A method for preparing a blood co-crosslinked biphasic gel and its application in stable hemostasis at the junction

Abstract

The application provides application of microgel particles taking cationic monomers as raw materials in preparation of blood crosslinking gel materials, and application of the microgel particles in preparation of interfacial stable hemostatic materials. The hemostatic gel prepared from the microgel particles has a sea-island belt structure based on blood cell bridging, and meanwhile, the strength and toughness of the material are improved. The gel can stably adhere to and support a wound, thereby achieving a dynamic hemostatic effect. The application fully utilizes the characteristics of large bleeding amount in severe bleeding, activates and anchors blood cells through an exogenous pathway based on electrostatic action. The application overcomes weakening effect of blood on a hemostatic material hemostatic interface, promotes formation of a blood enhanced phase, meets mechanical strength required for hemostasis at a severe bleeding site, and meets mechanical stability during patient transfer. The material used in the application has good biocompatibility after dialysis, has low toxicity and side effects on the human body, and provides great convenience for emergency hemostasis and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hemostatic materials, in particular to a blood co-crosslinked biphasic hemostatic gel for rapid hemostasis of severe bleeding at the junction and sustained hemostasis of wounds under dynamic stress environment such as stretching or compression during activities. BACKGROUND

[0002] Hemorrhagic shock caused by massive hemorrhage is one of the main causes of death in trauma cases, and is also the most preventable cause of death. Timely implementation of hemostatic measures can reduce mortality by up to 90%. The junctional sites, such as the groin, buttocks, shoulders, axillary fossae and the base of the neck, have the characteristics of difficult external compression, and the effect of traditional hemostatic devices (such as tourniquets, hemostatic forceps, etc.) in these areas is poor. In addition, since the junctional area is where large blood vessels run and branch, it is easy to form pulsatile bleeding when bleeding, and the bleeding rate can be as high as 110 cm / s. Rapid pulsatile bleeding can cause patients to enter a shock state within 3-5 minutes, with a mortality rate of 50%. In the environment of tactical first aid and long-distance transportation, preventing secondary bleeding is one of the key measures to prevent death. If the wound is not effectively protected during the treatment process, the patient will be exposed to the risk of other complications, of which 30% of patients may re-bleed, and 67% of patients die due to secondary bleeding. Therefore, the mechanical properties and mechanical stability of hemostatic materials are crucial for secondary bleeding caused by strong pulsatile flushing and dynamic environment, and are still an important challenge for junctional hemostasis.

[0003] In order to quickly stop bleeding, there are a variety of hemostatic materials on the market that are widely used in military and routine clinical practice, including zeolite powder, kaolin, sponge and biological adhesive, etc. However, some commercially available hemostatic materials, such as kaolin QuickClot and fibrin adhesive Fibrin glue, often exhibit relatively weak mechanical strength. For the sponge hemostat XStat, it is easy to cause excessive compression of the wound during the expansion process, which may cause secondary injury. The most effective protection for the wound during transportation is SAM-JT, however, its hook material may be degraded due to high temperature, humidity and blood exposure. As its strength gradually becomes difficult to withstand various forces during transportation, its effectiveness is only 24-48%. Therefore, the currently commercially available hemostatic materials cannot provide the appropriate mechanical properties and stability required by the wound.

[0004] To address the issue of mechanical performance mismatch of commercial hemostatic materials in dealing with large dose of bleeding such as arterial rupture, a variety of new hemostatic materials have been recently reported, including adhesives, sponges and zeolite gauze, etc. For example, a bioadhesive has been reported, which can stop high pressure bleeding of a 6 mm diameter heart penetrating hole within 20 seconds by mimicking the extracellular matrix in a mechanical plugging manner under ultraviolet irradiation, and its adhesive strength reaches 290 mmHg. In addition, a hemostatic gauze firmly bonded with zeolite of cotton fibers has an elution rate of only 1%. Combined with the method of compression hemostasis, the arterial amputation bleeding is controlled within 159 seconds. The high-strength chitosan sponge can stop the femoral artery bleeding by self-expanding and pressurizing through rapid blood absorption, and the hemostatic time is shortened to 58 seconds. These advanced materials can effectively stop severe arterial bleeding. However, the presence of blood often weakens the adhesive interface, or washes away the hemostatic components. In addition, they need additional operations (such as ultraviolet irradiation, long time pressurization) to achieve high strength hemostasis in use, or are difficult to adapt to irregular wound shapes, thereby limiting their wide application in severe bleeding wounds at the interface.

[0005] Powdered hemostatic materials can achieve rapid hemostasis and effective blockage of hemostasis without external stimulation, and have broad application prospects. Such hemostatic powders can quickly absorb blood and spontaneously form a gel barrier through strong physical interaction to effectively plug the wound. Some of these hemostatic powders, such as chitosan / diatom, skin secretions of Andrias davidianus and freeze-dried eggplant juice, form a hemostatic barrier by hydration through covalent / non-covalent bonding, have been developed and proven to effectively seal and hemostatic sites. These hemostatic powders still have some limitations in terms of liquid absorption capacity and mechanical strength after contacting blood, and are difficult to achieve rapid hemostasis and stable mechanical plugging for bleeding at the interface. In addition, because the presence of a large number of red blood cells hinders the formation of interaction between the expanded powders, the hydration process of the powders in blood is greatly weakened compared with pure water or PBS buffer.

[0006] Based on chitosan, a porous gel adhesive powder was prepared with four-arm polyethylene glycol amine, which can enrich blood cells in the pores of the hemostatic powder when bleeding, weaken the hindrance of blood to the hemostatic material, and form a structure of blood-filled micro-powder pores, which has good effect on rapid treatment of pig visceral bleeding. A hemostatic powder that can co-assemble with blood cells was prepared based on long alkyl quaternary ammonium chitosan, which improved its strength after co-assembly and achieved hemostasis of rabbit liver. However, the interface between the hemostatic powders is easily damaged by high-speed blood flow at the interface, making the powder interface a mechanically weak place. Therefore, in view of the problem of damage to the interface between the powders by high-speed blood flow at the interface, it is urgent to develop new hemostatic materials with a new mechanism and structure to reverse the damage of blood, and to provide excellent mechanical properties and stability to effectively deal with bleeding at the interface. SUMMARY

[0007] The present application aims to provide a blood co-crosslinked biphasic hemostatic gel capable of rapid hemostasis of severe bleeding at the junction and sustained hemostasis of the wound under dynamic stress environment such as stretching or compression during activity.

[0008] Junction, the junction of limbs refers to the area where the limbs are connected to the torso, including the shoulder joint area where the upper limbs are connected to the torso and the hip joint area where the lower limbs are connected to the torso. These parts are crucial in anatomy and function because they are not only connection points but also key areas for movement and weight bearing. The junction is also the area where large blood vessels run and branch. The present application is also called junction site, such as groin, hips, shoulders, armpits and neck bottom, etc.

[0009] In order to achieve the above-mentioned purpose, the present application provides the use of microgel particles prepared from cationic monomers in the preparation of blood crosslinked gel materials.

[0010] Preferably, the cationic monomer and hydrogen bond monomer are used as raw materials for polymerization to obtain a multiple valence bond crosslinked matrix gel; the matrix gel is dried, crushed and ground to obtain the microgel particles.

[0011] Preferably, the microgel particles are in contact with blood and crosslink with blood to form a hemostatic hydrogel in situ at the wound site, and the solid content of the hydrogel is 20-50wt%.

[0012] Preferably, the cationic monomer is an olefin cationic monomer. Preferably, the olefin cationic monomer of the present application refers to a monomer containing a double bond structure and carrying a positive charge. The olefin cationic monomer is polymerized by free radical initiation to form a high molecular material. Preferably, the olefin cationic monomer includes quaternary ammonium olefin cationic monomer; preferably, the olefin cationic monomer includes amine salt olefin cationic monomer.

[0013] The hydrogen bond monomer of the present application refers to a monomer that can be polymerized or form a structure through hydrogen bond interaction. Preferably, the hydrogen bond monomer contains amino functional group or carboxyl functional group, or other functional groups. Preferably, the cationic monomer includes at least one of 3-(isobutyrylamide) propyl trimethyl ammonium chloride, methacryloyloxyethyl trimethyl ammonium chloride or acryloyloxyethyl trimethyl ammonium chloride or 2-aminoethyl methacrylate hydrochloride.

[0014] Preferably, the hydrogen bond monomer includes at least one of carboxyl type methacrylic acid, imidazole ring type vinyl imidazole, amide group type acrylamide, N-vinyl pyrrolidone, and hydroxyl type hydroxypropyl methacrylate.

[0015] The present application also provides the use of the microgel particles of any one of the above in the preparation of an interfacial stable hemostatic material.

[0016] The present application also provides the microgel particles of any one of the above use.

[0017] The present application also provides the preparation method of the microgel particles of any one of the above use, comprising the following steps:

[0018] Step 1: preparation of a multiple valence bond crosslinking matrix gel: hydrogen bond monomers and cationic monomers are dissolved in physiological saline in a molar ratio of 10:1 to 1:10 to a solid content of 20wt% to 50wt%, preferably 40wt%, and stirred until the monomers are completely dissolved, then tetramethyl ethylenediamine and ammonium persulfate are added and mixed, and a polymerization reaction is carried out, after which the product is dialyzed to obtain a multiple valence bond crosslinking matrix gel;

[0019] Step 2: the matrix gel obtained in step 1 is dried, and the dried matrix gel is crushed and ground into fine particles to obtain the microgel particles.

[0020] Preferably in any one of the above, in step 1, the hydrogen bond monomers include at least one of carboxyl-based methacrylic acid, imidazole ring-based vinyl imidazole, amide-based acrylamide, N-vinyl pyrrolidone, and hydroxyl-based hydroxypropyl methacrylate.

[0021] Preferably in any one of the above, in step 1, the cationic monomers are at least one of 3-(isobutenyl amide) propyl trimethyl ammonium chloride, methacryloyloxy ethyl trimethyl ammonium chloride, or acryloyloxy ethyl trimethyl ammonium chloride, or MADQUAT.

[0022] Preferably in any one of the above, in step 1, the tetramethyl ethylenediamine is 1% to 10% of the total monomer moles, preferably 1%.

[0023] Preferably in any one of the above, in step 1, the ammonium persulfate is 1% to 10% of the total monomer moles, preferably 5%.

[0024] Preferably in any one of the above, in step 1, the dialysis bag has a molecular weight cut-off of 8000 to 14000 Da, and the dialysis is carried out in distilled water for 72 hours. Preferably, the dialysis bag has a molecular weight cut-off of 8000, 10000, 12000, 14000 Da, or a range therebetween.

[0025] Preferably in any one of the above, in step 1, the molar ratio of hydrogen bond monomers to cationic monomers is 10:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or a range therebetween.

[0026] Any of the above preferably, in step 2, the parent gel obtained in step 1 is dried at 40-80°C for 2-7 days, the higher the temperature, the shorter the time. Preferably, the parent gel obtained in step 1 is dried at 60°C for 48h;

[0027] Any of the above preferably, the particle size of the microgel particles is 120-180 μm. Preferably, the particle size of the microgel particles is 120, 140, 160, 180 μm and ranges therebetween.

[0028] In a preferred embodiment of the present application, a hemostatic gel is provided, the preparation raw materials include acrylamide (AAm, CAS: 79-06-1), 3-(isobutylene amide) propyl trimethyl ammonium chloride (MPTC, CAS: 51410-72-1). The hemostatic gel is a strong and tough blood gel formed by the synergistic crosslinking of the hydrogen bonding provided by AAm and the cationic positive charge provided by MPTC with blood cells. Severe bleeding at the junction often exceeds the limit of the body's natural hemostasis. The key role of red blood cells (RBCs) is often overlooked, but they can interact with platelets to form a hard blood clot tightly sealing the wound, which is crucial for blood coagulation. Based on the concept of design reversal, the characteristics of bleeding are fully utilized by introducing a large amount of bleeding into the structural design, successfully creating a unique and strong blood-bridged self-evolving sea-island belt structure. Unlike other studies, the present application innovatively synthesizes a poly- AAm / MPTC self-evolving sea-island belt structure precursor hemostatic powder, i.e. the microgel particles. The hemostatic powder has the characteristics of quickly absorbing water from blood and can enrich and anchor red blood cells. During the water absorption process, the soft and tough gel phase formed by the powder based on hydrogen bonding can dissipate energy when stressed, improving the toughness of the material. At the same time, through electrostatic interaction, the quaternary ammonium cation anchors the negatively charged red blood cells, bypasses the coagulation factor-related pathway to form a hard and tough blood phase, and connects adjacent powders to improve the interface strength. A reinforced "island belt structure" is formed by blood bridging. During the formation process, the cation promotes the formation of fibrin, not only strengthening the formation of blood crosslinking, but also forming a special structure of local interpenetration of blood fibrin and gel network at the interface, strengthening the gel-blood phase interface, and effectively avoiding interface failure. The unique island belt structure formed by blood can dissipate energy through the fracture of the island belt when stretched, further improving the toughness of the hemostatic material to resist the scouring of the blood flow at the junction. The self-evolving sea-island structure enhancement strategy based on blood bridging reverses the destruction of blood to the hemostatic material and achieves good mechanical properties and mechanical stability under the protection of blood, thereby solving the difficult problem of bleeding at the junction with large and fast bleeding volume and avoiding secondary bleeding.

[0029] In a preferred embodiment of the present application, the method for preparing the microgel particles comprises the following steps:

[0030] 1) Preparation of the multi-interactive crosslinking matrix gel

[0031] AAm and MPTC monomers were dissolved in physiological saline with a molar ratio of 10:1 to 1:10 to a solid content of 40 wt%, and stirred until the monomers were completely dissolved. Then the precursor solution was mixed with tetramethyl ethylenediamine (TEMED, CAS: 110-18-9) (1% of the total moles of monomers), ammonium persulfate (APS, CAS: 7727-54-0) (5% of the total moles of monomers), and polymerized at 37°C for 12 h. After the reaction was completed, the product was dialyzed in a dialysis bag (8000-14000 Da) in distilled water for 72 h to obtain the reversible interactive crosslinking matrix gel.

[0032] The reversible interaction of the present application preferably includes non-covalent interaction, such as hydrogen bonding and / or electrostatic interaction. The cationic monomer provides electrostatic interaction, and the hydrogen bonding monomer provides hydrogen bonding. The reversible interaction provides a driving force for the particles to interact with each other for the preparation of the matrix gel, and also provides a driving force for the gel particles to interact with blood for the subsequent formation of the sea-island band structure for the microgel particles.

[0033] The hydrogen bonding of the present application is preferably provided by hydrogen bond donors, such as carboxylic acid type methacrylic acid (MAAc, CAS: 79-41-4), amide type AAm, N-vinyl pyrrolidone (NVP, CAS: 88-12-0), imidazole ring type vinyl imidazole (VI, CAS: 106614-28-2), hydroxyl type hydroxypropyl methacrylate (HPMA, CAS: 27813-02-1), etc. Acrylamide is preferred.

[0034] The cationic interaction of the present application is preferably provided by quaternary ammonium type: MPTC, methacryloyloxyethyl trimethyl ammonium chloride (DMAEA-Q, CAS: 5039-78-1), acryloyloxyethyl trimethyl ammonium chloride (MATC, CAS: 44992-01-0); and / or amine salt type: 2-aminoethyl methacrylate hydrochloride (MADQUAT, CAS: 2420-94-2) as the cationic monomer.

[0035] 2) Preparation of the multi-interactive microgel particles

[0036] The matrix gel of step 1) was dried at 60°C for 48 h, and the dried matrix gel was ground into fine particles. A standard steel sieve was used to screen the microgel particles with uniform particle size. The particle size of the microgel particles is preferably in the range of 120-180 μm.

[0037] 3) In-situ formation of the multi-interactive gel

[0038] Put the microgel particles of step 2) into the cavity of a syringe, add a certain amount of whole blood to make the solid content 20-50wt%, push out the hydrogel and fill the mold to make the blood cell co-crosslinked blood gel.

[0039] The preferred solid content is 40wt%.

[0040] The beneficial effects of the present application are:

[0041] 1. The hemostatic gel prepared by the present application has high mechanical strength, and the sea-island belt structure based on blood cell bridging improves the strength and toughness of the material. The enhancement of the fibrin structure further improves the interfacial strength. A tough gel is formed to seal and stop bleeding at the junction for a long time, and the storage modulus of the blood cell co-crosslinked blood gel obtained is 3-5MPa. The compression strength can be maintained at 65% of the initial strength after 100 cycles of compression. The gel can stably adhere and support the wound, thereby achieving the effect of dynamic hemostasis.

[0042] 2. The present application makes full use of the characteristics of large amount of bleeding in severe bleeding, activates and anchors blood cells through external source based on electrostatic effect. Overcome the weakening effect of blood on the hemostatic interface of the hemostatic material, promote the formation of blood enhanced phase. In addition, based on the electrostatic repulsion phenomenon of molecular chain, the hemostatic material is endowed with rapid hydration and high porosity to enhance the water absorption characteristics, improve the blood cell enrichment effect, shorten the hemostasis time, and the hemostatic gel prepared under the action of multiple non-covalent bonds can quickly crosslink with blood cells to form a gel to block the bleeding point within 30s.

[0043] 3. The present application is based on the sea-island belt structure design scheme of blood cell bridging, which meets the mechanical strength required for hemostasis at the site of severe bleeding and the mechanical stability during patient transfer. Avoid complications such as hemorrhagic shock and secondary bleeding that threaten the lives of patients.

[0044] 4. The present application can meet the requirements of hemostasis time, irregular filling, portability, mechanical strength in different scenarios by adjusting the driving force of the microgel particles, and effectively stop bleeding at the affected area.

[0045] 5. The material used in the present application has good biocompatibility after dialysis, and has low toxic and side effects on the human body.

[0046] 6. The present application provides great convenience for emergency hemostasis, wound care and other processes. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The Fourier infrared spectrum of the microgel particles described in the present application in preferred embodiment 10 of the present application.

[0048] Figure 2Blood gel 100-cycle compression curve for preferred embodiment 11 of the present invention.

[0049] Figure 3 Scanning electron micrograph of blood gel red blood cell adhesion for preferred embodiment 13 of the present invention.

[0050] Figure 4 Scanning electron micrograph of blood gel fibrin network for preferred embodiment 14 of the present invention.

[0051] Figure 5 Blood gel adhesion muscle H&E stain for preferred embodiment 15 of the present invention.

[0052] Figure 6 Blood gel adhesion fat H&E stain for preferred embodiment 15 of the present invention.

[0053] Figure 7 Blood gel adhesion blood vessel H&E stain for preferred embodiment 15 of the present invention.

[0054] Figure 8 Sea-island band structure double stain for preferred embodiment 16 of the present invention.

[0055] Figure 9 Gel rheology test for different AAm to MPTC molar ratios for preferred embodiment 17 of the present invention.

[0056] Figure 10 Light microscope observation of P(AAm-MPTC)-blood structure for preferred embodiment 18 of the present invention.

[0057] Figure 11 Light microscope observation of celox-blood structure for preferred embodiment 18 of the present invention. DETAILED DESCRIPTION

[0058] The present invention is further described through the accompanying drawings and detailed description below.

[0059] Example 1

[0060] P(AAm-MPTC) parent gel preparation

[0061] AAm and MPTC monomers were mixed in a molar ratio of 10:1 to 1:10, and dissolved in physiological saline to a solid content of 40wt%, and stirred until the monomers were completely dissolved. Then the precursor solution was mixed with TEMED (1% of the total monomer moles) and APS (5% of the total monomer moles), and polymerized at 37°C for 12h. After the reaction was complete, the product was dialyzed in a dialysis bag (8000-14000Da) in distilled water for 72h to obtain a multiple valence bond interaction crosslinked parent gel.

[0062] Example 2

[0063] The P(AAm-MPTC) matrix gel from Example 1 was dried at 60°C for 48 hours. The dried P(AAm-MPTC) was then pulverized and ground into tiny particles. P(AAm-MPTC) microgel particles with a particle size of 120–180 μm were screened using a standard steel sieve.

[0064] Example 3

[0065] Preparation of P(AAm-MATC) master gel

[0066] AAM and MATC monomers were dissolved in physiological saline at a molar ratio of 10:1 to 1:10 until the solid content reached 40 wt%, and stirred until the monomers were completely dissolved. The precursor solution was then mixed with TEMED (1% of total monomer molar weight) and APS (5% of total monomer molar weight), and polymerized at 37°C for 12 h. After the reaction was complete, the product was dialyzed against distilled water for 72 h using a dialysis bag (8000–14000 Da) to obtain a multivalent crosslinked parent gel.

[0067] Example 4

[0068] In Example 3, the P(AAm-MATC) matrix gel was dried at 60°C for 48 hours. The dried P(AAm-MATC) was then pulverized and ground into tiny particles. P(AAm-MATC) microgel particles with a particle size of 120–180 μm were screened using a standard steel sieve.

[0069] Example 5

[0070] Preparation of P(AAm-MAAc-DMAEA-Q) matrix gel

[0071] The molar ratio of AAm to MAAc was 1:5 to 5:1, and the molar ratio of total hydrogen-bonded monomers to DMAEA-Q monomers was 10:1 to 1:10. The solutions were dissolved in physiological saline to a solid content of 40 wt%, and stirred until the monomers were completely dissolved. The precursor solution was then mixed with TEMED (1% of total monomer molar weight) and APS (5% of total monomer molar weight), and polymerized at 37°C for 12 h. After the reaction, the product was dialyzed against distilled water for 72 h using a dialysis bag (8000–14000 Da) to obtain a multivalent crosslinked parent gel.

[0072] Example 6

[0073] The P(AAm-MAAc-DMAEA-Q) parent gel from Example 5 was dried at 60°C for 48 hours. The dried P(AAm-MAAc-DMAEA-Q) was pulverized and ground into tiny particles. P(AAm-MAAc-DMAEA-Q) microgel particles with a particle size of 120-180 μm were screened using a standard steel sieve.

[0074] Example 7

[0075] Preparation of P(NVP-MPTC) microgel particles

[0076] The NVP and MPTC monomers were dissolved in physiological saline with a monomer molar ratio of 10:1 to 1:10 to a solid content of 40 wt%, and stirred until the monomers were completely dissolved. Then the precursor solution was mixed with TEMED (1% of the total monomer moles) and APS (5% of the total monomer moles), and polymerized at 37°C for 12 h. After the reaction was completed, the product was dialyzed in a dialysis bag (8000-14000 Da) in distilled water for 72 h to obtain the parent gel P(NVP-MPTC). The P(NVP-MPTC) parent gel was dried at 60°C for 48 h, and the dried P(NVP-MPTC) was ground into fine particles. The P(NVP-MPTC) microgel particles with a particle size of 120-180 μm were sieved using a standard steel sieve.

[0077] Example 8

[0078] Preparation of P(VI-MPTC) microgel particles

[0079] The VI and MPTC monomers were dissolved in physiological saline with a monomer molar ratio of 10:1 to 1:10 to a solid content of 40 wt%, and stirred until the monomers were completely dissolved. Then the precursor solution was mixed with TEMED (1% of the total monomer moles) and APS (5% of the total monomer moles), and polymerized at 37°C for 12 h. After the reaction was completed, the product was dialyzed in a dialysis bag (8000-14000 Da) in distilled water for 72 h to obtain the parent gel P(VI-MPTC). The P(VI-MPTC) parent gel was dried at 60°C for 48 h, and the dried P(VI-MPTC) was ground into fine particles. The P(VI-MPTC) microgel particles with a particle size of 120-180 μm were sieved using a standard steel sieve.

[0080] Example 9

[0081] Preparation of P(HPMA-MPTC) microgel particles

[0082] HPMA and MPTC monomers were dissolved in physiological saline at a molar ratio of 10:1 to 1:10 to a solid content of 40 wt%, and stirred until the monomers were completely dissolved. Then the precursor solution was mixed with TEMED (1% of the total monomer moles) and APS (5% of the total monomer moles), and polymerized at 37°C for 12 h. After the reaction was completed, the product was dialyzed in a dialysis bag (8000-14000 Da) in distilled water for 72 h to obtain the parent gel P(HPMA-MPTC). The P(HPMA-MPTC) parent gel was dried at 60°C for 48 h, the dried P(HPMA-MPTC) was ground into fine particles, and P(HPMA-MPTC) microgel particles with a particle size of 120-180 μm were sieved using a standard steel sieve.

[0083] Example 10

[0084] Preparation of P(VI-DMAEA-Q) microgel particles

[0085] VI and DMAEA-Q monomers were dissolved in physiological saline at a molar ratio of 10:1 to 1:10 to a solid content of 40 wt%, and stirred until the monomers were completely dissolved. Then the precursor solution was mixed with TEMED (1% of the total monomer moles) and APS (5% of the total monomer moles), and polymerized at 37°C for 12 h. After the reaction was completed, the product was dialyzed in a dialysis bag (8000-14000 Da) in distilled water for 72 h to obtain the parent gel P(VI-DMAEA-Q). The P(VI-DMAEA-Q) parent gel was dried at 60°C for 48 h, the dried P(VI-DMAEA-Q) was ground into fine particles, and P(VI-DMAEA-Q) microgel particles with a particle size of 120-180 μm were sieved using a standard steel sieve.

[0086] Example 11

[0087] Preparation of amine salt P(AAm-MADQUAT) microgel particles

[0088] AAm and MADQUAT monomers were dissolved in physiological saline at a molar ratio of 10:1 to 1:10 to a solid content of 40 wt%, and stirred until the monomers were completely dissolved. Then the precursor solution was mixed with TEMED (1% of the total monomer moles) and APS (5% of the total monomer moles), and polymerized at 37°C for 12 h. After the reaction was completed, the product was dialyzed in a dialysis bag (8000-14000 Da) in distilled water for 72 h to obtain the parent gel P(AAm-MADQUAT). The P(AAm-MADQUAT) parent gel was dried at 60°C for 48 h, the dried P(AAm-MADQUAT) was ground into fine particles, and P(AAm-MADQUAT) microgel particles with a particle size of 120-180 μm were sieved using a standard steel sieve.

[0089] Example 12

[0090] Chemical structure test of P(AAm-MPTC) microgel particles

[0091] Fourier infrared spectrum test: take appropriate amount of P(AAm-MPTC) hemostatic gel samples 1, 2, 3 in Example 2, respectively, mix with KBr powder uniformly and grind thoroughly. Use hydraulic tablet press to press the above mixture into a thin sheet, and then obtain the infrared spectrum of the three samples in the range of 4000-500 cm -1 -1 by Fourier transform infrared spectrometer. Sample 1 is the sample collected after the P(AAm-MPTC) parent gel is dried at 60°C for 48h, sample 2 is the sample collected after the P(AAm-MPTC) is crushed and ground into small particles after drying, and sample 3 is the P(AAm-MPTC) microgel particles with particle size of 120-180μm screened by using a standard steel sieve, i.e. the microgel particles according to the present application. Figure 1 The Fourier infrared spectrum of the microgel particles according to the present application is shown in Figure 3.

[0092] Example 13

[0093] Cyclic compression test

[0094] P(AAm-MPTC) microgel particles in Example 2 are made into compression test samples with a diameter of 10mm and a height of 10mm in a mold together with blood. The P(AAm-MPTC) hemostatic gel sample is placed on a universal mechanical testing machine, and a cyclic compression experiment is carried out. The sample is compressed to 20% strain, then unloaded and returned to the initial position of 0 strain, and so on for 100 times. The tensile speed is set to 50mm / min. As shown in Figure 4, the blood-treated P(AAm-MPTC) has fatigue resistance and can withstand 100 times of compression stress without breaking, and can provide stable support to the wound. Figure 2

[0095] Example 14

[0096] Red blood cell adhesion

[0097] P(AAm-MPTC) microgel particles are formed into a gel with a solid content of 40wt% with water, and whole blood (50μL) is added dropwise to the surface of the sample (10mg), and incubated at 37°C for 5min. Then the sample is washed with PBS for 3 times to remove the unattached blood cells, and is fixed with 2.5% glutaraldehyde for 2h, and is dehydrated with gradient concentration (30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%) ethanol solution. Put into a 37° oven overnight, and finally observe the cell morphology and material morphology by scanning electron microscopy. The morphology is shown in Figure 5. Figure 3 ​As shown, a large number of red blood cells were adsorbed on the surface of the gel, indicating the ability of P(AAm-MPTC) to adhere blood, and the cell morphology was normal, and no obvious cell rupture was found.

[0098] Example 15

[0099] Observation of blood gel structure

[0100] After the blood was frozen in the refrigerator, it was placed in a freeze dryer for low-temperature freeze-drying. Subsequently, the microgel particles and the reorganized hydrogel with the cross section facing up were pasted on the SEM sample table, and gold was sprayed for 50 s. Then, the surface of the microgel particles and the internal morphology of the reorganized gel sample were analyzed by scanning electron microscopy under the condition of 5 kV accelerating voltage. As shown in Figure 4 As shown, due to the positive stimulation of red blood cells by the hemostatic material, a fibrin network was formed across the coagulation cascade. The formation of the fibrin network is conducive to the bridging of blood to the gel, promoting the formation of the unique island-segment of the hemostatic material and improving its mechanical properties.

[0101] Example 16

[0102] Histological analysis

[0103] The blood gel and the adhered tissue (muscle, fat, or blood vessels, etc.) were decalcified in an ethylenediaminetetraacetic acid solution (pH = 7.2-7.4) for 2 weeks and embedded in paraffin, and then cut into 4 μm thick sections. Hematoxylin / eosin (H&E) staining was performed according to the manufacturer's instructions. Images were captured using an optical microscope. As shown in Figures 5 to 7 As shown, the hemostatic material played an adhesive role on the muscle, fat, blood vessels, and other tissues, and the interface was peeled off for observation. In addition, due to the effect of amino groups and other groups in the blood, the adhesion of the hemostatic material to the tissue was improved, enabling it to adhere to difficult-to-adhere sites such as fat.

[0104] Example 17

[0105] Verification of island-segment structure

[0106] After 5 ml of blood was blended with 2 mg of cyanine dye-Cy3 for 24 h, dialysis was performed in a physiological saline environment using a dialysis bag (3500 Da) for 72 h. CY3-stained blood was obtained. 0.347 g of P(AAm-MPTC) gel particles were soaked in 5 ml of fluorescein isothiocyanate (FITC, CAS: 3326-32-7) with a concentration of 5 ml / mg for 24 h to obtain FITC-stained P(AAm-MPTC). The two stained components were mixed to obtain a double-stained blood gel with a solid content of 40 wt%, and a confocal microscope was used to take pictures for observation. The results are shown in Figure 8The red color is the blood phase, forming an interconnected island band structure, and the large area of green is the P(AAm-MPTC) forming an ocean structure. The blood phase is distributed in the P(AAm-MPTC) and is interconnected, forming the island-ocean band structure proposed in the present application.

[0107] Example 18

[0108] Rheological analysis of different hydrogen bond cation ratios

[0109] P(AAm-MPTC) with AAm and MPTC monomer molar ratios of 5:1, 1:1, 1:5 and 1:10 were respectively mixed with water and blood to form gels with a solid content of 40wt%. The above gels were tested for their storage modulus (G') in the range of 0.1Hz-10Hz. As shown in Figure 9 with the decrease of AAm ratio, the G' of the gels formed under blood and water conditions decreased, representing the decrease of mechanical strength. Under the same ratio, the island band structure formed by blood enhances the gel structure, making its strength higher than that of the gel formed in water. For different parts, the junction has higher blood pressure and faster flow rate, which requires good strength to resist the scouring of blood flow, so 10:1 P(AAm-MPTC) is more suitable for this part of hemostasis; for small bleeding and deep bleeding, 1:5 P(AAm-MPTC) is more suitable for the strength matching of the surrounding tissue, preventing damage to the surrounding tissue. In addition, due to the lack of sufficient cross-linking structure of 1:10 P(AAm-MPTC), it cannot form a gel in water, so it cannot form an ocean structure under blood conditions, resulting in the failure of the island-ocean band structure hemostasis strategy.

[0110] Example 19

[0111] Comparison of the blood-bridging island-ocean band structure of the present application with the structure of the commercial cationic hemostatic material in the prior art

[0112] P(AAm-MPTC) and commercial hemostatic material celox were mixed with blood to form gels with a solid content of 40wt%. Both were placed under an optical microscope to observe their structures, with an objective lens of 20x and an eyepiece of 10x. The P(AAm-MPTC)-blood structure is shown in Figure 10 Similar to the observation of the structure in Example 16, the swollen P(AAm-MPTC) occupies a large area, forming an ocean structure. The island band structure of continuous blood fills between the particles, trapping and further fixing the ocean structure. The celox-blood structure is shown in Figure 11As shown, the field of view appears uniformly colored, with the celox particles absorbing blood into their interior, and thus no distinct blood phase is observed. Although the celox is positively charged, no sea-island banding is formed due to the lack of interaction between the celox particles, and the ability of blood cells to be absorbed into the interior of the particles.

[0113] The above embodiments are only examples for clearly illustrating the present application, and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Use of microgel particles based on cationic monomers for the preparation of a cross-linked gel material with blood, characterized in that, The multiple valence bond cross-linking mother gel is prepared by polymerization reaction of cationic monomer and hydrogen bond monomer; the microgel particles are obtained by drying, crushing and grinding the mother gel; the microgel particles cross-link with blood after contacting with blood, and form hemostatic hydrogel in situ at the wound site, the solid content of the hydrogel is 20-50 wt%; the cationic monomer is 3-(isobutyryl amide) propyl trimethyl ammonium chloride, the hydrogen bond monomer is acrylamide, and the molar ratio of the hydrogen bond monomer to the cationic monomer is 10:1-1:

10.

2. The use of the microgel particles in the application of claim 1 in the preparation of stable hemostatic materials at the interface.

3. The microgel particles for use according to claim 2.

4. The preparation method of the microgel particles of claim 3, comprising the following steps: Step 1: preparation of multiple valence bond cross-linking mother gel: the hydrogen bond monomer and the cationic monomer are dissolved in physiological saline at a molar ratio of 10:1-1:10 to a solid content of 40 wt%, stirred until the monomers are completely dissolved, then tetramethyl ethylenediamine and ammonium persulfate are added and mixed, polymerization reaction is carried out, after the polymerization reaction is completed, the product is dialyzed to obtain the multiple valence bond cross-linking mother gel; Step 2: the mother gel obtained in step 1 is dried, and the dried mother gel is crushed and ground into microgel particles.

5. The production method according to claim 4, wherein In step 1, the hydrogen bond monomer is acrylamide; the cationic monomer is 3-(isobutyryl amide) propyl trimethyl ammonium chloride; tetramethyl ethylenediamine is 1%-10% of the total monomer molar amount; ammonium persulfate is 1%-10% of the total monomer molar amount; the dialysis bag with a molecular weight cut-off of 8000-14000 Da is used for dialysis in distilled water for 72 h.

6. The production method according to claim 4, wherein In step 2, the mother gel obtained in step 1 is dried at 40-80℃ for 2-7 days; the particle size of the microgel particles is 120-180 μm.

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