Self-driven functionalized nucleic acid assembly inspired by blood coagulation mechanism and preparation method thereof

Abstract

The invention discloses a self-driven functionalized nucleic acid assembly inspired by a blood coagulation mechanism and a preparation method of the self-driven functionalized nucleic acid assembly, and belongs to the technical field of medical blood coagulation materials. The functionalized nucleic acid assembly is formed by rolling circle amplification of four blood coagulation related functionalized DNA (deoxyribonucleic acid) aptamers as shown in SEQ ID NO: 1-4. In addition, the functionalized nucleic acid assembly is loaded with the spearhead agkistrodon halys hemocoagulase, so that an exogenous material can be enhanced to trigger an organism to activate a blood coagulation cascade reaction. The Chinese herbal medicine hirudin is loaded in the functionalized nucleic acid assembly, so that thrombus formation and circulatory system diseases can be effectively prevented. The functional nucleic acid assembly blood coagulation material is reasonable in structural design and mature and feasible in manufacturing process, can be widely applied to treatment of complex wounds such as hidden deep-invasive hemorrhagic spots and circulatory system diseases, can also be further added into products such as medical dressings, hemostatic gel and hemostatic spray, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of medical coagulation materials technology, and in particular to a self-driven functionalized nucleic acid assembly inspired by the coagulation mechanism and its preparation method. Background Technology

[0002] Globally, approximately 1.5 million people die each year from traumatic hemorrhage, with over 85% of these deaths stemming from uncontrolled bleeding. Controlling complex, irregular, non-compressible, and deeply hidden fatal bleeding presents a significant and challenging problem in hemostasis. Currently, the gold standard clinical treatment is the transfusion of whole blood or blood components, which significantly improves the survival rate of bleeding patients. However, the supply of blood products is heavily dependent on donors and faces challenges such as immune reactions, contamination risks, limited shelf life, difficulties in blood type matching, and poor portability and storage, greatly limiting patient outcomes. In addition, clinical hemostasis strategies include direct compression, tourniquet application, combined application of local hemostatic agents and compression, and endovascular occlusion devices. However, these methods still face numerous challenges in practical application, such as the limitations of direct compression hemostasis, the need for standardized tourniquet application, and the uncertainty of the effectiveness of local hemostatic agents. Worse still, for bleeding in hidden, deep locations such as the abdominal cavity, retroperitoneum, or pelvis, traditional hemostasis methods are insufficient to directly target the bleeding point, requiring imaging examinations and interventional treatments to achieve precise hemostasis. The increasing demand for hemostatic dressings in traumatic bleeding patients, coupled with the difficulty of achieving rapid and safe hemostasis, has become a significant and pressing issue in emergency surgery. Therefore, future efforts should focus on further optimizing hemostasis protocols, incorporating advanced hemostatic techniques and materials, to improve the control of complex traumatic bleeding, thereby reducing mortality and improving patient outcomes.

[0003] From the perspective of the body's coagulation mechanism, traumatic hemostasis is based on the coordinated action of multiple cells capable of initiating hemostasis. The functionalized materials obtained, similar to platelet coagulation, can precisely target vWF (von Willebrand factor), fibrinogen, and erythrocytes, thereby accurately locating the bleeding point and stimulating a coagulation cascade reaction between platelets and erythrocytes, achieving efficient hemostasis. However, most existing clinical coagulation materials rely on "sealing" the surface of accessible and compressible bleeding, failing to penetrate deeper layers to activate the bleeding source and initiate the coagulation cascade reaction. Furthermore, "sealing" coagulation can lead to local blood pooling and thrombus formation. Prolonged blood pooling can cause atrophy, deformation, and even death of parenchymal cells, accompanied by the risk of interstitial fibrosis and reticular collagenization, ultimately leading to organ hardening. Controlling blood pooling while achieving hemostasis is crucial to prevent circulatory system diseases. Worse still, patients with severe trauma or postoperative complications experience a sharp increase in infection risk due to weakened immunity. Traumatic bleeding provides favorable conditions for microbial growth, and microbial infection increases the risk of bleeding, ultimately leading to a vicious cycle. Given the irregular, hidden, and deep-seated nature of severe and complex bleeding sites, the key to solving this problem lies in developing active substances that trigger the body's inherent coagulation cascade mechanism, thereby creating multifunctional hemostatic and antithrombotic materials.

[0004] Therefore, based on the body's coagulation cascade reaction, the required functional medical coagulation materials can ensure self-driven and precise delivery to the bleeding point to activate the body's adaptive regulation of hemostasis pathways. After hemostasis is completed, similar to the body's ability to activate negative feedback pathways to prevent thrombus formation and reduce the occurrence of circulatory diseases, this important principle will help to adopt reasonable medical technologies to truly mimic the body's coagulation pathways and solve the medical problems of bleeding in irregular, hidden, and complex wound sites as well as the consequences of excessive hemostasis. Summary of the Invention

[0005] The purpose of this invention is to provide a self-driven functionalized nucleic acid assembly inspired by coagulation mechanisms and its preparation method, thereby addressing the problems existing in the prior art. By loading a procoagulant drug (such as viper hemocoagulase) or an anticoagulant drug (such as hirudin) onto the functionalized nucleic acid assembly, DNA nanoflower coagulation materials are prepared, integrating self-driving, recognition, targeting, safety, and cyclic stability. After hemostasis, it triggers an in vivo coagulation cascade reaction (i.e., releasing only hirudin), preventing thrombus formation, while simultaneously preventing circulatory system diseases and promoting high-quality wound healing—a humane treatment that promotes truly perfect hemostasis and repair of hidden deep bleeding sources, making hemostatic materials more rational, safe, and effective.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a self-driven functionalized nucleic acid assembly inspired by the coagulation mechanism. The functionalized nucleic acid assembly is formed by rolling circle amplification using four coagulation-related functionalized DNA aptamers. The nucleotide sequences of the four coagulation-related functionalized DNA aptamers are shown in SEQ ID NO: 1-4.

[0008] Furthermore, the functionalized nucleic acid assembly can be presented in the form of DNA nanoflowers, DNA frameworks, or DNA hydrogels; in this embodiment of the invention, DNA nanoflowers are used as an example for illustration and functional verification.

[0009] The present invention also provides a method for preparing the functionalized nucleic acid assembly described above, comprising the following steps:

[0010] The template strand, primer strand, and sterile enzyme-free water are mixed and then annealed; wherein the nucleotide sequence of the template strand is shown in SEQ ID NO: 5-6, and the nucleotide sequence of the primer strand is shown in SEQ ID NO: 7-8;

[0011] After annealing, ligase was added to continue the reaction. After enzyme inactivation, phi29 DNA polymerase was added to carry out rolling circle amplification to obtain functionalized nucleic acid assemblies.

[0012] Optionally, the molar ratio of the two template strands, as shown in SEQ ID NO: 5-6, is (1-2):(3-5); by adjusting this ratio, it is intended to precisely target irregular, occult, and deep bleeding sites, and rapidly recruit fibrinogen and erythrocytes to initiate a coagulation cascade reaction.

[0013] And / or, the molar ratio of the two primer chains with nucleotide sequences as shown in SEQ ID NO: 7-8 is (2-4):(6-10).

[0014] Optionally, the volume ratio of the template strand, primer strand, and sterile enzyme-free water is (1-12):(2-24):(16-40).

[0015] And / or, when adding the ligase, 10 × Ligase Buffer is also added, wherein the volume ratio of the 10 × Ligase Buffer to the ligase is (3-9):(0.4-2.4); the reaction conditions are: 14-25 °C for 10-30 h;

[0016] And / or, when adding phi29 DNA polymerase, 10×Reaction Buffer and dNTP mixture are also added, wherein the volume ratio of phi29 DNA polymerase, 10×Reaction Buffer and dNTP mixture is (0.5-2):(3-18)-(1-12); the conditions for rolling circle amplification reaction are: 28-36 ℃ for 10-30 h.

[0017] Under the above conditions, by controlling the amount of template strand and primer strand added, as well as the rolling circle amplification time, elliptical DNA nanoflowers with a particle size of 300-450 nm can be prepared.

[0018] Further, the volume ratio of the template strand, primer strand, and sterile enzyme-free water is (0.5-10):(1-20):(16-32); and / or, when adding the ligase, 10× Ligase Buffer is also added, and the volume ratio of the 10× Ligase Buffer to the ligase is (2-8):(0.5-2); the reaction conditions are: 16-20 ℃ for 12-24 h; and / or, when adding the phi29 DNA polymerase, 10× Reaction Buffer and dNTP mixture are also added, and the volume ratio of the phi29 DNA polymerase, 10× Reaction Buffer, and dNTP mixture is (0.5-2):(4-16)-(2-8); the rolling circle amplification reaction conditions are: 30-35 ℃ for 12-24 h. Under these conditions, spherical nanoflowers with a particle size of 200-450 nm can be prepared by controlling the amount of template strand and primer strand added, as well as the rolling circle amplification time.

[0019] The present invention also provides a coagulation material containing a functionalized nucleic acid assembly with active substances, comprising the functionalized nucleic acid assembly and active substances loaded within the functionalized nucleic acid assembly, wherein the active substances include a procoagulant drug or an anticoagulant drug.

[0020] Optionally, the procoagulant includes viper hemocoagulase, and the anticoagulant includes hirudin.

[0021] The mechanism of action of procoagulant drugs lies in the fact that the lancehead viper's hemocoagulase is located in Ca... 2+ In the presence of specific hydrolysis of fibrinogen A α chain, fibrinopeptide A is released. The exposed fibrin monomer polymerization sites (EA sites) drive end-to-end polymerization of fibrin monomers, which crosslink into stable clots under the catalysis of coagulation factor XIIIa, thus achieving rapid "exogenous" hemostasis.

[0022] The mechanism of action of anticoagulant drugs lies in the fact that hirudin uses a "pseudo-substrate" strategy to irreversibly occupy both the active site and external site of thrombin, thereby causing it to lose its ability to cut fibrinogen and prevent platelet activation, thus blocking the common coagulation pathway.

[0023] The present invention also provides a method for preparing a coagulation material containing a functionalized nucleic acid assembly with active substances, comprising the step of adding hirudin solution dropwise to the functionalized nucleic acid assembly, mixing and reacting to obtain a coagulation material containing a nucleic acid assembly with active substances.

[0024] Optionally, the concentration of the functionalized nucleic acid assembly is 100-800 ng / μL, and the mass concentration of the hirudin solution is 20%-160%.

[0025] This invention also provides the use of the functionalized nucleic acid assembly or the coagulation material containing the functionalized nucleic acid assembly with active substance in any of the following:

[0026] (1) Use in the preparation of drugs for the diagnosis and / or treatment of acute traumatic hemorrhage;

[0027] (2) Use in the preparation of drugs for the diagnosis and / or treatment of occult deep wound hemorrhage;

[0028] (3) Application in the preparation of diagnostic and / or therapeutic preparations for patients with thrombocytopenic purpura and bleeding and circulatory disorders;

[0029] (4) Use in the preparation of drugs for the diagnosis and / or treatment of myocardial infarction or cerebral infarction.

[0030] This invention also provides the application of the functionalized nucleic acid assembly or the coagulation material containing the functionalized nucleic acid assembly with active substances in the preparation of hemostatic products, including medical dressings, hemostatic gels, and hemostatic sprays. These products can also be used for the repair and treatment of diabetic ulcers, burns, pressure ulcers, and various chronic, difficult-to-heal wounds, as well as for preventing myocardial infarction and cerebral infarction.

[0031] The present invention discloses the following technical effects:

[0032] (1) Inspired by the coagulation mechanism, this invention constructs a self-driven functionalized nucleic acid assembly coagulation material, mainly presented in the form of DNA nanoflowers, and verifies its multifunctionality. By precisely controlling the concentration, amount of substance and rolling circle amplification time of functionalized DNA segments, spherical and elliptical DNA nanoflowers are prepared, realizing platelet-like functions to rapidly and accurately target irregular, hidden and deep bleeding sites, activate the coagulation cascade reaction to induce safe, effective and rapid hemostasis, and prevent the occurrence of thrombosis.

[0033] (2) By introducing functionalized DNA aptamers (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3), this invention can precisely target irregular, hidden and deep bleeding sites, rapidly recruit fibrinogen and erythrocytes to trigger a coagulation cascade reaction, and effectively and precisely improve hemostasis efficiency.

[0034] (3) By introducing a functionalized DNA aptamer (SEQ ID NO: 4), this invention prevents the body from over-activating the hemostasis cascade reaction and generating a large amount of thrombin factors that could lead to thrombosis. The functionalized DNA aptamer SEQ ID NO: 4 can recognize thrombin exceeding the threshold and achieve precise release of hirudin on demand according to the concentration competition principle, thereby preventing or inhibiting the occurrence and formation of thrombi.

[0035] (4) The self-driven functionalized DNA nanoflower coagulation material prepared by the present invention can prepare spherical and elliptical DNA nanoflowers by adjusting the DNA segment concentration and rolling circle amplification time, which can meet the needs of detection and hemostasis of bleeding in different parts such as irregular, hidden and deep wounds, such as cerebral hemorrhage, liver injury and kidney injury.

[0036] (5) This invention achieves safe, efficient and long-circulating properties in vivo by adjusting the structure and particle size of self-driven functionalized DNA nanoflowers.

[0037] (6) The self-driven functionalized DNA nanoflower coagulation material prepared by the present invention is suitable for acute traumatic bleeding, hidden deep traumatic bleeding, bleeding and circulatory diseases in patients with platelet insufficiency (reducing the occurrence of cerebral infarction and myocardial infarction).

[0038] (7) The DNA nanoflower coagulation material of the present invention has a reasonable structural design and a mature and feasible manufacturing process. It can be widely used in the treatment of complex traumatic wounds such as hidden deep bleeding points and circulatory system diseases. It can also be further added to medical dressings, hemostatic gels, hemostatic sprays and other products for the repair and treatment of diabetic ulcers, burns, pressure sores and various chronic and difficult-to-heal wounds, as well as to prevent the occurrence of myocardial infarction and cerebral infarction. It has good application prospects. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1This is a schematic diagram illustrating the technical principle of the preparation and application of the self-driven functionalized DNA nanoflowers of this invention.

[0041] Figure 2 The microstructure of the self-driven functionalized DNA nanoflower in this embodiment of the invention;

[0042] Figure 3 This invention provides an evaluation of the ability of self-driven functionalized DNA nanoflowers loaded with drugs and to release hirudin in PBS buffer; A: Drug loading rate; B: Drug release rate;

[0043] Figure 4 For the safety assessment of the self-driven functionalized DNA nanoflowers in the embodiments of the present invention; A: hemolysis rate; B: DNA nanoflowers co-cultured with cells - live / dead staining results (100 nm).

[0044] Figure 5 For the evaluation of the precise targeting of self-driven functionalized DNA nanoflowers in this embodiment of the invention; A: Cell flow cytometry results; B: SEM results (10 μm).

[0045] Figure 6 To evaluate the in vitro hemostatic effect of the self-driven functionalized DNA nanoflowers in this invention and to explore their hemostatic mechanism; A: Blood coagulation in the control group and the DNA nanoflower group; B: Statistical results of blood coagulation index data; C: Adhesion of red blood cells (10 μm) between the control group and the DNA nanoflowers; D: Adhesion of platelets (10 μm) between the control group and the DNA nanoflowers.

[0046] Figure 7 This is an in vivo hemostasis evaluation of self-driven functionalized DNA nanoflowers in this embodiment of the invention.

[0047] Figure 8 Evaluation of the precise targeting of self-driven functionalized DNA nanoflowers to different bleeding sites in vivo in this embodiment of the invention; A: Imaging results; B: Fluorescence intensity statistics;

[0048] Figure 9 Evaluation of the prevention or reduction of thrombus formation by self-driven functionalized DNA nanoflowers in this invention (5μm); A: Brain; B: Heart;

[0049] Figure 10 This is an in vivo metabolic performance evaluation of the self-driven functionalized DNA nanoflowers in this embodiment of the invention.

[0050] Figure 11 For the in vivo safety assessment of self-driven functionalized DNA nanoflowers in this embodiment of the invention; A: blank control group; B: DNA nanoflower group. Detailed Implementation

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0054] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0055] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0056] This invention provides a self-driven functionalized DNA nanoflower inspired by coagulation mechanisms. It is constructed from coagulation-related functionalized DNA aptamers (functionalized DNA aptamers that precisely target irregular, occult, and deep-wound bleeding sites, with nucleotide sequences as shown in SEQ ID NO: 1; functionalized DNA aptamers that activate or trigger coagulation mechanisms, with nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3; and functionalized DNA aptamers that generate thrombi when thrombin levels in the blood exceed a threshold, with nucleotide sequences as shown in SEQ ID NO: 4), and formed into a nanoflower-like three-dimensional structure through rolling circle amplification. This DNA nanoflower can respond to changes in thrombin levels in the blood environment, enabling drug loading and on-demand release. A schematic diagram of the technical principle of the above solution is shown below. Figure 1 .

[0057] The following examples involve the main experimental materials:

[0058] (1) Functionalized DNA aptamers, whose nucleotide sequences are shown in Table 1.

[0059] Table 1. Functionalized DNA aptamer sequences

[0060]

[0061] (2) Template strands (DNA temple 1 and DNA temple 2) and primer strands (Primer 1 and Primer 2) required for the preparation of DNA nanoflowers, the nucleotide sequences of which are shown in Table 2.

[0062] Table 2 Template strand and primer strand

[0063]

[0064] (3) Hirudin solutions of different concentrations; PBS buffer solution (pH 7); enzyme-free sterile water; thrombin solutions of different concentrations.

[0065] Example 1: Construction of self-driven functionalized DNA nanoflowers

[0066] (1) Preparation of self-driven functionalized DNA nanoflowers (spherical): Take 5 μL of template strand (DNA temple 1 and DNA temple 2, molar ratio of 2:3) and place it in a 200 μL PE tube. Add 10 μL of primer strand (Primer 1 and Primer 2, molar ratio of 2:7) and mix evenly using a vortex mixer (Note: the speed should not be too high). Add 25 μL of DNase / RNase-free ddH2O, mix evenly again, and perform annealing treatment. The annealing procedure is shown in Table 3.

[0067] Table 3 PCR reaction annealing program

[0068]

[0069] Note: Decrease the temperature by 1-2 °C every 2-4 minutes until it reaches 4 °C, and stop at 106 °C for the lid.

[0070] After annealing, add 5 μL of 10× Ligase Buffer and 1 μL of T4 ligase and continue the PCR reaction at 18 °C for 20 h, with the cap temperature set to 4 °C or the cap temperature turned off.

[0071] After the reaction, the PCR program was set to 70 °C to inactivate T4 ligase (20 min). Then, 10 μL of 10 × Reaction Buffer and 5 μL of dNTP mixture were added and mixed thoroughly. Next, 1 μL of phi29 DNA polymerase was added. The PCR program was set to 32 °C for rolling circle amplification for 18 h.

[0072] (2) Preparation of self-driven functionalized DNA nanoflowers (elliptical): Take 6 μL of template strand (DNA temple 1 and DNA temple 2, molar ratio of 1:3) and place it in a 200 μL PE tube. Add 12 μL of primer strand (Primer 1 and Primer 2, molar ratio of 1:3) and mix evenly using a vortex mixer (Note: the speed should not be too high). Add 30 μL of DNase / RNase-free ddH2O and mix evenly again. Perform annealing treatment (decrease the temperature by 1-2 °C every 2-4 min until 4 °C is reached. Set the cap temperature to 106 °C, see Table 3).

[0073] After annealing, add 6 μL of 10 × Ligase Buffer and 1.4 μL of T4 ligase and continue the PCR reaction at 20 °C for 20 h, with the cap temperature set to 4 °C or the cap temperature turned off.

[0074] After the reaction, the PCR program was set to 65 ℃ to inactivate T4 ligase (20 min). Then, 11 μL of 10× Reaction Buffer and 6 μL of dNTP mixture were added and mixed thoroughly. Next, 1.5 μL of phi29 DNA polymerase was added. The PCR program was set to 32 ℃ for rolling circle amplification for 20 h. The morphology of the prepared DNA nanoflowers is as follows. Figure 2 As shown.

[0075] Example 2: Construction of self-driven functionalized DNA nanoflowers

[0076] (1) Preparation of self-driven functionalized DNA nanoflowers (spherical): Take 0.5 μL of template strand (DNA temple 1 and DNA temple 2, molar ratio 1:3) and place it in a 200 μL PE tube. Add 1 μL of primer strand (Primer 1 and Primer 2, molar ratio 3:7) and mix evenly using a vortex mixer (Note: the speed should not be too high). Add 16 μL of DNase / RNase-free ddH2O, mix evenly again, and perform annealing treatment. The annealing procedure is shown in Table 3.

[0077] After annealing, add 2 μL of 10 × Ligase Buffer and 0.5 μL of T4 ligase and continue the PCR reaction at 16 °C for 12 h, with the cap temperature set to 0 °C or the cap temperature turned off.

[0078] After the reaction, the PCR program was set to 60 °C to inactivate T4 ligase (30 min). Then, 4 μL of 10 × Reaction Buffer and 2 μL of dNTP mixture were added and mixed thoroughly. Next, 0.5 μL of phi29 DNA polymerase was added. The PCR program was then set to 30 °C for rolling circle amplification.

[0079] (2) Preparation of self-driven functionalized DNA nanoflowers (elliptical): Take 1 μL of template strand (DNA temple 1 and DNA temple 2, molar ratio of 1:4) and place it in a 200 μL PE tube. Add 2 μL of primer strand (Primer 1 and Primer 2, molar ratio of 3:10) and mix evenly using a vortex mixer (Note: the speed should not be too high). Add 16 μL of DNase / RNase-free ddH2O and mix evenly again. Perform annealing treatment (decrease the temperature by 1-2 °C every 2-4 min until 4 °C is reached. Set the cap temperature to 106 °C, see Table 3).

[0080] After annealing, add 3 μL of 10× Ligase Buffer and 0.4 μL of T4 ligase and continue the PCR reaction at 14 °C for 10 h, with the cap temperature set to 0 °C or the cap temperature turned off.

[0081] After the reaction, the PCR program was set to 55 ℃ to inactivate T4 ligase (10 min). Then, 3 μL of 10× Reaction Buffer and 1 μL of dNTP mixture were added and mixed thoroughly. Next, 0.5 μL of phi29 DNA polymerase was added. The PCR program was set to 28 ℃ for rolling circle amplification. The morphology of the prepared DNA nanoflowers was similar to... Figure 2 same.

[0082] Example 3: Construction of self-driven functionalized DNA nanoflowers

[0083] (1) Preparation of self-driven functionalized DNA nanoflowers (spherical): Take 10 μL of template strand (DNA temple 1 and DNA temple 2, molar ratio of 1:5) and place it in a 200 μL PE tube. Add 20 μL of primer strand (Primer 1 and Primer 2, molar ratio of 2:3) and mix evenly using a vortex mixer (Note: the speed should not be too high). Add 32 μL of DNase / RNase-free ddH2O, mix evenly again, and perform annealing treatment. The annealing procedure is shown in Table 3.

[0084] After annealing, add 8 μL of 10 × Ligase Buffer and 2 μL of T4 ligase and continue the PCR reaction at 20 °C for 24 h, with the cap temperature set to 0-4 °C or the cap temperature turned off.

[0085] After the reaction, the PCR reaction program was set to 75 ℃ to inactivate T4 ligase (10–30 min). Then, 16 μL of 10 × Reaction Buffer and 8 μL of dNTP mixture were added and mixed thoroughly. Next, 2 μL of phi29 DNA polymerase was added. The PCR reaction program was set to 35 ℃ for rolling circle amplification.

[0086] (2) Preparation of self-driven functionalized DNA nanoflowers (elliptical): Take 12 μL of template strand (DNA temple 1 and DNA temple 2, molar ratio of 2:5) and place it in a 200 μL PE tube. Add 24 μL of primer strand (Primer 1 and Primer 2, molar ratio of 2:5) and mix evenly using a vortex mixer (Note: the speed should not be too high). Add 40 μL of DNase / RNase-free ddH2O and mix evenly again. Perform annealing treatment (decrease the temperature by 1-2 °C every 2-4 min until 4 °C is reached. Set the cap temperature to 106 °C, see Table 3).

[0087] After annealing, add 9 μL of 10 × Ligase Buffer and 2.4 μL of T4 ligase and continue the PCR reaction at 25 °C for 30 h, with the cap temperature set to 4 °C or the cap temperature turned off.

[0088] After the reaction, the PCR reaction program was set to 75 ℃ to inactivate T4 ligase (20 min). Then, 18 μL of 10× Reaction Buffer and 12 μL of dNTP mixture were added and mixed thoroughly. Next, 2 μL of phi29 DNA polymerase was added. The PCR reaction program was set to 28-36 ℃ for rolling circle amplification. The morphology of the prepared DNA nanoflowers was similar to... Figure 2 same.

[0089] Example 4: Self-driven functionalized DNA nanoflowers loaded with hirudin

[0090] The prepared self-driven functionalized DNA nanoflowers (using Example 1 as an example) were prepared to a concentration of 100-800 ng / μL (200 ng / μL was used in this example) and placed on a shaker at 30-37 ℃, labeled as solution A. Different concentrations of hirudin (20%, 40%, 60%, 80%, 100%, 120%, 140%, and 160%) were prepared in cooled, sterile, enzyme-free water (4-10 ℃, 4 ℃ was used in this example), labeled as solution B. Solution B was then gradually added dropwise to solution A and thoroughly mixed. After 12-24 h (12 h was used in this example), a single sample was tested for drug loading using a NanoDrop spectrophotometer to determine the maximum loading capacity of the self-driven functionalized DNA nanoflowers. Figure 3 (A) Ensure sufficient hirudin loading to prevent thrombus formation and reduce the occurrence of circulatory diseases.

[0091] Self-driven functionalized DNA nanoflowers loaded with hirudin were added to preheated PBS buffer (pH 7, 35°C) and placed in a shaker at 35°C to simulate the release of hirudin during in vivo circulation, thus determining their stability (Note: mimicking the body's hemostasis process, the amount of thrombin gradually increases, while no thrombin accumulation occurs at non-bleeding sites). Sample release was then tested using a NanoDrop spectrophotometer. The results showed that hirudin was essentially not released from the DNA nanoflowers in non-thrombin-accumulating regions. Figure 3 (B)

[0092] Self-driven functionalized DNA nanoflowers loaded with hirudin were added to thrombin (30-42 μg / mL) at a specific concentration prepared with sterile, enzyme-free water to simulate the on-demand release of hirudin from the self-driven functionalized DNA nanoflowers in vivo. Sample release was tested using a NanoDrop spectrophotometer. The hirudin drug release at a concentration mimicking in vivo thrombin (0.01 mg / mL) was 6 ± 0.96% (Table 4). For anti-venous thrombosis, such as in clinical acute thromboembolism, the effective single dose of heparin is 400 U / kg, where 1 mg = 125 U. Therefore, the expected dosage in mouse in vivo experiments is shown in Table 4.

[0093] Table 4. Evaluation results of the amount of hirudin released by self-driven functionalized DNA nanoflowers when the in vivo thrombin threshold is too high.

[0094]

[0095] Note: The effective dose of intravenous anticoagulation for acute bleeding is 400 U / kg. A mouse weighs approximately 20g, so the injection dose = (400 / 1000) × 20g = 8 U.

[0096] 4. Performance characterization of self-driven functionalized DNA nanoflowers

[0097] Biosafety: A certain number of L929 cells were cultured and placed in a laser confocal dish, and co-incubated with DNA nanoflowers for 24 h. Cell status and proliferation were observed using a live-dead staining kit. The biosafety results are as follows: Figure 4 As shown, at the co-culture time points, compared with the control group, the cell proliferation rate of the DNA nanoflowers was significantly accelerated, and the cell number increased significantly. This result indicates that the self-driven DNA nanoflowers have good biocompatibility and can provide a favorable environment for cell growth and proliferation.

[0098] 5. Functional validation of self-driven functionalized DNA nanoflowers

[0099] (1) Performance evaluation of accurate bleeding point location:

[0100] In vitro: A Transwell chamber was placed on top of a laser confocal dish. A certain amount of erythrocytes / platelets were mixed into Opti DMEM medium and thoroughly mixed by pipetting. Then, 100-200 μL of the erythrocyte mixture was added to the chamber, and 200-400 μL of self-driven DNA nanoflowers (mixed with PBS solution) were added to the laser confocal dish. After incubation for 24 h, the recruitment efficiency was verified by flow cytometry and SEM. Flow cytometry results showed that the number of platelets recruited by the self-driven DNA nanoflowers (271,680 / platelet) was 1 ± 0.25 times higher than that of the control group (217,174 / platelet). Figure 5 (A). Furthermore, SEM results further confirmed that a large number of platelets aggregated around the DNA nanoflowers, indicating their highly efficient platelet recruitment capacity. Figure 5 (B)

[0101] (2) Assessment of hemostasis and prevention of circulatory system diseases:

[0102] In vitro: The hemostasis process is closely related to the enhancement / activation of the intrinsic and extrinsic coagulation cascade, with erythrocytes and platelets being the main components in clot formation. Erythrocytes and platelets were co-incubated with self-driven DNA nanoflowers, and the in vitro hemostatic effect was observed using an inverted PE tube test. After 20 seconds of interaction between blood cells and DNA nanoflowers... Figure 6 In Figure A, the blood in the blank control group did not coagulate, while the blood in the DNA nanoflower group and the DNA nanoflower loaded with Batroxobin (BA-DNA nanoflower) group completely lost its fluidity and essentially coagulated. This phenomenon indicates that specific segments in the DNA nanoflower can mediate the interaction between blood cells, thereby inducing blood coagulation. Furthermore, the DNA nanoflower loaded with the active substance Batroxobin (BA-DNA nanoflower) also induced blood coagulation, and its coagulation index was lower than the other two groups. Figure 6 (B). Furthermore, the DNA nanoflowers exhibited better adhesion to erythrocytes compared to the control group (…). Figure 6 (as shown in C) and platelets ( Figure 6 The ability of D).

[0103] In vivo: Hemostasis was measured using a mouse liver biopsy model as a bleeding model. In the liver biopsy model, blood gushed out once the wound formed. The filter paper in the control group was significantly soaked; in contrast, [the control group's filter paper was much more saturated]. Figure 7 As shown, when DNA nanoflowers were injected into bleeding points, the blood-stained filter paper area in this group was smaller.

[0104] Occult traumatic hemorrhage often occurs in the brain, heart, liver, kidneys, and abdominal cavity. Mice were anesthetized with isoflurane gas, and their abdomens were shaved and disinfected three times with iodine, alcohol, and iodine, then draped with a sterile drape. Occult hemorrhage was simulated by creating models in the gastrointestinal tract and other internal organs. This was achieved by using a 26G sterile injection needle (0.45 mm outer diameter) to perform a full-thickness penetration-withdrawal maneuver perpendicular to the long axis of the gastrointestinal tract, repeated three times with a 2mm interval between needle insertions. The puncture point was located between the greater and lesser curvatures of the gastrointestinal tract, avoiding the vascular arch. After puncture, the stomach wall was gently pressed for 5 seconds to confirm the absence of overt bleeding (observing whether a pinpoint hemorrhage immediately appeared in the gastrointestinal tract after puncture, but without blood flowing from the needle puncture site (indicating that the bleeding was confined by the serous membrane, meeting the definition of "occult"). The area was then disinfected with iodine. Subsequently, self-driven DNA nanoflowers (10-20 μL) were injected via the tail vein, and their targeting performance was observed using small animal imaging. Figure 8 The DNA nanoflowers demonstrate that they can self-drive and precisely target bleeding sites.

[0105] To construct a system for treating occult bleeding points (especially brain and heart bleeding), the specific steps are as follows: (1) Occult brain bleeding points: A 34 G insulin injection needle (outer diameter 0.18 mm) is installed in a microsyringe. The needle is inserted vertically 3.0 mm, held at the back of the head for 10 s, rotated 180° to enlarge the needle path, and then withdrawn at a constant speed (within 20 s) with no fresh blood spilling out of the needle path. (2) Occult heart bleeding points: A 30 G needle is connected to a 1 mL syringe, inserted vertically 2 mm along the intercostal space, and the resistance suddenly decreases when it touches the epicardium. The needle is then advanced 1 mm [(total depth 3 mm, only penetrating the parietal pericardium and epicardium), held for 5 s, and then gently pressed with a micro-cotton ball for 10 s at the moment of withdrawal to confirm that no fresh blood flows out of the needle hole]. During the hemostasis process, thrombi formed due to excessive coagulation eventually form thrombi and block blood vessels, thereby causing cerebral infarction or myocardial infarction, which has become the most serious fatal threat to patients. Positron emission tomography (PET-CT) was used for imaging. In the control group, the left side of the brain region of mice showed a distinctly lighter color, indicating infarction; the right side of the brain of mice in the DNA nanoflower-loaded with 40% hirudin group also showed an infarct area. In contrast, the brains of mice in the DNA nanoflower-loaded with 60% hirudin group showed a uniform ocean color on both sides, indicating no cerebral infarction. Figure 9 (A). In the later stages of the experiment, the mice were returned to a normal diet and observed one day later for any signs of myocardial infarction. Figure 9 As shown in Figure B, no myocardial infarction was observed in any of the mice in the group.

[0106] (3) Safety and immunogenicity assessment

[0107] Injection of biomaterials into the body may trigger an immune response. Observing whether an immune response, such as inflammation or allergic reaction, occurs during the metabolism of the biomaterial is crucial for assessing its biocompatibility. Furthermore, different individuals may metabolize biomaterials differently; observing metabolic processes helps determine the suitability and safety of the material in different patients. The metabolic performance and survival of mice after cardiac puncture were continuously monitored. The results showed that all mice that underwent cardiac puncture survived during the 21-day observation period, and the DNA nanoflowers had largely completed their metabolic process in the mice. Figure 10 As shown, from day 3 to day 18, imaging results in mice revealed that the DNA nanoflowers were primarily metabolized by the kidneys and excreted in the urine. Furthermore, the thymus, kidneys, and lymph nodes of the dissected mice were removed and stained with H&E. Figure 11 As shown, no inflammatory immune response was observed in the DNA nanoflowers compared to the blank control group. Note: Three mice were used in each group for the parallel experiment.

[0108] The self-driven functionalized DNA nanoflower coagulation material prepared by this invention not only mimics the platelet conformation in structure and function, but also integrates multiple functions such as precise targeting of bleeding sites, activation of coagulation cascade reactions, and prevention of thrombosis. It exhibits excellent comprehensive performance and broad clinical translation prospects in the field of tissue repair and regenerative medicine.

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A self-driven functionalized nucleic acid assembly inspired by coagulation mechanisms, characterized in that, The functionalized nucleic acid assembly is formed by rolling circle amplification using four coagulation-related functionalized DNA aptamers, and the nucleotide sequences of the four coagulation-related functionalized DNA aptamers are shown in SEQ ID NO: 1-4.

2. A method for preparing a functionalized nucleic acid assembly as described in claim 1, characterized in that, Includes the following steps: The template strand, primer strand, and sterile enzyme-free water are mixed and then annealed; wherein the nucleotide sequence of the template strand is shown in SEQ ID NO: 5-6, and the nucleotide sequence of the primer strand is shown in SEQ ID NO: 7-8; After annealing, ligase was added to continue the reaction. After enzyme inactivation, phi29 DNA polymerase was added to carry out rolling circle amplification to obtain functionalized nucleic acid assemblies.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the two template strands, as shown in SEQ ID NO: 5-6, is (1-2):(3-5); And / or, the molar ratio of the two primer strands with nucleotide sequences as shown in SEQ ID NO: 7-8 is (2-4):(6-10).

4. The preparation method according to claim 2, characterized in that, The volume ratio of the template strand, primer strand, and sterile enzyme-free water is (1-12):(2-24):(16-40). And / or, when adding the ligase, 10 × Ligase Buffer is also added, wherein the volume ratio of the 10 × Ligase Buffer to the ligase is (3-9):(0.4-2.4); the reaction conditions are: 14-25 °C for 10-30 h; And / or, when adding phi29 DNA polymerase, 10×Reaction Buffer and dNTP mixture are also added, wherein the volume ratio of phi29 DNA polymerase, 10×Reaction Buffer and dNTP mixture is (0.5-2):(3-18)-(1-12); the conditions for rolling circle amplification reaction are: 28-36 ℃ for 10-30 h.

5. A coagulation material containing an active substance-functionalized nucleic acid assembly, characterized in that, It includes the functionalized nucleic acid assembly of claim 1, and an active substance loaded within the functionalized nucleic acid assembly, the active substance including a procoagulant or an anticoagulant.

6. The coagulation material containing functionalized nucleic acid assemblies of active substances as described in claim 5, characterized in that, The procoagulant includes viper hemocoagulase, and the anticoagulant includes hirudin.

7. A method for preparing a coagulation material containing an active substance-functionalized nucleic acid assembly as described in claim 5 or 6, characterized in that, The method includes the step of adding a solution of lancehead viper hemocoagulase or hirudin to a functionalized nucleic acid assembly, mixing and reacting to obtain a coagulation material containing the functionalized nucleic acid assembly of the active substance.

8. The preparation method according to claim 8, characterized in that, The concentration of the functionalized nucleic acid assembly is 100-800 ng / μL, and the mass concentration of the hirudin solution is 20%-160%.

9. The use of the functionalized nucleic acid assembly as described in claim 1 or the coagulation material containing the functionalized nucleic acid assembly with active substance as described in any one of claims 5-6 in any of the following: (1) Use in the preparation of drugs for the diagnosis and / or treatment of acute traumatic hemorrhage; (2) Use in the preparation of drugs for the diagnosis and / or treatment of occult deep wound hemorrhage; (3) Use in the preparation of drugs for the diagnosis and / or treatment of bleeding and circulatory diseases in patients with thrombocytopenic purpura; (4) Use in the preparation of drugs for the diagnosis and / or treatment of myocardial infarction or cerebral infarction.

10. The use of the functionalized nucleic acid assembly as described in claim 1 or the coagulation material containing the functionalized nucleic acid assembly with active substance as described in any one of claims 5-6 in the preparation of hemostatic products, characterized in that, The hemostatic products include medical dressings, hemostatic gels, and hemostatic sprays.

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