DNA molecular glue for moist wound hemostasis and its preparation method and application

Abstract

The present invention discloses a DNA molecular glue for wet wound hemostasis and its preparation method and application, which relates to the technical field of biomedical materials. The method is inspired by the wet adhesion mechanism of mussels. The tannic acid-dopamine phenolic hydroxyl structure is grafted onto the DNA double helix chain using a genetically encoded "click" reaction to prepare a wet adhesion hemostatic DNA molecular glue. The present invention adds a coagulation factor (Ca) into 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 through multiple pathways.

Description

Technical Field

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

[0002] Currently, the mortality rate due to blood loss is approximately 30%, and rapid hemostasis is the key to reducing this mortality rate. Although hemostatic agents, sealants, and adhesives suitable for a variety of bleeding wounds are emerging in an endless stream, effective hemostasis in a wet environment remains a challenge. Currently, a single hemostatic material can no longer meet the hemostasis needs of complex wounds, and composite intelligent hemostatic materials that coordinate multiple coagulation pathways are the development trend. The current global 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 amount of water molecules are present, the existing gel hemostatic materials have a slow gelation rate, reduced adhesion properties, poor adhesion, and cannot effectively adhere to and seal the wound. In addition, gel adhesive hemostatic materials prepared by chemical synthesis have problems such as organic residues, poor degradability, and poor biocompatibility, which are not conducive to wound healing and repair. In other words, 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-adhesive polyphenols can be spontaneously grafted onto DNA chains through hydrogen bonds in deionized water to form a three-dimensional network structure. DNA has precise base pair recognition properties, and the base pairing reaction in the double strand has the same characteristics as other "click" reactions, such as fast rate, high base pairing selectivity and no by-products. Compared with traditional "click" reactions, the advantages of genetically encoded "click" reactions are more prominent.

[0004] After the DNA double helix is ​​unwound, it polymerizes hydroxyl groups with 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. Their application in hemostatic materials can significantly improve the efficiency of hemostasis. 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 in fields such as biomedicine and tissue engineering. However, their adhesion properties in a wet environment are limited, and their hemostatic effect in large bleeding wounds is average. Meanwhile, the prior art discloses DNA nanocomposite hydrogel adhesive materials with hemostatic properties based on clay, deoxyribonucleic acid (DNA), and methacrylic anhydride-modified gelatin. While these materials exhibit hemostasis, tissue healing, and wound sealing effects when applied to wounds, their preparation process is complex and requires modified gelatin and UV crosslinking to form a gel, making them unsuitable for large-scale production and posing a risk of organic residue. Also disclosed in the prior art is a hydrophobic chain-driven mussel-like wet-adhesive hydrogel that exhibits wet adhesion to arteries and internal organs and rapid hemostasis. However, the preparation process requires the use of an initiator and a crosslinker, resulting in high cost, organic reagent residue, and complex operation, with limited biocompatibility. Summary of the Invention

[0005] In view of the deficiencies in the above-mentioned background technology, the present invention mainly solves the following problems: the existing hemostatic hydrogels have poor wet adhesion properties, and after contact with water, the adhesion strength decreases, and it cannot achieve effective blocking and hemostatic effect; the existing hemostatic adhesives have a single hemostatic effect, and the effect is general only through the physical adhesion pathway, and they do not have the function of activating the body's own coagulation pathway; and the existing hemostatic adhesives need to add initiators, cross-linking agents or organic reagents to trigger gelation. The organic matter 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 its preparation method and application. 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 using a genetically encoded "click" reaction to prepare a wet adhesion hemostatic DNA molecular glue. The present invention adds a coagulation factor (Ca) into the DNA molecular glue. 2+ ) silicate bioactive glass rapidly activates the body's intrinsic and extrinsic coagulation pathways, synergizing with the wet adhesion of DNA molecular glue to achieve multi-pathway rapid hemostasis. The DNA molecular glue prepared by this invention is green and biofriendly. The preparation and gelation process does not require initiators or crosslinking agents, and gelation can be achieved 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:

[0007] Dispersing tannic acid uniformly in an aqueous solvent, adding dopamine hydrochloride, and mixing uniformly to obtain a tannic acid-dopamine solution;

[0008] The mesoporous bioactive glass was evenly dispersed in a water solvent, and DNA was added, and the mixture was stirred at 85-95°C for 10-15 minutes to obtain MBG@DNA solution.

[0009] Add tannic acid-dopamine solution to the MBG@DNA solution and stir at room temperature for 20 to 40 minutes. 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.

[0010] Preferably, the mass ratio of the tannic acid to the dopamine hydrochloride is 0.5-1.5:0.675.

[0011] Preferably, the mass ratio of the mesoporous bioactive glass to DNA is 1:3 to 8.

[0012] Preferably, the process of preparing the tannic acid-dopamine solution comprises:

[0013] Add tannic acid to 0.8 to 1.5 mL of deionized water, heat in a water bath at 40 to 42°C, and stir until completely dissolved. Then, lower the water bath temperature to 36.5 to 37.5°C, add dopamine hydrochloride, and stir for 5 to 10 minutes.

[0014] Preferably, when preparing the DNA gel, the tannic acid-dopamine solution is added and stirred at room temperature for 20 to 40 minutes while the temperature drops from 85 to 95° C. to room temperature, i.e., during the DNA annealing process.

[0015] Preferably, when preparing DNA molecular glue, the concentration of mesoporous bioactive glass in the MBG@DNA solution is 0.8 to 1.6 w / v; the volume ratio of tannic acid-dopamine solution to MBG@DNA solution is 0.1 to 0.2:1.

[0016] Preferably, the mesoporous bioactive glass is prepared according to the following steps:

[0017] CTAB is evenly dispersed in a mixed solvent, the pH value is adjusted with ammonia water, tetraethyl orthosilicate is added dropwise, and then calcium nitrate is added. After the reaction is completed, the product is obtained;

[0018] The product is calcined to remove the structural template CTAB, thereby obtaining mesoporous bioactive glass.

[0019] Preferably, the mixed solvent is prepared from deionized water, ethanol and ether in a volume ratio of 7.5:1:2.

[0020] The second purpose of the present invention is to provide a DNA molecular glue for hemostasis of moist wounds.

[0021] The third object of the present invention is to provide an application of DNA molecular glue in the preparation of moist wound hemostatic drugs.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a DNA molecular glue for moist wound hemostasis, as well as its preparation method and application. Based on a genetically encoded "click" reaction, the invention forms hydrogen bonds with the phenolic hydroxyl groups of tannic acid (TA) and dopamine (DOPA) during DNA base complementation, introducing TA and DOPA into the DNA nucleic acid chain. Furthermore, through physical doping with mesoporous bioactive glass (MBG), a composite MBG@DNA / TA-DOPA hydrogel for moist wound hemostasis is prepared. This DNA molecular glue exhibits wet adhesion, accelerates hemostasis, aggregates red blood cells, and forms a fibrin network, and exhibits good biocompatibility and degradability.

[0024] Inspired by the wet adhesion mechanism of mussels, the present invention grafts the tannic acid-dopamine phenolic hydroxyl structure onto the DNA double helix 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 through multiple pathways.

[0025] The DNA molecular gel prepared by the present 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

[0026] Figure 1 Schematic diagram of the synthesis of DNA molecular glue;

[0027] Figure 2 SEM results of DNA molecular gel, where A: SEM of MBG@DNA / TA-DOPA; B: SEM of the white box area at 25 times magnification;

[0028] Figure 3 Water-gelling properties of DNA molecular glue. A: MBG@DNA-DOPA / TA adheres to a 1 mL pipette tip after gelling in water; B: MBG@DNA-DOPA / TA adheres to a rubber glove and an EP tube; C: MBG@DNA-DOPA / TA adheres to a rubber glove and pork; D: MBG@DNA-DOPA / TA adheres to a rubber glove and pig skin.

[0029] Figure 4 MBG@DNA-DOPA / TA composite molecular glue's adhesive sealing effect on wet tissues. A: Pig small intestine filled with water; B: Blade puncture 0.5 cm; C: Apply MBG@DNA-DOPA / TA composite hydrogel to block the water outlet; D: Blocked for 2 h.

[0030] Figure 5 FTIR results of DNA molecular glue;

[0031] Figure 6 DNA molecule glue 1 H NWR results;

[0032] Figure 7 GPC results of DNA molecular gel;

[0033] Figure 8 Rheological properties of DNA molecular glue;

[0034] Figure 9 Adhesion of DNA molecular glue;

[0035] Figure 10 Liquid absorption of DNA molecular glue, where A: liquid absorption rate; B: expansion rate;

[0036] Figure 11 In vitro coagulation time of DNA molecular gel, where A: coagulation image; B: coagulation time bar graph;

[0037] Figure 12 SEM results of DNA molecular gel-aggregated red blood cells, where A: DNA; B: MBG@DNA; C: MBG@DNA / TA; D: MBG@DNA / TA-DOPA;

[0038] Figure 13 BCI results of DNA molecular gel, where A: sample BCI image; B: BCI result bar graph;

[0039] Figure 14 Liver hemostasis results of DNA molecular gel, where A: hemostasis picture of liver injury model; B: bleeding time of liver injury model;

[0040] Figure 15 Hemolysis results of DNA molecular gel, where A: hemolysis picture of sample; B: hemolysis rate bar graph of sample;

[0041] Figure 16Cell compatibility of DNA molecular glue, including: A: cell viability measured by MTT; B: number of living cells counted by Image-J; C: live-dead cell staining after co-culture of sample extracts with L929 cells for 24 and 48 h. DETAILED DESCRIPTION

[0042] 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 with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0043] The first aspect of the present invention provides a method for preparing a DNA molecular glue for moist wound hemostasis, comprising the following steps:

[0044] Tannic acid (TA) is uniformly dispersed in a water solvent, and dopamine hydrochloride (DOPA) is added and mixed to obtain a tannic acid-dopamine solution (TA-DOPA).

[0045] Mesoporous bioactive glass (MBG) was uniformly dispersed in water, DNA was added, and the mixture was stirred at 85-95°C for 10-15 minutes to obtain MBG@DNA solution.

[0046] Add tannic acid-dopamine solution to the MBG@DNA solution and stir at room temperature for 20 to 40 minutes. 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).

[0047] Inspired by the wet adhesion mechanism of mussels, the present invention grafts the tannic acid-dopamine phenolic hydroxyl structure onto the DNA double helix 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 through multiple pathways.

[0048] In one embodiment, mesoporous bioactive glass (MBG) is uniformly dispersed in an aqueous solvent, and DNA is added thereto. The mixture is stirred at 85°C to 95°C for 10 to 15 minutes to obtain a mesoporous bioactive glass and DNA mixture (MBG@DNA). The MBG@DNA is prepared and ready for immediate use.

[0049] Wherein, the mass ratio of the tannic acid to the dopamine hydrochloride is 0.5-1.5:0.675.

[0050] The mass ratio of the mesoporous bioactive glass to DNA is 1:3 to 8.

[0051] The process for preparing the tannic acid-dopamine solution comprises:

[0052] Add tannic acid to 0.8 to 1.5 mL of deionized water, heat in a water bath at 40 to 42°C, and stir until completely dissolved. Then, lower the water bath temperature to 36.5 to 37.5°C, add dopamine hydrochloride, and stir for 5 to 10 minutes.

[0053] 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.

[0054] 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.

[0055] The mesoporous bioactive glass is prepared according to the following steps:

[0056] CTAB is evenly dispersed in a mixed solvent, and after adjusting the pH value with ammonia water, tetraethyl orthosilicate is added dropwise, followed by calcium nitrate. After the reaction is completed, the product is obtained;

[0057] The product is calcined to remove the structural template CTAB, thereby obtaining mesoporous bioactive glass.

[0058] The mixed solvent is prepared from deionized water, ethanol and ether in a volume ratio of 7.5:1:2.

[0059] The second aspect of the present invention provides a DNA molecular glue for hemostasis of moist wounds.

[0060] The third aspect of the present invention provides an application of DNA molecular glue in the preparation of a moist wound hemostatic drug.

[0061] 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.

[0062] The DNA used in the following examples, namely high molecular weight thread-bound salmon sperm DNA, was purchased from Shenzhen Cellma Biotechnology Co., Ltd.

[0063] In the following examples, MBG@DNA / TA was prepared by adding 1.0 g of TA to 1 mL of deionized water, heating in a 40–42°C waterbath with stirring until completely dissolved. 40 μL of the TA solution was then added to 400 μL of the MBG@DNA solution and stirred at room temperature for 20–40 minutes.

[0064] Example 1

[0065] This embodiment involves the synthesis of DNA molecular glue

[0066] MBG preparation: 150 mL of deionized water, 20 mL of ethanol, and 40 mL of co-solvent ether were added sequentially to a round-bottom flask containing 2.0 g of CTAB. Stir at high speed at room temperature until the CTAB was completely dissolved. An appropriate amount of aqueous ammonia was added dropwise to the solution to adjust the pH. After 10 minutes, TEOS was added dropwise to the solution while stirring at room temperature. After 10 minutes, a predetermined amount of Ca(NO₃)₂·4H₂O was added to the solution and the reaction was allowed to proceed at room temperature with magnetic stirring at 1200 rpm for 4 hours. After the reaction, the precipitate was collected by centrifugation at 10,000 rpm for 5 minutes to obtain the sample. The sample was washed three times with anhydrous ethanol and three times with deionized water, then dried under vacuum at 50°C for 24 hours. The sample was calcined in a muffle furnace at 550°C for 5 hours to remove the template, at a heating rate of 1°C / min. The sample was then sealed under vacuum and used for storage.

[0067] Preparation of TA-DOPA: Weigh 0.5 g of tannic acid (TA) powder and add it to 1 mL of deionized water. Heat in a 40°C waterbath and stir until completely dissolved. Then, lower the waterbath temperature to 37°C. Weigh 0.675 g of dopamine hydrochloride (DOPA) powder and stir vigorously for 5 minutes. The prepared TA-DOPA can be stored at 4°C and used within 1 week. Incubate at 37°C for 10 minutes before each use.

[0068] Preparation of MBG@DNA: Weigh 10 mg of prepared MBG into 1 mL of deionized water. Ultrasonicate at 100 Hz for 2 minutes to obtain a MBG dispersion. Add 50 mg of DNA to the MBG dispersion and stir at 90°C, 200 rpm, for 10 minutes to fully dissolve the DNA. MBG@DNA is ready for use.

[0069] Preparation of MBG@DNA / TA-DOPA: 100 μL of TA-DOPA solution was added to the MBG@DNA solution and stirred continuously at room temperature for 30 minutes to allow uniform gel formation. During this process, the temperature of the MBG@DNA decreased from 90°C to room temperature, a process known as DNA annealing. TA-DOPA then hydrogen-bonded to the deoxynucleotide duplex of the DNA, producing MBG@DNA / TA-DOPA, or DNA molecular glue.

[0070] See also Figure 1 Figure 2 shows the synthesis mechanism of DNA glue. MBG is pre-dispersed in deionized water. After dissolving the DNA, it is mixed with TA-DOPA at high temperature, followed by cooling and stirring at room temperature. During DNA annealing, TA-DOPA is grafted onto the nucleic acid double helix through hydrogen bonds, resulting in MBG-doped DNA glue.

[0071] Figure 2 This 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 Figure A, MBG particles are dispersed within the pores or scaffold of a DNA gel. The introduction of TA-DOPA into the DNA molecules creates a pore structure with pores ranging from approximately 14.8 to 30.0 μm after freeze-drying. Figure 2 In image B, MBG can be observed within the DNA gel scaffold. MBG particle sizes range from approximately 400 to 800 μm, with distinct pores. MBG is uniformly incorporated into the DNA gel.

[0072] Example 2

[0073] Same as Example 1, except that:

[0074] Preparation of TA-DOPA: Weigh 1.0 g of tannic acid (TA) powder and add it to 1 mL of deionized water. Heat in a 40°C waterbath and stir until completely dissolved. Then, lower the waterbath temperature to 37°C. Weigh 0.675 g of dopamine hydrochloride (DOPA) powder and stir vigorously for 5 minutes. The prepared TA-DOPA can be stored at 4°C and used within 1 week. Incubate at 37°C for 10 minutes before each use.

[0075] Example 3

[0076] Same as Example 1, except that:

[0077] Preparation of TA-DOPA: Weigh 1.5 g of tannic acid (TA) powder and add it to 1 mL of deionized water. Heat in a 40°C waterbath and stir until completely dissolved. Then, lower the waterbath temperature to 37°C. Weigh 0.675 g of dopamine hydrochloride (DOPA) powder and stir vigorously for 5 minutes. The prepared TA-DOPA can be stored at 4°C and used within 1 week. Incubate at 37°C for 10 minutes before each use.

[0078] To illustrate the properties of the DNA adhesive provided by the present invention, the following figures illustrate the preparation of MBG@DNA / TA: 1.0 g of TA was added to 1 mL of deionized water, heated in a 40°C waterbath, and stirred until completely dissolved. 40 μL of the TA solution was then added to 400 μL of the MBG@DNA solution and stirred at room temperature for 30 minutes.

[0079] Example 4

[0080] This example involves wet adhesion testing of DNA molecular glue

[0081] 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 gelation ability 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, upon stirring, the DNA glue immediately changes from a fluid state to a white gel-like substance, which adheres to the blue tip of the pipette. Furthermore, considering the flushing and soaking effects of blood during extensive bleeding, the adhesion properties of the DNA glue after soaking in water were further investigated. Even after soaking in water for 1 hour, it could still adhere to a 2.0 g centrifuge tube ( 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 molecular 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 had strong adhesion.

[0082] Example 5

[0083] This embodiment relates to the blocking performance of DNA molecular glue in a wet environment

[0084] Taking the DNA molecular glue prepared in Example 1 as an example, the ability of DNA molecular glue to form a gel and block tissue in a wet environment in vitro was tested, indicating that DNA molecular glue has wet adhesion and blocking properties. The pig small intestine was filled with water, and a 0.5 cm water outlet was prepared with a blade, as shown in FIG. Figure 4As shown in 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. Once the hole is completely sealed with the DNA glue, no water will flow out even when pressed, and the hole is intact. These results demonstrate that the DNA glue has the ability to gel when exposed to water. Once gelled, it can seal tissue pores by adhesion in the presence of water molecules (i.e., in a wet environment), preventing water from escaping. It is expected to be used to seal wounds and bleeding in wet environments.

[0085] Example 6

[0086] This example involves the characterization of characteristic groups of DNA molecular glue (FTIR)

[0087] 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 gel. When DNA is not mixed with TA / DOPA, the wave number of the phosphate group's initial antisymmetric vibration 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 , which is 1095.49 cm -1 The peaks at 1081.99 and 1033.77 cm -1 This result indicates an enhanced interaction between TA, DOPA, and DNA. The overall shift of the peaks to lower wavenumbers indicates an increase in DNA-TA / DOPA interactions, particularly with the OH-C (phenyl) groups from the acyl groups in TA.

[0088] Example 7

[0089] This example involves the characterization of hydrogen bonds in DNA molecule glue ( 1 H NMR)

[0090] 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 6As shown, the resulting DNA gel exhibits characteristic H absorption spectra of benzene rings and phosphate groups. The peaks at 1.97 ppm and 2.69 ppm represent the methyl proton peaks of thymine on the DNA molecule. When DNA is mixed with TA / DOPA, the proton peaks of the benzene rings of the catechol groups in TA-DOPA are observed at 6.3 to 6.62 ppm. This demonstrates that TA-DOPA is grafted onto the deoxyribonucleotide double helix through hydrogen bonding.

[0091] Example 8

[0092] This example involves the molecular weight characterization of DNA molecules by gel permeation chromatography (GPC).

[0093] 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 7 As can be seen from the data, the PDIs of DNA, DNA / TA, and DNA / TA-DOPA are 1.071, 1.074, and 1.069, respectively, and their 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 the DNA gel gradually increases. Furthermore, the peak onset and end times of DNA are 9.237 and 10.278 min, respectively; the peak onset and end times of DNA / TA are 9.235 and 10.253 min, respectively; and the peak onset and end times of DNA / TA-DOPA are 9.213 and 10.208 min, respectively. These results indicate that although the introduction of TA-DOPA alters the state and viscosity of DNA, its essence remains a DNA molecular chain. DNA / TA and DNA / TA-DOPA represent the introduction of TA into DNA and the introduction of TA and DOPA into DNA, respectively.

[0094] Example 9

[0095] This example involves the rheological characteristics of DNA molecular glue

[0096] Taking the DNA molecular glue prepared in Example 1 as an example, the rheological results are as follows: Figure 8 The results show that the DNA hydrogel has stable rheological and gelling properties. The storage modulus (G') and loss modulus (G'') were measured from 20°C to 80°C under oscillatory mode, constant strain, and temperature sweep conditions, with a constant force-strain of 100 Pa and a constant frequency of 0.1-10 Hz. As the temperature changes, the gel state shows that G'' remains greater than G', and the rheological properties do not change with increasing temperature. This indicates that the hydrogel is in a stable gel state and has good temperature stability.

[0097] Example 10

[0098] This example involves the adhesion strength of DNA molecular glue

[0099] Taking the DNA molecular glue prepared in Example 1 as an example, the adhesion strength of the DNA molecular glue was tested on pig skin. Before adhesion, fresh pig skin purchased from the market was soaked in 1 mol / L NaOH for 15 minutes to remove the fat tissue in the pig skin, and then washed with deionized water three 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. Figure 9 As shown, Figure 9 The tensile stress curve on the left shows the DNA glue exhibiting the highest tensile stress (9.85 N). Pigskin adhered to pure DNA fractured at 0.36 N, while the commercial CS glue exhibited a tensile stress of 1.03 N. Based on the force and adhesion strength calculation formula, the adhesion strength of the DNA glue was 43.45 ± 2.70 kPa, while the adhesion strengths of pure DNA and commercial CS glue were 1.82 ± 0.42 and 4.51 ± 2.58 kPa, respectively. These results demonstrate that the DNA glue exhibits excellent adhesion properties; compared to commercial CS glue and pure DNA, the DNA glue exhibits the highest tensile stress and adhesion strength.

[0100] Example 11

[0101] This example relates to the liquid absorption performance of DNA molecular glue

[0102] Taking the DNA molecular glue prepared in Example 1 as an example, it is illustrated that the DNA molecular glue has both liquid absorption and expansion properties. Figure 10 A represents the sample's water absorption rate. The sample was immersed in Tris buffer to measure its water absorption rate. The detailed steps are as follows: After vacuum drying at 60°C for 24 hours, the sample was removed and weighed (W0). 5 mL of Tris solution was added (V mL). The sample was magnetically stirred at 37°C, 250 rpm, for 10 minutes and allowed to stand for 20 minutes. The sample was filtered until the last drop of liquid dripped into a filter flask, and the sample weight was weighed (W1). The results showed that the DNA gel had a significantly higher water absorption rate than DNA, reaching approximately 6.19%.

[0103] Figure 10B represents the swelling rate of the sample. The detailed procedure is as follows: First, the sample was dried in a vacuum at 60°C for 24 hours. 0.2 g of the sample was added to 10 mL of Tris buffer and incubated at 37°C for 10, 20, 30, 40, 50, and 60 minutes, respectively. The sample was then centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was recorded as W (g). The results showed that the DNA molecular glue exhibited swelling properties, which gradually stabilized between 50 and 60 minutes. However, DNA and MBG@DNA, which were not grafted with TA-DOPA and did not form a gel, gradually dissolved after immersion in Tris buffer at 37°C for 40 minutes. These results demonstrate that the DNA molecular glue possesses both adhesive hemostatic properties and the ability to absorb blood at the wound surface, swelling and hemostasis, demonstrating its multi-pathway hemostatic potential.

[0104] Example 12

[0105] This example involves the coagulation properties of DNA molecular gel

[0106] Taking the DNA molecular gel prepared in Example 1 as an example, the in vitro coagulation performance of DNA, MBG@DNA, MBG@DNA / TA and MBG@DNA / TA-DOPA series samples was tested using sodium citrate anticoagulated whole blood of SD rats. Figure 11 Among 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.

[0107] Results showed that the DNA gel, MBG@DNA / TA-DOPA, exhibited the shortest in vitro clotting time, 16.3 ± 3.2 s. Pure DNA exhibited no procoagulant activity, with a clotting time (86.7 ± 7.6 s) not significantly different from that of the blank control (90.0 ± 5.0 s). The addition of MBG improved the clotting properties of DNA, resulting in a clotting time of 71.7 ± 2.9 s for MBG@DNA. The introduction of TA into MBG@DNA resulted in a gel-like structure of the DNA molecules, accelerating red blood cell aggregation, resulting in an in vitro clotting time of 50.0 ± 5.0 s. In summary, the addition of both the hemostatic active factor (MBG) and the wet-adhesive phenolic hydroxyl group (TA-DOPA) to the DNA gel resulted in a gradual increase in clotting performance, resulting in the composite DNA gel exhibiting optimal clotting properties.

[0108] Example 13

[0109] This example involves the aggregation of red blood cells and the formation of fibrin networks using DNA molecular glue

[0110] 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 anticoagulated, and 10.0 mg of the sample to be tested was added. 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 Figure 12 As shown, a clear fibrin network was observed in the DNA gel group, while a large number of activated erythrocytes were observed in the MBG@DNA / TA group, but the fibrin network had not yet formed. Neither activated platelets nor a clear fibrin network was observed in the DNA or MBG@DNA groups. Only a small amount of fibrin, which had not yet formed a network, was observed in the MBG@DNA group. These results demonstrate that DNA gel can significantly activate platelets, form a fibrin network, and promote erythrocyte aggregation.

[0111] Example 14

[0112] This example involves the binding properties of DNA molecular glue to hemoglobin

[0113] 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 ( Figure 13 ), indicating that the DNA molecule 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 minutes. 50 μL of SD rat whole blood was injected and incubated (37°C, 30 rpm, 5 minutes). Then, 30 mL of deionized water was added (37°C, 30 rpm, 1 hour). After the blood diffused, the solution was collected and its absorbance was measured at 540 nm. Pure blood was used as a negative control, n = 3. The lower the blood coagulation index (BCI) value, the better the sample's coagulation ability and the less hemoglobin diffusion in deionized water. Figure 13 Results from test B show that DNA glue (67.2 ± 0.9%) exhibits the best coagulation performance, while hemoglobin diffusion is weak. In deionized water, however, numerous red blood cells rupture upon contact, dispersing hemoglobin into the solution, resulting in a BCI value of 96.7 ± 2.5%. MBG@DNA / TA exhibits a slightly higher BCI value (76.9 ± 1.8%) than the DNA glue. These results demonstrate that DNA glue possesses the optimal coagulation index.

[0114] Example 15

[0115] This example involves in vivo liver hemostasis using DNA molecular glue

[0116] Taking the DNA molecular glue prepared in Example 1 as an example, a liver injury and bleeding model of SD rats was prepared. The statistical results of bleeding time after treatment are as follows: Figure 14 As shown. After treatment, the DNA gel group had the shortest hemostatic time (11.0 ± 3.6 s), which was approximately 92% shorter than the blank group. The MBG@DNA / TA group had a longer bleeding time than the DNA gel, indicating that the introduction of DOPA enhanced the hemostatic effect of the DNA gel on the liver, which may be related to the wet adhesion properties of DOPA. Note: Compared with the blank group, the differences between each group were significant **P ≤ 0.01****P ≤ 0.0001; the DNA gel group had the best hemostatic effect on the SD liver, with the shortest hemostatic time.

[0117] Example 16

[0118] This example involves the blood compatibility of DNA molecular glue

[0119] Taking the DNA molecular gel prepared in Example 1 as an example, the sample was incubated with SD whole blood dilution solution, and the absorbance of the supernatant at 540 nm was tested to detect whether the sample would cause red blood cell rupture. Figure 15 As shown, the hemolysis rate of the DNA series samples is lower than the national standard (5%). This means that the DNA series samples have good blood compatibility and will not cause red blood cell rupture. Note: Figure 15 In the figure, the sample concentrations on the horizontal axis are 2000, 1000, 500, and 250 μg / mL, respectively. The numbers 1, 2, 3, and 4 represent DNA, MBG@DNA, MBG@DNA-TA, and MBG@DNA-TA / DOPA, respectively. The DNA samples have good blood compatibility, with hemolysis rates below 5%.

[0120] Example 17

[0121] This example relates to the cell compatibility of DNA molecular glue

[0122] 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 hours, and the sample toxicity and live-dead cell tests were tested. The results showed that the DNA series samples had good compatibility with L929 cells and had the effect of promoting cell proliferation. Figure 16 shown. Figure 16 A. The results show that the cell viability of DNA molecular gel is greater than 95%, except for Zeolite used as a control. Figure 16Image-J analysis (B) shows that DNA glue significantly promotes cell proliferation after 48 hours of co-culture, consistent with the results of 16C live-dead staining. These results demonstrate that DNA glue is non-cytotoxic and has good cytocompatibility.

[0123] The present invention describes preferred embodiments and their effects. However, those skilled in the art, once informed of the basic inventive concept, may make additional changes and modifications to these embodiments. Therefore, it is intended that the appended claims be interpreted to include the preferred embodiments as well as all changes and modifications that fall within the scope of the invention.

[0124] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the 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 uniformly in an aqueous solvent, adding dopamine hydrochloride, and mixing uniformly to obtain a tannic acid-dopamine solution; The mesoporous bioactive glass was uniformly dispersed in an aqueous solvent, and DNA was added, and the mixture was stirred at 85-95°C for 10-15 minutes 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 minutes. 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 a 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 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 to 8.

4. The method for preparing a DNA molecular glue for moist wound hemostasis according to claim 1, characterized in that: The tannic acid-dopamine solution is prepared by 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. The water bath temperature is then lowered to 36.5 to 37.5°C, and dopamine hydrochloride is added and stirred for 5 to 10 minutes.

5. The method for preparing 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 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 the 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 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, the pH value is adjusted with ammonia water, tetraethyl orthosilicate is added dropwise, and then calcium nitrate is added. 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 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 moist wound hemostatic drug.

Images