DNA (deoxyribonucleic acid) molecular glue for stopping bleeding of wet wound surface as well as preparation method and application of DNA molecular glue

Abstract

The invention discloses DNA (deoxyribonucleic acid) molecular glue for stopping bleeding of wet wounds as well as a preparation method and application of the DNA molecular glue, and relates to the technical field of biomedical materials. The method is inspired by a mussel wet adhesion mechanism, and a tannic acid-dopamine phenolic hydroxyl structure is grafted into a DNA double helix chain by adopting a gene coding'click 'reaction to prepare the wet adhesion hemostasis DNA molecular glue. Silicate bioactive glass with a blood coagulation factor (Ca < 2 + >) is added into the DNA molecular glue, endogenous and exogenous blood coagulation pathways of an organism can be rapidly activated, the wet adhesion effect of the DNA molecular glue is cooperated, and multi-pathway rapid hemostasis is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a DNA molecular glue used for hemostasis of wet wounds and a preparation method and application thereof. Background Art

[0002] At present, the mortality rate caused by blood loss is about 30%, and rapid hemostasis is the key to reducing the mortality rate. Although there are endless hemostatic agents, sealants and adhesive products suitable for various bleeding wounds, it is still challenging to effectively stop bleeding in a wet environment. At present, a single hemostatic material can no longer meet the hemostatic needs of complex wounds, and composite intelligent hemostatic materials that coordinate multiple coagulation pathways are the development trend. The current world environment and scientific and technological progress have laid the foundation for the future development direction of ideal materials, and the intersection of basic disciplines can obtain more favorable composite materials. When a large number of water molecules are present, the existing gel hemostatic materials have a slow gelation speed, reduced adhesion performance, poor adhesion, and cannot effectively adhere to and seal the wound. In addition, the gel adhesive hemostatic materials prepared by chemical synthesis have problems such as organic residues, poor degradability, and poor biocompatibility, which are not conducive to the healing and repair of wounds. That is, the potential toxic hazards of chemical synthesis limit its application.

[0003] Research on the wet adhesion mechanism of mussels has shown that the proteins that play the main role in underwater adhesion are various polyphenols related to dopamine, tannic acid and other adjacent components. Natural biopolymer nucleic acids have the characteristics of high molecular weight, easy modification, hydrophilicity and hydrophobicity, injectability and good biocompatibility. By replacing traditional chemically synthesized polymers with DNA polymers, wet adhesion polyphenols can be spontaneously grafted into DNA chains through hydrogen bonds in deionized water to form a three-dimensional network structure. DNA has precise base pair recognition characteristics, and the base pairing reaction in the double chain has the same characteristics as other "click" reactions, such as fast rate, high base pairing selectivity and no by-products. Compared with the traditional "click" reaction, the advantages of genetically encoded "click" reactions are more prominent.

[0004] After the DNA double helix is ​​unwound, it achieves polymerization of hydroxyl and phosphate groups through hydrogen bonds with polyphenols such as dopamine and tannic acid during base pairing. The phenolic hydroxyl groups enable hydrogel materials to still have adhesion properties in a wet environment. Silicate nanoporous particles have the function of activating the body's own coagulation pathway. Applying them in hemostatic materials can significantly improve the hemostatic efficiency. The silicate-based hydrogel materials disclosed in the prior art have good mechanical properties and self-healing properties, and have good adhesion and coagulation properties to tissues. They can be used for coagulation and hemostasis of bleeding wounds, and are expected to be applied to fields such as biomedicine and tissue engineering. However, their adhesion properties in a wet environment are limited, and the hemostatic effect in large bleeding wounds is average. At the same time, the DNA nanocomposite hydrogel adhesive material with hemostatic properties based on clay, deoxyribonucleic acid DNA and methacrylic anhydride modified gelatin disclosed in the prior art has the effects of hemostasis, tissue healing and wound sealing when used on the wound surface, but its preparation process is complicated and requires modified gelatin and ultraviolet cross-linking before it can form a gel, which is not conducive to large-scale production and has the risk of organic residues. A hydrophobic chain-driven mussel-like wet adhesion hydrogel is also disclosed in the prior art, which has the effects of wet adhesion to arteries and internal organs and rapid hemostasis, but the preparation process requires the use of initiators and cross-linking agents to have wet adhesion, high cost, organic reagent residues and complex operation, and its biocompatibility is general. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned background technology, the present invention mainly solves the problems that the existing hemostatic hydrogel has poor wet adhesion performance. After contact with water, the adhesion strength decreases and it cannot achieve effective blocking and hemostatic effect; the existing hemostatic adhesive has a single hemostatic effect, and the effect is general only through the physical adhesion pathway, and it does not have the function of activating the body's own coagulation pathway; and the existing hemostatic adhesive needs to add initiators, cross-linking agents or organic reagents to trigger gelation. The organic residues in the preparation or gelation process of the hydrogel are serious, which is harmful to the body and the environment. The present invention provides a DNA molecular glue for wet wound hemostasis and a preparation method and application thereof. The method is inspired by the wet adhesion mechanism of mussels, and the tannic acid-dopamine phenol hydroxyl structure is grafted into the DNA double helix chain by a genetically encoded "click" reaction to prepare a wet adhesion hemostatic DNA molecular glue. The present invention adds a coagulation factor (Ca) to the DNA molecular glue. 2+ ) silicate bioactive glass can quickly activate the body's endogenous and exogenous coagulation pathways, cooperate with the wet adhesion of DNA molecular glue, and quickly stop bleeding in multiple ways. The DNA molecular glue prepared by the present invention is green and bio-friendly. The preparation and gelation process does not require initiators or cross-linking agents, etc., and can be gelled in deionized water.

[0006] The first object of the present invention is to provide a method for preparing a DNA molecular glue for moist wound hemostasis, comprising the following steps: Dispersing tannic acid evenly in a water solvent, adding dopamine hydrochloride, and mixing evenly to obtain a tannic acid-dopamine solution; The mesoporous bioactive glass was uniformly dispersed in a water solvent, and then DNA was added and stirred at 85-95°C for 10-15 minutes to obtain a MBG@DNA solution; Add tannic acid-dopamine solution to the MBG@DNA solution and stir at room temperature for 20 to 40 min. The tannic acid-dopamine phenolic hydroxyl structure will be grafted to the deoxynucleotide double strand of DNA through hydrogen bonding to obtain DNA molecular glue.

[0007] Preferably, the mass ratio of the tannic acid to the dopamine hydrochloride is 0.5 ~ 1.5:0.675.

[0008] Preferably, the mass ratio of the mesoporous bioactive glass to DNA is 1:3 to 8.

[0009] Preferably, the process of preparing the tannic acid-dopamine solution comprises: Add tannic acid to 0.8 ~ 1.5 mL of deionized water, heat in a 40 ~ 42℃ water bath and stir until completely dissolved, then lower the water bath temperature to 36.5 ~ 37.5℃, add dopamine hydrochloride, and stir for 5 ~ 10 min.

[0010] Preferably, when preparing the DNA molecular gel, the tannic acid-dopamine solution is added and stirred at room temperature for 20 to 40 minutes, and the temperature is dropped from 85 to 95° C. to room temperature, that is, during the DNA annealing process.

[0011] Preferably, when preparing DNA molecular glue, the concentration of mesoporous bioactive glass in MBG@DNA solution is 0.8 ~ 1.6w / v; the volume ratio of tannic acid-dopamine solution to MBG@DNA solution is 0.1 ~ 0.2:1.

[0012] Preferably, the mesoporous bioactive glass is prepared according to the following steps: CTAB is uniformly dispersed in a mixed solvent, and tetraethyl orthosilicate is added dropwise after adjusting the pH value with ammonia water, followed by calcium nitrate. After the reaction is completed, the product is obtained; The product is calcined to remove the structural template CTAB, thereby obtaining mesoporous bioactive glass.

[0013] Preferably, the mixed solvent is prepared from deionized water, ethanol and ether in a volume ratio of 7.5:1:2.

[0014] The second purpose of the present invention is to provide a DNA molecular glue for hemostasis of wet wounds.

[0015] The third object of the present invention is to provide an application of DNA molecular glue in the preparation of moist wound hemostatic drugs.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a DNA molecular glue for wet wound hemostasis and its preparation method and application. The present invention is based on the gene-encoded "click" reaction. In the process of DNA base complementation, hydrogen bonds are formed between the phenolic hydroxyl groups of tannic acid (TA) and dopamine (DOPA), and TA and DOPA are introduced into the DNA nucleic acid chain. Then, by physical doping of mesoporous bioactive glass (MBG), a composite MBG@DNA / TA-DOPA hydrogel that can be used for wet wound hemostasis is prepared. The DNA molecular glue has the effects of wet adhesion, accelerated hemostasis, aggregation of red blood cells and fibrin network formation, and has good biocompatibility and degradability.

[0017] Inspired by the wet adhesion mechanism of mussels, the present invention grafts the tannic acid-dopamine phenolic hydroxyl structure onto the DNA double helix chain using a gene-encoded "click" reaction to prepare a wet adhesion hemostatic DNA molecular glue. 2+ ) silicate bioactive glass can quickly activate the body's endogenous and exogenous coagulation pathways, cooperate with the wet adhesion of DNA molecular glue, and quickly stop bleeding in multiple ways.

[0018] The DNA molecular gel prepared by the invention is green and bio-friendly. No initiator or cross-linking agent is required in the preparation and gelling process, and the gel can be formed in deionized water. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the synthesis of DNA molecular glue; Figure 2 SEM results of DNA molecular gel, where A: SEM of MBG@DNA / TA-DOPA; B: SEM of the white frame area magnified 25 times; Figure 3 The gelation of DNA molecular glue when it comes into contact with water, among which A: MBG@DNA-DOPA / TA adheres to the 1 mL pipette tip after it comes into contact with water; B: MBG@DNA-DOPA / TA adheres to rubber gloves and EP tube; C: MBG@DNA-DOPA / TA adheres to rubber gloves and pork; D: MBG@DNA-DOPA / TA adheres to rubber gloves and pig skin; Figure 4MBG@DNA-DOPA / TA composite molecular glue has an adhesive and sealing effect on wet tissues, where A: the pig small intestine is filled with water; B: the blade pierces 0.5 cm; C: the MBG@DNA-DOPA / TA composite hydrogel is applied to block the water outlet; D: the water outlet is blocked for 2 h; Figure 5 FTIR results of DNA molecular gel; Figure 6 DNA molecule glue 1 H NWR results; Figure 7 GPC results of DNA molecular gel; Figure 8 Rheological properties of DNA molecule glue; Fig. 9 Adhesion of DNA molecule glue; Fig.10 Liquid absorption of DNA molecular glue, where A: liquid absorption rate; B: expansion rate; Fig.11 In vitro coagulation time of DNA molecular gel, where A: coagulation picture; B: coagulation time bar graph; Fig.12 SEM results of DNA molecular glue aggregating red blood cells, where A: DNA; B: MBG@DNA; C: MBG@DNA / TA; D: MBG@DNA / TA-DOPA; Fig.13 BCI results of DNA molecular gel, where A: sample BCI image; B: BCI result bar graph; Fig.14 The results of liver hemostasis of DNA molecular gel, where A: hemostasis picture of liver injury model; B: bleeding time of liver injury model; Fig.15 Hemolysis results of DNA molecular gel, where A: hemolysis picture of sample; B: hemolysis rate bar graph of sample; Fig.16 Cell compatibility of DNA molecular glue, where A: cell survival rate measured by MTT; B: number of living cells counted by Image-J; C: live and dead cell staining of sample extracts co-cultured with L929 cells for 24 and 48 h. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0021] The first aspect of the present invention provides a method for preparing a DNA molecular glue for moist wound hemostasis, comprising the following steps: Dispersing tannic acid (TA) uniformly in a water solvent, adding dopamine hydrochloride (DOPA), and mixing uniformly to obtain a tannic acid-dopamine solution (TA-DOPA); The mesoporous bioactive glass (MBG) was uniformly dispersed in a water solvent, and then DNA was added and stirred at 85-95°C for 10-15 min to obtain the MBG@DNA solution. Add tannic acid-dopamine solution to the MBG@DNA solution and stir at room temperature for 20 to 40 min. The tannic acid-dopamine phenolic hydroxyl structure will be grafted to the deoxynucleotide double strand of DNA through hydrogen bonding to obtain DNA molecular glue (MBG@DNA / TA-DOPA).

[0022] Inspired by the wet adhesion mechanism of mussels, the present invention grafts the tannic acid-dopamine phenolic hydroxyl structure onto the DNA double helix chain using a gene-encoded "click" reaction to prepare a wet adhesion hemostatic DNA molecular glue. 2+ ) silicate bioactive glass can quickly activate the body's endogenous and exogenous coagulation pathways, cooperate with the wet adhesion of DNA molecular glue, and quickly stop bleeding in multiple ways.

[0023] In one embodiment, mesoporous bioactive glass (MBG) is uniformly dispersed in a water solvent, and then DNA is added and stirred at 85-95° C. for 10-15 min to obtain a mixed solution of mesoporous bioactive glass and DNA (MBG@DNA), wherein the MBG@DNA is prepared and used immediately.

[0024] Wherein, the mass ratio of the tannic acid to the dopamine hydrochloride is 0.5 ~ 1.5:0.675.

[0025] The mass ratio of the mesoporous bioactive glass to DNA is 1:3-8.

[0026] The process of preparing the tannic acid-dopamine solution includes: Add tannic acid to 0.8 ~ 1.5 mL of deionized water, heat in a 40 ~ 42℃ water bath and stir until completely dissolved, then lower the water bath temperature to 36.5 ~ 37.5℃, add dopamine hydrochloride, and stir for 5 ~ 10 min.

[0027] When preparing DNA molecular gel, add tannic acid-dopamine solution and stir at room temperature for 20 to 40 minutes. The temperature drops from 85 to 95°C to room temperature, which is the process of DNA annealing.

[0028] When preparing DNA molecular glue, the concentration of mesoporous bioactive glass (MBG) in the MBG@DNA solution is 0.8 ~ 1.6w / v; the volume ratio of tannic acid-dopamine solution to MBG@DNA solution is 0.1 ~ 0.2:1.

[0029] The mesoporous bioactive glass is prepared according to the following steps: CTAB is uniformly dispersed in a mixed solvent, and tetraethyl orthosilicate is added dropwise after adjusting the pH value with ammonia water, followed by calcium nitrate. After the reaction is completed, the product is obtained; The product is calcined to remove the structural template CTAB, thereby obtaining mesoporous bioactive glass.

[0030] The mixed solvent is prepared from deionized water, ethanol and ether in a volume ratio of 7.5:1:2.

[0031] The second aspect of the present invention provides a DNA molecular glue for hemostasis of wet wounds.

[0032] The third aspect of the present invention provides an application of a DNA molecular glue in the preparation of a wet wound hemostatic drug.

[0033] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified; the reagents and materials used are all commercially available unless otherwise specified.

[0034] The DNA used in the following examples, namely high molecular weight thread-bound salmon sperm DNA, was purchased from Shenzhen Cellma Biotechnology Co., Ltd.

[0035] In the following example, the preparation method of MBG@DNA / TA is as follows: TA is added to 1 mL of deionized water, heated in a water bath at 40 to 42°C and stirred until completely dissolved, wherein the mass of TA is 1.0 g. Then, 40 μL of TA solution is added to 400 μL of MBG@DNA solution, and stirred at room temperature for 20 to 40 min.

[0036] Example 1 This example involves the synthesis of DNA molecule glue Preparation of MBG: 150 mL of deionized water, 20 mL of ethanol and 40 mL of co-solvent ether were added to a round-bottom flask containing 2.0 g of CTAB in sequence. Stir at high speed at room temperature until CTAB is completely dissolved, and add an appropriate amount of ammonia water to the above solution to adjust the pH value. After 10 min, TEOS was added dropwise to the above system and stirred at room temperature. After 10 min, a certain amount of Ca(NO3)2·4H2O was added to the above solution, and the solution was stirred at 1 200 rpm for 4 h at room temperature. After the reaction, the precipitate was collected by centrifugation at 10 000 rpm for 5 min to obtain the sample. Wash with anhydrous ethanol and deionized water three times each, and then vacuum dry at 50°C for 24 h. The sample was calcined at 550°C in a muffle furnace for 5 h to remove the template, with a heating rate of 1°C / min, and vacuum sealed for use.

[0037] Preparation of TA-DOPA: Weigh 0.5 g of tannic acid (TA) powder, add it to 1 mL of deionized water, heat in a 40°C water bath and stir until completely dissolved. Then, lower the water bath temperature to 37°C. Weigh 0.675 g of dopamine hydrochloride (DOPA) powder and stir vigorously for 5 min. The prepared TA-DOPA can be stored at 4°C and used within 1 week. Warm it at 37°C for 10 min before each use.

[0038] Preparation of MBG@DNA: Weigh the prepared MBG (10 mg) into 1 mL of deionized water. Ultrasonicate and mix for 2 min at 100 Hz to obtain MBG dispersion. Weigh 50 mg of DNA and add it to the above MBG dispersion. Stir at 90°C, 200 rpm for 10 min to fully dissolve the DNA. MBG@DNA is ready for use.

[0039] Preparation of MBG@DNA / TA-DOPA: 100 μL of TA-DOPA solution was added to the above MBG@DNA, and stirred continuously for 30 min at room temperature to make the gel uniform. During this process, the temperature of MBG@DNA dropped from 90°C to room temperature, that is, during the DNA annealing process, TA-DOPA was grafted to the double-stranded deoxynucleotide of DNA through hydrogen bonds, and MBG@DNA / TA-DOPA, i.e., DNA molecular glue, was obtained.

[0040] See also Figure 1 As shown in the figure, the synthesis mechanism of DNA molecular glue, MBG is pre-dispersed in deionized water, and after dissolving DNA, it is mixed with TA-DOPA at high temperature, and then cooled and stirred at room temperature. During the DNA annealing process, TA-DOPA is grafted into the nucleic acid double helix chain through hydrogen bonds to obtain MBG-doped DNA molecular glue.

[0041] Figure 2This is the SEM result of DNA molecular gel. Note: MBG is evenly doped into the DNA molecular gel. Figure 2 A: SEM of MBG@DNA / TA-DOPA; Figure 2 B: SEM of the white box area at 25 times magnification. Figure 2 In A, MBG particles are dispersed in the pores or scaffolds of DNA molecule gel. TA-DOPA is introduced into DNA molecules, and the resulting DNA molecule gel has a porous structure after freeze-drying, with a pore size of about 14.8 ~30.0 μm. Figure 2 In B, MBG can be observed in the support of DNA molecular gel. The particle size of MBG is about 400 ~ 800 μm, and the pore size is obvious. MBG is uniformly doped into the DNA molecular gel.

[0042] Example 2 Same as Example 1, except that: Preparation of TA-DOPA: Weigh 1.0 g of tannic acid (TA) powder, add it to 1 mL of deionized water, heat in a 40°C water bath and stir until completely dissolved. Then, lower the water bath temperature to 37°C. Weigh 0.675 g of dopamine hydrochloride (DOPA) powder and stir vigorously for 5 min. The prepared TA-DOPA can be stored at 4°C and used within 1 week. Warm it at 37°C for 10 min before each use.

[0043] Example 3 Same as Example 1, except that: Preparation of TA-DOPA: Weigh 1.5 g of tannic acid (TA) powder, add it to 1 mL of deionized water, heat in a 40°C water bath and stir until completely dissolved. Then, lower the water bath temperature to 37°C. Weigh 0.675 g of dopamine hydrochloride (DOPA) powder and stir vigorously for 5 min. The prepared TA-DOPA can be stored at 4°C and used within 1 week. Warm it at 37°C for 10 min before each use.

[0044] In order to illustrate the relevant performance of the DNA molecular glue provided by the present invention, it is described in conjunction with the accompanying drawings, wherein the preparation method of MBG@DNA / TA is to add TA to 1 mL of deionized water, heat in a water bath at 40°C and stir until completely dissolved, wherein the mass of TA is 1.0 g. Then, 40 μL of TA solution is drawn and added to 400 μL of MBG@DNA solution, and stirred at room temperature for 30 min.

[0045] Example 4 This example involves wet adhesion test of DNA molecule glue Taking the DNA molecular glue prepared in Example 1 as an example, the gelation process and state of the DNA molecular glue in the presence of water molecules were observed, and its ability to gel in a wet environment was intuitively analyzed, indicating that the DNA molecular glue has wet adhesion; the results are as follows Figure 3 As shown, Figure 3 A, in deionized water, stirring, DNA molecular glue immediately changes from a flowing state to a white gel-like substance, and adheres to the blue gun tip. On this basis, considering the blood flushing and soaking effect during large-area bleeding, the adhesion performance of DNA molecular glue after soaking in water was further investigated. That is, after soaking in water for 1 hour, it can still stick to a centrifuge tube weighing 2.0 g ( Figure 3 B), 5.1 g of pork ( Figure 3 C) and 3.2 g of pig skin ( Figure 3 D) The above results once again verified that the DNA molecule glue still had adhesion properties when it came into contact with water, and intuitively proved that the synthesized DNA molecular glue had the ability to form gels when it came into contact with water and the adhesion strength.

[0046] Example 5 This example relates to the blocking performance of DNA molecular glue in a wet environment Taking the DNA molecular glue prepared in Example 1 as an example, the ability of DNA molecular glue to gel and plug tissue in a wet environment in vitro was tested, indicating that DNA molecular glue has wet adhesion and plugging properties. Fill the pig small intestine with water and use a blade to prepare a 0.5 cm water outlet, such as Figure 4 A and 4B. Water flows out of the hole, and the DNA molecule glue is applied to the hole, as shown in Figure 4 C. After 10 s, the pig intestine was squeezed again, and no water flowed out of the holes, indicating that the DNA molecule gelled when it met water, effectively blocking the holes and forming a sticky white film-like substance at the holes, as shown in Figure 2. Figure 4 As shown in D. After the hole is completely sealed by DNA molecular glue, no water flows out even when pressed, and the hole is intact. The above results show that DNA molecular glue has the property of gelling when it meets water, and after gelling, it can seal the tissue holes by adhesion in the presence of water molecules (i.e., wet environment) to prevent water molecules from flowing out. It is expected to be used for bonding and sealing wound bleeding in a wet environment.

[0047] Example 6 This example involves the characterization of characteristic groups of DNA molecular glue (FTIR) Taking the DNA molecular glue prepared in Example 1 as an example, the FTIR results of the tested DNA molecular glue are as follows: Figure 5 As shown in the figure, characteristic groups such as benzene ring, phosphate, and phenolic hydroxyl groups can be observed in the DNA molecular glue. When DNA is not mixed with TA / DOPA, the wave number of the initial antisymmetric vibration of the phosphate group is 1238.22 cm -1, DNA-TA / DOPA mixture, at 1095.49 cm -1 The ribose vibration mode (CC sugar) detected at 1085.85 cm -1 , that is 1095.49 cm -1 The peaks at 1081.99 and 1033.77 cm -1 The results indicate an enhanced interaction between TA, DOPA, and DNA. The overall shift of the peaks to lower wavenumbers indicates an increased DNA-TA / DOPA interaction, especially with the OH-C (phenyl) from the acyl group in TA.

[0048] Example 7 This example involves the characterization of hydrogen bonds in DNA molecules ( 1 H NMR) Taking the DNA molecular glue prepared in Example 1 as an example, the DNA molecular glue tested 1 H NMR results are as follows Figure 6 As shown, the formed DNA molecular glue has characteristic absorption H spectra of benzene ring and phosphate group. 1.97 ppm and 2.69 ppm are the methyl proton peaks of thymine on DNA molecules. When DNA is mixed with TA / DOPA, the proton peaks in the benzene ring of the catechol group in TA-DOPA can be observed at 6.3 ~ 6.62 ppm. It proves that TA-DOPA is grafted onto the double helix chain of deoxyribonucleotides through hydrogen bonds.

[0049] Example 8 This example involves the molecular weight characterization of DNA molecule gel (GPC) Taking the DNA molecular gel prepared in Example 1 as an example, the GPC peak spectrum and molecular weight distribution are as follows: Figure 7 As shown in the figure, with the introduction of small molecules, the molecular weight of DNA molecule gel gradually increases. Figure 7It can be seen that the PDI of DNA, DNA / TA and DNA / TA-DOPA are 1.071, 1.074 and 1.069, respectively, and the average molecular weights are approximately 13286.7, 14333.6 and 14966.4 kDa, respectively. With the introduction of TA and DOPA, the molecular weight of DNA molecular glue is gradually increasing. In addition, the peak time and end time of DNA are 9.237 and 10.278 min, respectively; the peak time and end time of DNA / TA are 9.235 and 10.253 min, respectively; the peak time and end time of DNA / TA-DOPA are 9.213 and 10.208 min, respectively; the above results show that although the introduction of TA-DOPA changes the state and viscosity of DNA, its essence is still a DNA molecular chain. Among them, DNA / TA and DNA / TA-DOPA represent the introduction of TA into DNA and the introduction of TA and DOPA into DNA, respectively.

[0050] Example 9 This example involves the rheological characteristics of DNA molecular glue Taking the DNA molecular gel prepared in Example 1 as an example, the rheological results are as follows: Figure 8 As shown, it shows that DNA molecular glue has stable rheology and gelation. In oscillation mode, under the temperature scanning conditions of fixed strain and frequency, the storage modulus (G') and loss modulus (G'') at 20~80℃ were tested with a constant force strain of 100Pa and a constant frequency of 0.1~10 Hz. As the temperature changes, in the gel state, G'' is always greater than G', and the rheology does not change with the increase in temperature. This shows that the hydrogel is in a stable gel state and has good temperature stability.

[0051] Example 10 This example involves the adhesion strength of DNA molecular glue Taking the DNA molecular glue prepared in Example 1 as an example, pig skin was used to test the adhesion strength of the DNA molecular glue. Before adhesion, fresh pig skin purchased from the market was soaked in 1 mol / L NaOH for 15 min to remove the fat tissue in the pig skin, and then washed with deionized water for 3 times. Subsequently, the treated pig skin was cut into rectangular pieces (30 × 15 mm 2 The specific operation process is as follows: smear 500 μL of DNA molecular glue on the surface of pig skin, and then cover it with another layer of pig skin, with a bonding area of ​​225 mm 2 The adhered pigskin was immersed in deionized water, PBS, SBF and other liquids for a certain period of time, and the shear strength during stretching was measured at a stretching rate of 10 mm / min until the pigskin bond was broken. Fig. 9 As shown, Fig. 9The left side shows the tensile stress curve. The DNA molecular glue has the maximum tensile stress (9.85 N). The pig skin adhered to pure DNA breaks at 0.36 N, while the tensile stress of the commercial CS glue is 1.03 N. According to the force and adhesion strength calculation formula, the adhesion strength of the DNA molecular glue is 43.45 ± 2.70 kPa, and the adhesion strengths of pure DNA and commercial CS glue are 1.82 ± 0.42 and 4.51 ± 2.58 kPa, respectively. The above results show that the DNA molecular glue has excellent adhesion properties; compared with the commercial CS glue and pure DNA, the DNA molecular glue has the maximum tensile stress and adhesion strength.

[0052] Embodiment 11 This example relates to the liquid absorption performance of DNA molecular glue Taking the DNA molecular glue prepared in Example 1 as an example, it is explained that the DNA molecular glue has both liquid absorption and swelling properties. Fig.10 A is the sample's water absorption rate. The sample was immersed in Tris buffer to determine its water absorption rate. The detailed operation steps are as follows: After vacuum drying at 60℃ for 24 h, take out the sample and weigh it to record W0. Add 5 mL of Tris liquid and record it as V mL. Magnetic stirring temperature is 37℃, 250 rpm, 10 min, and let it stand for 20 min. Filter until the last drop of liquid drips into the filter bottle, and weigh the sample weight as W1. The results show that the water absorption rate of DNA molecular glue is significantly higher than that of DNA, and the water absorption rate is about 619%.

[0053] Fig.10 B is the expansion rate of the sample. The detailed operation steps are as follows: First, the sample was dried in a vacuum at 60℃ for 24 h, 0.2 g of the sample was added to 10 mL of Tris buffer, and incubated at 37℃ for 10, 20, 30, 40, 50 and 60 min, respectively, and centrifuged at 5 000 rpm for 10 min. The supernatant was discarded and the precipitate was recorded as W (g). The results showed that DNA molecular glue has swelling properties and gradually stabilized at 50-60 min. However, DNA and MBG@DNA did not form gels because they were not grafted with TA-DOPA. They gradually dissolved after being immersed in Tris buffer at 37℃ for 40 min. The above results show that DNA molecular glue has both adhesion and hemostasis properties, and can absorb blood at the wound surface, expand and stop bleeding, and has multi-pathway hemostasis potential.

[0054] Example 12 This example involves the coagulation properties of DNA molecular gel Taking the DNA molecular gel prepared in Example 1 as an example, the in vitro coagulation properties of DNA, MBG@DNA, MBG@DNA / TA and MBG@DNA / TA-DOPA series samples were tested using sodium citrate anticoagulated whole blood of SD rats. Fig.11Among them, the significant differences between the samples and the Blank group were **P<0.05 ***P<0.001****P<0.0001; the in vitro coagulation effect of DNA molecular gel was significantly higher than that of blank and pure DNA.

[0055] The results showed that the DNA molecular glue, MBG@DNA / TA-DOPA, had the shortest in vitro coagulation time, i.e., 16.3 ± 3.2 s. Pure DNA had no procoagulant effect, and there was no significant difference between its in vitro coagulation time (86.7 ± 7.6 s) and that of the blank group (90.0 ± 5.0 s). The addition of MBG improved the coagulation performance of DNA, and the coagulation time of MBG@DNA was 71.7 ± 2.9 s. The introduction of TA into MBG@DNA made the DNA molecules gel, accelerating the aggregation of red blood cells, and its in vitro coagulation time was 50.0 ± 5.0 s. In summary, with the addition of hemostatic active factors (MBG) and wet adhesion phenolic hydroxyl groups (TA-DOPA) in the DNA molecular glue, its coagulation performance gradually increased, and the composite DNA molecular glue had the best coagulation performance.

[0056] Example 13 This example involves red blood cell aggregation and fibrin network generation of DNA molecular glue Taking the DNA molecular glue prepared in Example 1 as an example, SD rat sodium citrate anticoagulated whole blood was used, 1 mL of anticoagulated whole blood was added with 10.0 mg of the sample to be tested. After the blood coagulated, it was washed 3 times with PBS to remove excess red blood cells. 2.5% glutaraldehyde was added to the blood clot and fixed at 4°C for 2.5 h. The sample was dehydrated and washed 3 times with a series of gradient ethanol (25%, 50%, 75%, 85% and 100%) to eliminate the adhesion of red blood cells to the surface of the material, and each concentration of ethanol was soaked for 15 min. After drying, gold was sprayed for 60 s at a voltage of 10 mV, and the aggregation of red blood cells in vitro was observed by SEM. The results are as follows Fig.12 As shown in the figure, a clear fibrin network structure can be observed in the DNA molecular glue group, and a large number of activated red blood cells can be observed in the MBG@DNA / TA group, but the fibrin network structure has not yet formed. Neither activated platelets nor obvious fibrin networks were observed in the DNA and MBG@DNA groups, and only a small amount of fibrin that had not yet formed a network was observed in the MBG@DNA group. The above results show that DNA molecular glue can significantly activate platelets, form a fibrin network, and promote red blood cell aggregation.

[0057] Embodiment 14 This example involves the binding properties of DNA molecular glue to hemoglobin Taking the DNA molecular gel prepared in Example 1 as an example, in order to further test the coagulation ability of the sample, a hemoglobin coagulation test was performed ( Fig.13), indicating that DNA molecular glue has excellent hemoglobin binding ability. In the hemoglobin binding test, the sample was placed in a 9 cm culture dish and preheated at 37°C for 10 min. SD rat whole blood (50 μL) was injected and incubated (37°C, 30 rpm, 5 min). Then, 30 mL of deionized water (37°C, 30 rpm, 1 h) was added, and the solution was collected after the blood diffused, and the absorbance of the solution was detected at 540 nm. Pure blood was used as a negative control, n = 3. The lower the coagulation index (BCI) value, the better the coagulation ability of the sample and the less the diffusion of hemoglobin in deionized water. Fig.13 The results of B show that DNA molecular glue (67.2± 0.9%) has the best coagulation performance, and hemoglobin diffusion is weak. In deionized water, a large number of red blood cells rupture after contact with water, and hemoglobin is dispersed in the solution, with a BCI value of 96.7 ± 2.5%. The BCI value of MBG@DNA / TA (76.9 ±1.8%) is slightly higher than that of DNA molecular glue. The above results show that DNA molecular glue has the best coagulation index.

[0058] Embodiment 15 This example involves in vivo liver hemostasis using DNA molecular glue Taking the DNA molecular gel prepared in Example 1 as an example, a liver injury bleeding model of SD rats was prepared. The statistical results of the bleeding time after treatment are as follows: Fig.14 As shown. After treatment, the hemostasis time of the DNA molecular glue group was the shortest (11.0 ± 3.6 s), which was shortened by about 92% compared with the blank group. The bleeding time of the MBG@DNA / TA group was longer than that of the DNA molecular glue, that is, the introduction of DOPA improved the liver hemostasis effect of the DNA molecular glue, which may be related to the wet adhesion property of DOPA. Note: Compared with Blank, the differences between each group and it were significant **P ≤ 0.01****P ≤ 0.0001; the SD liver hemostasis effect of the DNA molecular glue was the best and had the shortest hemostasis time.

[0059] Example 16 This example involves the blood compatibility of DNA molecular glue Taking the DNA molecular gel prepared in Example 1 as an example, the sample was incubated with SD whole blood dilution, and the absorbance of the supernatant at 540 nm was tested to detect whether the sample would cause red blood cell rupture. Fig.15 As shown, the hemolysis rate of the DNA series samples is lower than the national standard (5%). That is, the DNA series samples have good blood compatibility and will not cause red blood cells to rupture. Note: Fig.15In the figure, the sample concentrations on the horizontal axis are 2000, 1000, 500, and 250 μg / mL from large to small; the numbers 1, 2, 3, and 4 represent DNA, MBG@DNA, MBG@DNA-TA, and MBG@DNA-TA / DOPA, respectively; the DNA series samples have good blood compatibility, and the hemolysis rates are all lower than 5%.

[0060] Embodiment 17 This example relates to the cell compatibility of DNA molecular glue Taking the DNA molecular glue prepared in Example 1 as an example, the sample extract was co-cultured with L929 cells for 24 and 48 h, and the sample toxicity and live-dead cell tests were tested. It was shown that the DNA series samples had good compatibility with L929 cells and had a cell proliferation promoting effect. The results are as follows: Fig.16 shown. Fig.16 A The results show that the cell activity of DNA molecular gel is greater than 95%, except for Zeolite used as a control. Fig.16 B is the statistical result of Image-J, showing that DNA molecular glue has a significant effect on promoting cell proliferation at 48 h of co-culture, which is consistent with the live-dead staining result of 16C. The above results show that DNA molecular glue has no cytotoxicity and good cell compatibility.

[0061] The present invention describes preferred embodiments and their effects. However, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0062] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a DNA molecular glue for moist wound hemostasis, characterized in that: The following steps are involved: Dispersing tannic acid evenly in a water solvent, adding dopamine hydrochloride, and mixing evenly to obtain a tannic acid-dopamine solution; The mesoporous bioactive glass was uniformly dispersed in a water solvent, and then DNA was added and stirred at 85 to 95°C for 10 to 15 minutes to obtain a MBG@DNA solution; Add tannic acid-dopamine solution to the MBG@DNA solution and stir at room temperature for 20 to 40 min. The tannic acid-dopamine phenolic hydroxyl structure will be grafted to the deoxynucleotide double strand of DNA through hydrogen bonding to obtain DNA molecular glue.

2. The method for preparing the DNA molecular glue for moist wound hemostasis according to claim 1, characterized in that: The mass ratio of the tannic acid to the dopamine hydrochloride is 0.5-1.5:0.

675.

3. The method for preparing the DNA molecular glue for moist wound hemostasis according to claim 1, characterized in that: The mass ratio of the mesoporous bioactive glass to DNA is 1:3-8.

4. The method for preparing the DNA molecular glue for moist wound hemostasis according to claim 1, characterized in that: The process of preparing the tannic acid-dopamine solution includes: adding tannic acid to 0.8 to 1.5 mL of deionized water, heating in a water bath at 40 to 42° C. and stirring until completely dissolved, lowering the water bath temperature to 36.5 to 37.5° C., adding dopamine hydrochloride, and stirring for 5 to 10 minutes.

5. The method for preparing the DNA molecular glue for moist wound hemostasis according to claim 1, characterized in that: When preparing DNA molecular gel, add tannic acid-dopamine solution and stir at room temperature for 20 to 40 minutes. The temperature drops from 85 to 95°C to room temperature, which is the process of DNA annealing.

6. The method for preparing the DNA molecular glue for moist wound hemostasis according to claim 1, characterized in that: When preparing DNA molecular glue, the concentration of mesoporous bioactive glass in MBG@DNA solution is 0.8 ~ 1.6 w / v; the volume ratio of tannic acid-dopamine solution to MBG@DNA solution is 0.1 ~ 0.2:

1.

7. The method for preparing the DNA molecular glue for moist wound hemostasis according to claim 1, characterized in that: The mesoporous bioactive glass is prepared according to the following steps: CTAB is evenly dispersed in a mixed solvent, and tetraethyl orthosilicate is added dropwise after adjusting the pH value with ammonia water, followed by calcium nitrate. After the reaction is completed, the product is obtained; The product is calcined to remove the structural template CTAB, thereby obtaining mesoporous bioactive glass.

8. The method for preparing the DNA molecular glue for moist wound hemostasis according to claim 7, characterized in that: The mixed solvent is prepared from deionized water, ethanol and ether in a volume ratio of 7.5:1:

2.

9. A DNA molecular glue for moist wound hemostasis prepared by the method according to any one of claims 1 to 8.

10. Use of the DNA molecular glue according to claim 9 in preparing a wet wound hemostatic drug.

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