A tPA delivery system based on tetrahedral DNA origami structure, its preparation method and application

Abstract

This invention proposes a tPA delivery system based on a tetrahedral DNA origami structure, its preparation method, and its applications, relating to the biomedical field. It includes a carrier composed of a tetrahedral DNA origami structure, a thrombus-targeting peptide, and short-chain DNA. The thrombus-targeting peptide and short-chain DNA are pre-coupled to form a peptide-short-chain complex, which is grafted onto the tetrahedral DNA origami structure. tPA and short-chain DNA are pre-coupled and loaded onto the edges of the tetrahedral DNA origami structure. Using the tetrahedral DNA origami structure as the core carrier, the vertices can be precisely modified with thrombus-targeting peptides to achieve targeting of the thrombus site. The tPA molecule is loaded onto the edges of the DNA origami structure via sulfo-SMCC linkage, and its release is controlled by introducing a thrombin-responsive DNA locking mechanism. This invention overcomes the limitations of current tPA thrombolytic therapy, enabling on-demand release of tPA during treatment, achieving local thrombolysis while reducing the risk of systemic bleeding.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a tPA delivery system based on a tetrahedral DNA origami structure, its preparation method, and its application. Background Technology

[0002] The core mechanism of thrombosis in ischemic stroke involves multiple steps, including vascular endothelial injury, platelet activation and aggregation, and activation of the coagulation system. Following the rupture or erosion of atherosclerotic plaques, subendothelial collagen and von Willebrand factor (vWF) are exposed. Platelets rapidly adhere and activate via surface glycoprotein receptors (such as GPIb-IX-V and GPVI), releasing mediators such as ADP and thromboxane A2 (TxA2), further recruiting and activating peripheral platelets to form initial platelet aggregates (white thrombi). Simultaneously, exposed tissue factor (TF) initiates the extrinsic coagulation pathway, generating thrombin, which promotes the conversion of fibrinogen into fibrin, reinforcing the platelet thrombus and forming a stable mixed thrombus.

[0003] Currently, the standard treatment for ischemic stroke mainly includes intravenous thrombolysis and endovascular mechanical thrombectomy. Among these, recombinant tissue plasminogen activator (rt-PA / tPA) is the only widely approved drug for acute-phase intravenous thrombolysis, effectively degrading fibrin and dissolving thrombi. However, the clinical application of tPA still faces many serious challenges: a narrow treatment window (usually within 4.5 hours of symptom onset), poor systemic targeting, high bleeding risk (especially intracranial hemorrhage), and re-occlusion after recanalization, limiting its efficacy and applicable population.

[0004] DNA origami technology, with its precisely programmable nanoscale spatial arrangement capabilities, has become a core platform for targeted delivery and precision medicine in the biomedical field. Peptide functionalization is a crucial step in endowing DNA origami with functions such as targeted recognition and bioactivity regulation. However, directly loading peptides onto DNA origami still presents many drawbacks. For example: Traditional direct covalent coupling methods typically crosslink peptides directly to active groups such as amino and thiol groups on DNA origami. Because active sites are widely distributed on the origami, problems such as random grafting sites and uncontrollable numbers can easily occur, and it may even destroy the secondary folding structure of DNA origami. Non-specific adsorption methods cannot achieve site-specific modification at all, and the grafting position and number are uncontrollable. Chemical cross-linking requires extreme pH and high temperature reaction conditions, which can easily cause DNA origami base pairing disorder and structural disintegration, and can also lead to irreversible damage to the higher-order structure of peptides. Traditional non-specific adsorption methods have extremely low grafting efficiency, and peptides are prone to desorption in physiological environments, resulting in extremely poor stability. Direct covalent coupling methods are prone to producing multimeric byproducts of peptides and DNA, resulting in the mixing of free raw materials and target products. Subsequent purification steps are cumbersome, product recovery rates are low, and batch-to-batch stability is poor.

[0005] Therefore, developing novel delivery systems that can precisely target thrombus sites, control drug release on demand, and have good safety profiles is of great significance to current research on stroke treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a tPA delivery system based on a tetrahedral DNA origami structure, its preparation method, and its applications. The system uses a tetrahedral DNA origami structure as its core carrier, with thrombus-targeting peptides precisely modified at its vertices to achieve targeted delivery to the thrombus site. tPA molecules are loaded onto the edges of the DNA origami structure via sulfo-SMCC linkages, and their release is controlled by introducing a thrombin-responsive DNA locking mechanism. This overcomes the limitations of current tPA thrombolytic therapy, enabling on-demand release of tPA during treatment, achieving local thrombolysis while reducing the risk of systemic bleeding.

[0007] The technical problem solved by this invention is achieved by the following technical solution.

[0008] On one hand, embodiments of this application provide a tPA delivery system based on a tetrahedral DNA origami structure, comprising a carrier composed of a tetrahedral DNA origami structure, a thrombosis-targeting polypeptide, and short-chain DNA, with tPA loaded on the edges of the carrier.

[0009] Furthermore, the thrombosis-targeting peptide and short-chain DNA are pre-coupled to form a peptide-short-chain complex, which is then grafted onto a tetrahedral DNA origami structure.

[0010] Furthermore, tPA and short-chain DNA are pre-coupled and then loaded onto the edges of a tetrahedral DNA origami structure via chain-to-chain complementarity.

[0011] Furthermore, it is characterized in that the short-chain DNA sequence is as shown in SEQ ID No. 1.

[0012] Secondly, embodiments of this application provide a method for preparing a tPA delivery system based on a tetrahedral DNA origami structure, comprising the following steps: S1: Select short chains, scaffold chains, buffer, magnesium chloride and ultrapure water in a volume ratio of 20-30:10-20:5-10:8-12:15-25 respectively, mix them in an ice bath, centrifuge them, and then perform three-stage annealing by PCR to obtain tetrahedral DNA origami structures. S2: Prepare thiol-modified short-chain DNA stock solution, thrombosis-targeting peptide stock solution, TCEP solution, SPDP solution and PBS buffer respectively; then mix the thiol-modified short-chain DNA stock solution and TCEP solution at a volume ratio of 1:1 and incubate at a constant temperature for 12 h to obtain activated thiol-modified short-chain DNA stock solution. The thrombus-targeting peptide stock solution, PBS buffer and SPDP solution were mixed at a volume ratio of 10-20:1-3:5-8 and incubated in the dark for 12 hours to obtain the modified thrombus-targeting peptide stock solution. After mixing the activated thiol-modified short-chain DNA stock solution and the modified thrombus-targeting peptide stock solution, the mixture was incubated at room temperature for 16 hours to obtain the peptide-short-chain complex. S3: Mix the ssDNA stock solution and TCEP solution at a volume ratio of 1:1 and incubate at a constant temperature for 12 hours to obtain the activated ssDNA stock solution. Prepare a sulfo-SMCC stock solution and a tPA solution. Then, add the sulfo-SMCC stock solution dropwise to the tPA solution at a volume ratio of 1:10. After mixing, react at 4°C in the dark for 3 hours. After the reaction is complete, centrifuge to obtain the tPA-SMCC conjugate. The tPA-SMCC conjugate and activated ssDNA stock solution were mixed and incubated at 4°C in the dark for 12 h. After the reaction was completed, the mixture was centrifuged to obtain the tPA-ssDNA conjugate. S4: Mix the tPA-ssDNA conjugate and the polypeptide-short chain complex, and incubate at room temperature to obtain a tPA delivery system based on a tetrahedral DNA origami structure.

[0013] Furthermore, in step S1, the scaffold strand is a p7249 single-stranded circular DNA scaffold strand.

[0014] Furthermore, the product after annealing is purified by centrifugation. The purification steps are as follows: centrifuge the annealed product at 3000 rpm for 5 min, and repeat 8 times.

[0015] Furthermore, the concentration of the TCEP solution was 8 mM.

[0016] Thirdly, embodiments of this application provide the application of the above-mentioned tPA delivery system based on a tetrahedral DNA origami structure in the preparation of in vitro thrombolytic agents.

[0017] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. This invention employs a two-step design of "short-chain DNA pre-coupling - base complementary pairing anchoring," ensuring that the grafting site is entirely determined by the pre-defined extended complementary sequence of the tetrahedral DNA origami structure. This allows for the precise positioning of thrombosis-targeting peptides or other peptides at specific sites such as the vertices of the tetrahedral DNA origami structure. The number of grafts can be quantitatively controlled at the single-molecule level through the copy number of the complementary sequence, avoiding product heterogeneity caused by random modifications, ensuring the uniformity of the DNA origami structure, and laying a structural foundation for the synergistic function of the peptides. 2. The reaction conditions of this invention are mild. The covalent coupling of peptides and short-chain DNA is carried out at room temperature and in a neutral pH environment. The base complementary pairing step can be completed efficiently under physiological conditions. There is no heavy metal catalysis or extreme environmental stimulation. It can preserve the bioactivity of peptides and the integrity of tetrahedral DNA origami structure to the greatest extent, and is suitable for application scenarios with strict requirements for bioactivity, such as in vivo targeted drug delivery. 3. This invention enables the pre-coupling of peptides with different functions to short-chain DNA with different complementary sequences, and then the simultaneous, site-specific, and quantitative grafting of multiple peptides through multiple sets of complementary sequences preset on a tetrahedral DNA origami structure. The grafting processes of different peptides do not interfere with each other, and can flexibly achieve the synergistic integration of multiple functions such as targeting, thrombolysis, and anti-inflammation, greatly expanding the functional design space of DNA origami nanostructures. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a gel electrophoresis image of TDNs and control p7249 in Example 1 of the present invention; Figure 2 This is a TEM image of the TDNs in Example 1 of the present invention; Figure 3 This is a gel electrophoresis analysis diagram of the successful synthesis of KQAGDV-L18 in Example 1 of this invention; Figure 4 This is the time-of-flight mass spectrum of tPA grafted onto DNA tetrahedrons in Example 1 of the present invention; Figure 5 This is a comparison of gel electrophoresis analysis of the synthesis efficiency of KQAGDV-L18 at different TCEP concentrations in Example 2 of the present invention; Figure 6 The images show the in vitro thrombolysis of TDNs and the changes in thrombolysis rate and absorbance in Example 3 of this invention. Detailed Implementation

[0020] 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0022] In all embodiments of the present invention, the following English characters represent: L18: A DNA oligonucleotide containing 18 bases; tPA: Tissue plasminogen activator; KQAGDV: Thrombosis-targeting polypeptide; TDNs: Tetrahedral DNA origami structure; sulfo-SMCC: sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester; DMSO: Dimethyl sulfoxide; TCEP: Tris(2-carboxyethyl)phosphine; SPDP: Succinimide 3-(2-pyridyldimercapto)-propionate; BSA: Bovine serum albumin.

[0023] Example 1 This embodiment provides a detailed method for preparing a tPA delivery system based on a tetrahedral DNA origami structure, including the following steps: S1: Preparation of tetrahedral DNA origami structures (TDNs): Weigh 26.12 μL of short-chain (490 nM / μL), 16 μL of scaffold chain 7249 (100 nM / μL), 8 μL of TAE buffer (10X), 9.6 μL of magnesium chloride (100 mM / μL), and 20.28 μL of ultrapure water. Mix well on ice, briefly centrifuge in a PCR centrifuge tube, and then perform PCR preparation according to the following parameters: The TDNs structural assembly employs a three-stage programmed annealing strategy, with the following specific steps: The first stage is isothermal preheating, where the reaction system temperature is adjusted to 65°C and maintained for 15 minutes to lay the foundation for the orderly folding of the nucleic acid chains. The second stage involves slow annealing, where the temperature is initially lowered to 50°C, and then gradually reduced at a rate of 0.1°C every 39.6 minutes until the system temperature drops to 40°C. This slow cooling process takes a total of 66 hours to ensure that the nucleic acid chains are precisely paired according to the preset structure. The third stage is rapid annealing, where, after the temperature reaches 40°C, a rapid cooling mode is switched to, continuously reducing the temperature at a rate of 1°C every 2.4 minutes until it reaches 15°C. This stage lasts for a total of 1 hour.

[0024] After annealing, the product was characterized by agarose gel electrophoresis. The results are as follows: Figure 1 As shown, the electrophoretic migration distance of TDNs is slightly shorter than that of the control p7249, and no obvious polymeric stray bands appear in the electrophoretic pattern. This characterization result confirms that the target TDNs have successfully achieved precise folding. The annealed product was then observed using transmission electron microscopy (TEM), as shown... Figure 2 As shown, it has a distinct tetrahedral structure with a projected side length of approximately 60 nm, which is consistent with the theoretically designed side length of TDNs, proving the successful synthesis of the product.

[0025] S2: Thrombus-targeting polypeptide KQAGDV grafted onto TDNs: Reagent preparation includes: rinsing short-chain DNA (L18) with TAE buffer (10 mM Mg²⁺). + Dissolve the following reagents in anhydrous DMSO (pH 8.0) to prepare a 200 μM stock solution of thiol-modified short-chain DNA (L18), aliquot and store at -20°C for later use; dissolve KQAGDV in anhydrous DMSO and adjust the concentration to 2 mM for later use; prepare a 10 mM TCEP solution using ultrapure water for later use; prepare a 7.78 mM SPDP solution using anhydrous DMSO for later use; additionally prepare a 0.5 mol / L phosphate buffer (PB, pH 7.4) to adjust the pH of the reaction system and ensure coupling efficiency.

[0026] The short DNA sequence is shown in SEQ ID No. 1: AAA ACT CCA ACA CAT AAT AAT-SH.

[0027] Take 1.5 μL of thiol-modified short-chain DNA stock solution and place it in a centrifuge tube. Add 1.5 μL of 10 mM TCEP solution, mix gently, and incubate at 37°C in a metal bath for 12 hours to obtain activated thiol-modified short-chain DNA stock solution. This step utilizes the reducing effect of TCEP to break potential endogenous disulfide bonds in the DNA chain, fully exposing free thiol groups (-SH), providing active sites for subsequent covalent coupling with peptides.

[0028] Take 15 μL of 2 mM KQAGDV solution into a new centrifuge tube, add 2.33 μL of 0.5 mol / L PB buffer and 6 μL of 7.78 mM SPDP solution sequentially, gently vortex to mix, and incubate at room temperature in the dark for 12 hours to obtain the modified thrombus-targeting peptide stock solution. This step utilizes the cross-linking effect of SPDP to introduce active disulfide bonds onto the peptide molecule, constructing a coupling reaction site between the peptide and DNA.

[0029] Then, the activated thiol-modified short-chain DNA stock solution and the modified thrombus-targeting peptide stock solution were transferred to the same centrifuge tube, thoroughly mixed, and incubated at room temperature for 16 hours. Through the specific exchange reaction between thiol (-SH) and disulfide bonds, KQAGDV and L18 were covalently coupled to form a peptide-short-chain complex.

[0030] Furthermore, in this embodiment, to ensure the coupling effect, SDS-PAGE electrophoresis with a 20% separating gel was used to verify the coupling effect. The electrophoresis conditions were a constant voltage of 100 V until the loading baffer migrated to the bottom of the gel. The electrophoresis results are as follows: Figure 3 As shown, the migration rate of the KQAGDV-L18 conjugate band is significantly slower than that of the L18 short-chain DNA and KQAGDV bands alone. Combined with the negative correlation between molecular weight and electrophoretic migration rate, this indicates that the molecular weight of the product is significantly larger than that of the single component, confirming that KQAGDV and L18 short-chain DNA have been successfully covalently conjugated. This lays the foundation for subsequent specific binding and targeting function construction with TDNs.

[0031] S3: tPA load is applied to the edge of TDNs: Referring to S2, the principle and grafting strategy of KQAGDV with TDNs are similar. The coupling of tPA and TDNs also adopts a "pre-coupling" form, that is, a short DNA chain (ssDNA) is first linked to the tPA molecule, and then it binds to the extended sequence on the TDN structure through complementary base pairing. The specific steps are as follows: Take 6 μL of 200 mM thiol-modified ssDNA stock solution, add 6 μL of 10 mM TCEP solution, and incubate for 12 hours in a metal bath at room temperature to obtain activated ssDNA stock solution. This step breaks the potential disulfide bonds in the DNA chain through the reducing effect of TCEP, fully exposing free thiol groups (-SH), providing active sites for subsequent coupling reactions with [specific enzyme name missing].

[0032] Dissolve 70 μg of tPA in 50 μL of 1×PBS buffer (pH 7.4), and separately dissolve 2.7 μg of sulfo-SMCC reagent in 5 μL of anhydrous DMSO to prepare a stock solution. Slowly add the sulfo-SMCC solution dropwise to the BSA solution, gently vortex to mix, and incubate at 4°C in the dark for 3 hours. After the reaction is complete, use an ultrafiltration tube with a molecular weight cutoff of 30 kD and centrifuge at 3000 g for 10 minutes to remove unreacted free sulfo-SMCC from the system, obtaining the purified tPA-SMCC conjugate.

[0033] The purified tPA-SMCC conjugate was then mixed with activated ssDNA stock solution, gently vortexed, and incubated at 4°C in the dark for 12 hours. Covalent binding was achieved through the specific reaction between the maleimide group and the thiol group. After the reaction was complete, the mixture was centrifuged again at 3000 g for 10 minutes using a 30 kD ultrafiltration tube to remove unbound free ssDNA, finally yielding the pure tPA-ssDNA conjugate.

[0034] In this step, because the tPA structure is unstable and may degrade, time-of-flight mass spectrometry was used to verify its synthesis results. The experimental results are as follows: Figure 4 As shown, since the mass of tPA is around 68 kDa and the mass of the bound L18 sequence is around 6 kDa, and the peak shifts to a higher mass, it indicates that the tPA-ssDNA conjugate was successfully synthesized.

[0035] S4: Formal coupling of tPA and TDNs: After mixing the tPA-ssDNA conjugate and the polypeptide-short chain complex at a ratio of 1:5, reacting at 37°C for 30 minutes, and then allowing it to cool naturally overnight, tPA can be conjugated to TDNs.

[0036] Example 2 The steps in this embodiment are basically the same as those in Embodiment 1, except that in step S3, bovine serum albumin (BSA), which has a structure similar to tPA, is used as a substitute model for the study.

[0037] During the experiment, the inventors discovered through repeated experiments that although gel electrophoresis results showed successful synthesis of KQAGDV-L18, the synthesis efficiency was low. Furthermore, too low a TCEP concentration led to incomplete reduction of disulfide bonds on the protein / peptide, resulting in low efficiency and poor reproducibility of subsequent labeling with thiol-based reactive dyes such as Texas Red; while too high a concentration caused excessive reduction, potentially damaging the protein's native structure and leading to protein aggregation and inactivation.

[0038] Therefore, in this embodiment, the concentration of TCEP was readjusted. Three concentrations of 5, 8, and 10 mM were set, and gel electrophoresis was performed after the reaction. The intensity percentage of the corresponding band in the BSA gel electrophoresis (with stable results) was used to observe whether the TCEP concentration could affect the synthesis efficiency of KQAGDV-L18. The experimental results are as follows: Figure 5 As shown, when the TCEP concentration is adjusted to 8 mM, the synthesis efficiency increases from 54% to 78.5%.

[0039] Example 3 This embodiment validates the in vitro thrombolytic effect of the tPA delivery system (TDNs-tPA group, 27 μg / ml TDNs-tPA solution) based on tetrahedral DNA origami structure prepared in Examples 1 and 2, and sets up a pure tPA group (10 μg / ml tPA solution) and a saline group as controls, including the following steps: Several mice with similar growth patterns were selected. Blood was collected from their eyeballs and quickly transferred to the corresponding wells of a sterile 96-well plate, 50 μl per well. To prevent premature blood coagulation, 50 μl of thrombin solution at a concentration of 50 U / ml was added first. The 96-well plate was placed in a 37°C, 5% CO2 constant temperature shaker and incubated at 100 rpm for 30 min to allow the blood to fully coagulate and form a thrombus. After incubation, the supernatant in the wells was carefully aspirated, and 200 μl of sterile physiological saline was added to each well. This washing step was repeated twice.

[0040] Completely transfer the thrombus from the 96-well plate to the corresponding wells of a sterile 24-well plate, adding 1 ml of experimental material precisely to each well. After adding the material, gently shake the 24-well plate to ensure the material evenly coats the thrombus. Place the 24-well plate back into a 37°C, 5% CO2 constant-temperature shaker and incubate at 100 rpm. Samples are taken at six time points: 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 3 h. At each time point, 20 μl of the supernatant from each well is transferred to a new 96-well plate, and the absorbance (A value) at 415 nm and 540 nm wavelengths is measured using a microplate reader. Each sample is tested in triplicate, and the average A value is recorded (reflecting the amount of hemoglobin released during fibrinolysis, indirectly indicating fibrinolytic activity). Simultaneously, at each time point, the thrombus from each well is removed with tweezers and placed on pre-weighed sterile filter paper, and excess moisture is blotted off the filter paper. Immediately weigh the thrombus using an analytical balance, record the data, calculate the rate of change in thrombus weight for each group, and comprehensively evaluate the fibrinolytic effect of the material based on the absorbance results. Experimental results are as follows: Figure 6 As shown, tPA and TDNs-tPA have clear fibrinolytic activity, which can promote thrombus dissolution and release hemoglobin, while reducing thrombus weight and achieving a thrombolysis rate as high as 47%.

[0041] Based on the above experimental conclusions, it can be clearly concluded that TDNs-tPA can be applied to thrombolytic therapy. Under normal conditions, tPA prepared from it is tightly sealed to avoid premature activation. When TDNs-tPA accumulates in the thrombus microenvironment (local thrombin concentration increases), thrombin binds to the aptamer, triggering a conformational change, which allows tPA to be released on demand, achieving local thrombolysis while reducing the risk of systemic bleeding.

[0042] In summary, this invention provides a tPA delivery system based on a tetrahedral DNA origami structure, its preparation method, and its application. Through a two-step design of "short-chain DNA pre-coupling - base complementary pairing anchoring," the grafting site is entirely determined by the pre-defined extended complementary sequence of the tetrahedral DNA origami structure. This allows for precise positioning of thrombosis-targeting peptides or other peptides at specific sites such as the vertices of the tetrahedral DNA origami structure. The grafting number can be quantitatively controlled at the single-molecule level by adjusting the copy number of the complementary sequence, avoiding product heterogeneity caused by random modifications, ensuring the uniformity of the DNA origami structure, and laying a structural foundation for the synergistic function of the peptides. The reaction conditions of this invention are mild. The covalent coupling of peptides and short-chain DNA is carried out at room temperature and in a neutral pH environment. The base complementary pairing step can be completed efficiently under physiological conditions. There is no heavy metal catalysis or extreme environmental stimulation. It can preserve the bioactivity of peptides and the integrity of tetrahedral DNA origami structure to the greatest extent, and is suitable for application scenarios with strict requirements for bioactivity, such as in vivo targeted drug delivery. This invention enables the pre-coupling of peptides with different functions to short-chain DNA with different complementary sequences. Then, through multiple sets of complementary sequences preset on the tetrahedral DNA origami structure, it achieves site-specific and quantitative synchronous grafting of various peptides. Moreover, the grafting processes of different peptides do not interfere with each other, which can flexibly realize the synergistic integration of multiple functions such as targeting, thrombolysis, and anti-inflammation, greatly expanding the functional design space of DNA origami nanostructures.

[0043] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A tPA delivery system based on tetrahedral DNA origami structure, characterized in that, The vector comprises a tetrahedral DNA origami structure, a thrombosis-targeting polypeptide, and short-chain DNA, with tPA loaded on the edges of the vector.

2. The tPA delivery system based on tetrahedral DNA origami structure according to claim 1, wherein, The thrombosis-targeting polypeptide and the short-chain DNA are pre-coupled to form a polypeptide-short-chain complex, which is then grafted onto the tetrahedral DNA origami structure.

3. The tPA delivery system based on tetrahedral DNA origami structure according to claim 1, wherein, The tPA and the short-chain DNA are pre-coupled and then loaded onto the edges of the tetrahedral DNA origami structure through chain complementarity.

4. The tetrahedral DNA origami structure-based tPA delivery system according to any one of claims 1-3, wherein, The short-chain DNA sequence is shown in SEQ ID No.

1.

5. A method for preparing a tPA delivery system based on a tetrahedral DNA origami structure as described in claim 4, characterized in that, Includes the following steps: S1: Select short chains, scaffold chains, buffer, magnesium chloride and ultrapure water in a volume ratio of 20-30:10-20:5-10:8-12:15-25 respectively, mix them in an ice bath, centrifuge them, and then perform three-stage annealing by PCR to obtain tetrahedral DNA origami structures. S2: Prepare thiol-modified short-chain DNA stock solution, thrombosis-targeting peptide stock solution, TCEP solution, SPDP solution and PBS buffer respectively; then mix the thiol-modified short-chain DNA stock solution and TCEP solution at a volume ratio of 1:1 and incubate at a constant temperature for 12 h to obtain activated thiol-modified short-chain DNA stock solution. The thrombus-targeting peptide stock solution, PBS buffer and SPDP solution were mixed at a volume ratio of 10-20:1-3:5-8 and incubated in the dark for 12 hours to obtain the modified thrombus-targeting peptide stock solution. After mixing the activated thiol-modified short-chain DNA stock solution and the modified thrombus-targeting peptide stock solution, the mixture was incubated at room temperature for 16 hours to obtain the peptide-short-chain complex. S3: Mix the ssDNA stock solution and TCEP solution at a volume ratio of 1:1 and incubate at a constant temperature for 12 hours to obtain the activated ssDNA stock solution. Prepare a sulfo-SMCC stock solution and a tPA solution. Then, add the sulfo-SMCC stock solution dropwise to the tPA solution at a volume ratio of 1:

10. After mixing, react at 4°C in the dark for 3 hours. After the reaction is complete, centrifuge to obtain the tPA-SMCC conjugate. The tPA-SMCC conjugate and activated ssDNA stock solution were mixed and incubated at 4°C in the dark for 12 h. After the reaction was completed, the mixture was centrifuged to obtain the tPA-ssDNA conjugate. S4: Mix the tPA-ssDNA conjugate and the polypeptide-short chain complex and incubate at room temperature for 12 h to obtain a tPA delivery system based on a tetrahedral DNA origami structure.

6. The preparation method according to claim 5, characterized in that, In step S1, the scaffold chain is a p7249 single-stranded circular DNA scaffold chain.

7. The preparation method according to claim 6, characterized in that, It also includes centrifugal purification of the annealed product, wherein the purification step is: centrifuging the annealed product at a rate of 3000 rpm for 5 min, and repeating 8 times.

8. The preparation method according to claim 7, characterized in that, The concentration of the TCEP solution is 8 mM.

9. The application of the tPA delivery system based on a tetrahedral DNA origami structure as described in claim 1 in the preparation of in vitro thrombolytic agents.

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