophage clearance and prolonging the blood circulation half-life. In vitro thrombus penetration experiments have confirmed that EcN-PN / rt-PA can effectively penetrate into the thrombus, reaching deep thrombus tissue at both 1 and 3 hours, thus enabling deep thrombolysis.

[0024] (4) Highly effective thrombolytic effect, inhibiting secondary thrombosis In vitro thrombolysis experiments showed that the thrombolytic volume in the EcN-PN / rt-PA group was significantly higher than that in the free rt-PA group and the PN / rt-PA group within 6 hours. In vivo carotid artery thrombosis model experiments further confirmed that the EcN-PN / rt-PA group had a faster thrombolysis rate, with significant blood flow restoration observed within 30 minutes, and no secondary thrombosis occurred within 210 minutes, while the free rt-PA group developed blood flow obstruction (secondary thrombosis) at 180 minutes. H&E and Masson staining results also confirmed that the EcN-PN / rt-PA group had the best thrombolytic therapeutic effect.

[0025] (5) Good blood compatibility In vitro hemolysis experiments showed that the hemolysis rate of EcN-PN / rt-PA was less than 5%, which meets the blood compatibility requirements of biomaterials and provides a safety basis for intravenous administration.

[0026] (6) Dual targeting of platelets and neutrophil membranes enables precise thrombus identification. This invention innovatively hybridizes platelet membranes and neutrophil membranes to prepare nanovesicles, endowing the microrobots with dual thrombus targeting capabilities. The platelet membrane can specifically bind to activated platelets at the thrombus site via surface receptors, while the neutrophil membrane responds to the inflammatory chemokine gradient at the thrombus site. The synergistic effect of the two cell membranes significantly enhances the targeting capability compared to a single cell membrane camouflage strategy. In vivo small animal imaging experiments showed that EcN-PN / rt-PA exhibited significant fluorescence enrichment at the thrombus site within 10 minutes of administration, with the fluorescence intensity gradually increasing over time, significantly higher than the free rt-PA group and the single-membrane vesicle group.

[0027] (7) Surface modification significantly improves the biosafety of probiotics This invention effectively inhibits the proliferation of probiotics by modifying the surface of EcN with hybrid membrane nanovesicles. OD600nm measurement and agar plate colony counting experiments both showed that the proliferation rate of PN / rt-PA modified EcN was significantly reduced, while maintaining a high survival rate. In vivo safety evaluation showed that the EcN-PN / rt-PA group had no significant differences in blood routine and blood biochemical indicators compared to the control group, and no obvious pathological damage was observed in the H&E staining of major organs; while the unmodified EcN group showed significant inflammatory reactions and liver and kidney toxicity. This indicates that the surface modification strategy successfully solves the biosafety issues of intravenous administration of probiotics, providing an important guarantee for clinical translation.

[0028] (8) PLA2-responsive drug release reduces the risk of bleeding. This invention utilizes PLA2, which is highly expressed at the thrombus site, as a triggering factor to achieve responsive drug release at the thrombus site. In the thrombus microenvironment, PLA2 hydrolyzes platelet membranes, causing vesicle rupture and releasing loaded rt-PA. In normal blood circulation, the drug is stably encapsulated within the vesicles, reducing drug exposure to non-target sites. In vivo bleeding time experiments confirmed that the bleeding time in the EcN-PN / rt-PA group was significantly shorter than that in the free rt-PA group, indicating that this system can significantly reduce the risk of bleeding complications while ensuring thrombolytic efficacy.

[0029] In summary, the blood cell membrane camouflaged bacteria-driven microrobot delivery system of the present invention achieves efficient and safe thrombolytic therapy through multiple synergistic effects of bio-self-driving, dual-target recognition, deep-penetration delivery, responsive release, and high biocompatibility, providing a novel technical solution for the treatment of thrombotic diseases. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0031] In this embodiment, the present invention provides a blood cell membrane disguised bacteria-driven microrobot drug delivery system. The system uses engineered probiotic Escherichia coli Nissle1917 (EcN) as a self-driven motor and drug carrier. Platelet-neutrophil membrane hybrid nanovesicles loaded with the thrombolytic drug rt-PA are modified on its surface to construct a thrombosis treatment microrobot (EcN-PN / rt-PA) with dual-targeting, self-driven, and deep-penetrating characteristics.

[0032] I. Extraction and purification of neutrophil membrane (NEM) Step 1: Isolation of neutrophils Neutrophils were isolated from mouse peripheral blood using a discontinuous density gradient method. Specifically, peripheral blood was collected from 8-week-old male C57BL / 6 mice and resuspended in RPMI-1640 medium to prepare a blood cell suspension. A gradient interface was formed by adding 1 mL of Histopaque-1119 and 1 mL of Histopaque-1077 sequentially to a 15 mL sterile centrifuge tube. 2 mL of the blood cell suspension was carefully added dropwise to the upper layer of the gradient medium, and the mixture was centrifuged at 700×g at room temperature for 45 min. After centrifugation, the solution within 0.5 cm above the bottom opaque layer was collected, washed with 10 mL of PBS, centrifuged at 800×g for 5 min, and the supernatant was discarded to obtain the purified neutrophil pellet.

[0033] Step 2: Extraction of neutrophil membranes Resuspend the neutrophil pellet in PBS and adjust the concentration to 1×10⁻⁶. 91 mL of cell suspension was added to a 2 mL cryovial and rapidly frozen in liquid nitrogen for 10 min. Immediately after freezing, the mixture was transferred to a 37°C water bath and thawed for 15 min. This freeze-thaw cycle was repeated three times. After the final thawing, the ruptured cell suspension was centrifuged at 2000 × g for 5 min, and the cell membrane vesicles in the supernatant were collected. The obtained neutrophil membrane vesicles were resuspended in PBS containing protease inhibitors, centrifuged at 2000 × g for 5 min, and washed three times to remove organelles and other impurities, yielding purified neutrophil membranes (NEM).

[0034] II. Extraction and Purification of Platelet Membrane (PLM) Step 1: Platelet separation Platelets were isolated using a human peripheral blood platelet separation kit. Specifically, fresh anticoagulated blood was collected from mice and diluted with an equal volume of PBS. 4 mL of platelet separation buffer was added to a centrifuge tube, followed by 2 mL of 60% separation buffer (1200 μL platelet separation buffer + 800 μL tissue diluent) to form a separation interface. Then, 4 mL of diluted anticoagulated blood was added, and the mixture was centrifuged at 350 × g for 20 min at room temperature. After centrifugation, the platelet layer was carefully transferred to a 15 mL centrifuge tube, 10 mL of PBS was added, and the mixture was centrifuged at 500 × g for 20 min. The supernatant was discarded, and the platelet pellet was resuspended in 1 mL of PBS to obtain a platelet suspension.

[0035] Step 2: Platelet membrane extraction Take 1 mL of platelet suspension (concentration 1×10⁻⁶) 9 Platelet vesicles (platelets / mL) were added to a 2 mL cryovial and rapidly frozen in liquid nitrogen for 10 min. Immediately after freezing, the vesicles were transferred to a 37°C water bath and thawed for 15 min. This freeze-thaw cycle was repeated three times. After the final thawing, platelet membrane vesicles were prepared by centrifugation at 2000×g for 5 min. The obtained platelet membrane vesicles were resuspended in PBS containing protease inhibitors, centrifuged at 2000×g for 5 min, and washed three times to obtain purified platelet membranes (PLM).

[0036] III. Preparation of drug-loaded hybrid membrane nanovesicles (PN / rt-PA) Step 1: Preparation of platelet-neutrophil hybridization membrane Take an equal amount of neutrophil pellet (1×10⁻⁶) 9 (cells / mL) and platelet precipitation (1×10⁻⁶) 9(cells / mL), resuspended in 2 mL PBS and mixed thoroughly. The mixed cell suspension was flash-frozen in liquid nitrogen for 1 min, then rapidly thawed at 37°C for 3 min, repeated 3 times. After the last thawing, the supernatant was collected by centrifugation at 4°C, 1500 rpm for 10 min; the precipitate was resuspended in PBS and centrifuged again at 4°C, 14000 rpm for 30 min to obtain the mixed cell membrane vesicle precipitate. The hybrid membrane vesicles were resuspended in PBS containing protease inhibitors, centrifuged at 2000g for 5 min, and washed 3 times to obtain purified platelet-neutrophil hybrid membrane vesicles (PN).

[0037] Step 2: Packet loading of rt-PA Purified hybrid membrane vesicles were mixed with the thrombolytic drug rt-PA at a mass ratio of 10:1 and sonicated at 20% power for 10 min to allow the drug to enter the membrane vesicles. Then, using an Avestin micro-extruder, the vesicles were extruded five times through a 200 nm polycarbonate porous membrane to homogenize the vesicle size, ultimately obtaining drug-loaded hybrid membrane nanovesicles PN / rt-PA. The obtained PN / rt-PA nanovesicles had a particle size of approximately 105.71 ± 2.8 nm, exhibited a distinct core-shell structure, a zeta potential of -11.32 mV, an encapsulation efficiency of 87.27% for rt-PA, and a drug loading of 28.52%.

[0038] IV. Preparation of Blood Cell Membrane-Made Probiotic Microrobots (EcN-PN / rt-PA) Step 1: Cultivation of EcN probiotics Escherichia coli Nissle1917 (EcN) was inoculated into LB liquid medium and cultured at 37°C with shaking at 220 rpm until the logarithmic growth phase, yielding a large number of highly active EcN probiotics. The obtained EcN were rod-shaped, approximately 2-3 μm in length, and possessed a natural flagellar structure.

[0039] Step 2: Biotin-streptavidin-mediated surface modification PN / rt-PA nanovesicles were modified onto the EcN surface using a biotin-streptavidin binding strategy: ① Preparation of biotinylated PN / rt-PA: sulfo-NHS-biotin (1 mL, 500 μM PBS solution) was mixed with PN / rt-PA solution (1 mL, 1 mg / mL), and the mixture was stirred in the dark for 30 min. After the reaction, the mixture was dialyzed in PBS for 12 h using a 2 kDa dialysis bag to obtain biotinylated (PN / rt-PA)-Biotin, which was dispersed in 2 mL PBS and stored at 4 °C.

[0040] ② Biotinylation and modification of EcN: Take EcN (1×10 4CFU was sequentially incubated with sulfo-NHS-biotin (1 mM, 500 μL), streptavidin (1 mM, 500 μL), and biotinylated (PN / rt-PA)-Biotin (1 mg, 500 μL) at 37 °C for 1 h each. After each incubation, the cells were washed three times with PBS to remove unbound material. The resulting probiotic microrobot EcN-PN / rt-PA, disguised as a blood cell membrane, was stored at 4 °C for later use. The encapsulation efficiency of rt-PA in the obtained EcN-PN / rt-PA was approximately 65.73%, the drug loading was approximately 21.71%, and the rt-PA activity retention rate was approximately 68%.

[0041] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions that fall within the scope of the present invention are within the scope of protection of the present invention.

[0042] The terms "first" and "second" in this invention are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0043] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0044] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

Claims

1. A blood cell membrane-masked microrobot drug delivery system, characterized in that, include Engineered probiotics, and drug-loaded blood cell membrane hybrid nanovesicles modified on the surface of the engineered probiotics; The drug-loaded blood cell membrane hybrid nanovesicles include hybrid membrane vesicles of platelet membrane and neutrophil membrane, and thrombolytic drugs loaded in the hybrid membrane vesicles of platelet membrane and neutrophil membrane.

2. The delivery system according to claim 1, characterized in that, The engineered probiotic is Escherichia coli Nissle1917.

3. The delivery system according to claim 1, characterized in that, The thrombolytic drug is a recombinant tissue plasminogen activator.

4. The delivery system according to claim 1, characterized in that, The drug-loaded blood cell membrane hybrid nanovesicles are modified on the surface of the engineered probiotics via biotin-streptavidin coupling.

5. The delivery system according to claim 1, characterized in that, The particle size of the drug-loaded blood cell membrane hybrid nanovesicles is 100–110 nm.

6. A method for preparing a blood cell membrane-masked bacterial-driven microrobot drug delivery system as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Neutrophil membranes and platelet membranes were extracted separately. S2. Mix the neutrophil membrane and platelet membrane to prepare platelet-neutrophil hybrid membrane vesicles; S3. Encapsulate the thrombolytic drug inside the platelet-neutrophil hybrid membrane vesicle to obtain drug-loaded blood cell membrane hybrid nanovesicles; S4. Modify the drug-loaded hybridization membrane nanovesicles onto the surface of engineered probiotics.

7. The preparation method according to claim 6, characterized in that, The preparation of the platelet-neutrophil hybrid membrane vesicles includes: Step 1, resuspending equal amounts of neutrophil and platelet precipitates in PBS and repeatedly freezing and thawing them; Step 2: Centrifuge the lysed suspension and collect the supernatant and precipitate separately. Resuspend the precipitate in PBS and centrifuge to obtain a mixed cell membrane vesicle precipitate. Step 3: Resuspend the mixed cell membrane vesicle pellet in PBS containing protease inhibitors, centrifuge and wash to obtain purified platelet-neutrophil hybrid membrane vesicles.

8. The preparation method according to claim 6, characterized in that, The preparation of the drug-loaded hybrid membrane nanovesicles includes: mixing the platelet-neutrophil hybrid membrane nanovesicles with a thrombolytic drug at a mass ratio of 10:1, ultrasonicating, and then homogenizing by extrusion through a 200nm polycarbonate porous membrane.

9. The preparation method according to claim 6, characterized in that, The modification specifically includes: biotinylating the drug-loaded hybrid membrane nanovesicles, sequentially biotinylating and streptavidin-modifying the engineered probiotics, and then incubating and combining the biotinylated drug-loaded hybrid membrane nanovesicles with the engineered probiotics sequentially modified with biotinyl and streptavidin.

10. The use of a delivery system according to any one of claims 1 to 5 in a medicament for treating thrombotic diseases.