Injectable aggregate adhesive with postoperative adhesion prevention characteristic as well as preparation method and application of injectable aggregate adhesive

Abstract

The invention discloses an injectable aggregate adhesive with a postoperative adhesion prevention characteristic as well as a preparation method and application of the injectable aggregate adhesive. Dynamic nanogel TNGD is used as a construction unit, and temperature-sensitive phase change is utilized to induce the nanogel to generate aggregation gelation, so that the supramolecular aggregate is prepared. The aggregation behavior is influenced by the concentration and temperature of the nanogel. The mechanical strength of the aggregate can be further enhanced by prolonging the heating time. The aggregate has excellent injectability, adhesion and cell adhesion resistance, can be attached to the surface of complex soft tissue, is still stable under the action of dynamic deformation, and can be used for preventing and treating postoperative adhesion.

Description

Technical Field

[0001] The present invention relates to the field of polymer technology, and in particular to an injectable aggregate adhesive with the property of preventing postoperative adhesion, and a preparation method and application thereof. Background Art

[0002] Postoperative adhesions (POA) refer to abnormal fibrous connections formed between organs and tissues during the process of wound repair after surgery. This pathological phenomenon is common in areas such as the abdomen, pericardium, and uterus, and is a key cause of long-term surgical complications. Tissue adhesions not only cause chronic abdominal discomfort and pain, but may also lead to a series of serious clinical consequences such as intestinal obstruction and infertility, which have a profound impact on patients' quality of life and impose a heavy medical and economic burden. In recent years, with the advancement of biomaterials and clinical medicine, strategies for preventing postoperative adhesions have gradually diversified, covering multiple aspects such as mechanical dissection, chemical drug regulation, and physical barriers.

[0003] Mechanical dissection usually refers to the use of scissors, lasers or electric knives to perform adhesion lysis and directly remove adhesion tissue. However, simple mechanical dissection may lead to the formation of new adhesions, especially in areas of severe trauma, and the adhesion tissue formed by the second surgery will also be thicker than the initial one. Adhesion lysis is prone to cause secondary damage and increase patient pain, and usually needs to be combined with other preventive measures to achieve better therapeutic effects. Chemical drugs mainly prevent adhesions by regulating inflammation, coagulation and fibrosis pathways, and are mainly divided into the following categories: anti-inflammatory drugs, anticoagulant and fibrinolytic system regulating drugs, anti-fibrotic drugs, and growth factor regulating drugs. However, systemic administration may cause adverse reactions such as bleeding tendency, immunosuppression and gastrointestinal damage in the body, which limits its clinical application. Physical barriers are one of the most effective strategies currently, which is to isolate the damaged tissue surface by applying materials such as films, polymer solutions, gel dressings, etc. to the affected area, reduce fibroblast adhesion, and block fibrin bridging. Polymer anti-postoperative adhesion materials mainly include super-hydrophilic (such as polyethylene glycol (PEG), polyvinyl alcohol (PVA)) and super-hydrophobic polymers (such as polycaprolactone (PCL), polydimethylsiloxane (PDMS)). The low surface energy of super-hydrophobic polymers allows proteins to stretch on their surface and lose their biological and functional structures, which can reduce the colonization of fibroblasts on the surface. It also has self-cleaning and anti-fouling functions, and can also reduce the risk of bacterial contamination during preparation or operation. Super-hydrophilic polymers quickly absorb moisture and swell at the application site to form a hydration layer, preventing the adhesion of fibronectin and fibroblasts. Some negatively charged polymers (such as alginate, oxidized regenerated cellulose, etc.) can also resist protein adsorption through electrostatic repulsion and exert a good anti-adhesion effect. However, the polymer solutions currently developed lack strength and cannot maintain their own shape to remain in the treatment site; large pieces of film or hydrogel shape adaptability are poor, making it difficult to fit complex wounds and adapt to tissue peristalsis. In addition, most anti-adhesion materials have difficulty balancing tissue adhesion and anti-adhesion properties, and require the use of sutures and other means for fixation or complex asymmetric structural design, which greatly limits their application in the body.

[0004] Therefore, there is still much room for improvement in the development of biomedical anti-adhesion materials. It is of great significance to develop injectable supramolecular hydrogels with tissue adhesion through chemical design of materials, precisely control their physicochemical properties, and achieve good anti-postoperative adhesion effects. Summary of the Invention

[0005] To address the lack of adhesion and poor soft tissue compatibility of current anti-adhesion materials, an injectable supramolecular hydrogel aggregate could be developed to prevent postoperative tissue adhesion. Its injectability, dynamic environmental adaptability, tissue adhesion, and inhibitory properties against fibroblast and bacterial adhesion will be investigated to provide a new material strategy for addressing postoperative tissue adhesion.

[0006] One objective of the present invention is to provide a method for preparing injectable aggregates with both tissue adhesion and anti-adhesion properties. In this method, arginine (Arg), which contains a guanidine cationic group, is modified with methacrylic anhydride (MA) to prepare a super-hydrophilic zwitterionic monomer (M-Arg). Furthermore, using the thermosensitive monomer N-isopropylacrylamide (NIPAM) and the zwitterionic ion (M-Arg) as monomers, N,N'-methylenebisacrylamide (MBA) as a crosslinker, and cetyltrimethylammonium bromide (CTAB) as a surfactant, a high-molecular-weight dynamic nanogel (TNG) is obtained through aqueous precipitation polymerization initiated by 2,2'-azobisisobutylamidine dihydrochloride (V-50). Dopamine (DA) is then introduced into the TNG system via coupling with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to produce a TNGD nanogel. Viscous, injectable supramolecular aggregates (TAHs) are synthesized by self-assembly through a temperature-induced hydrophobic phase transition of TNGD nanogels. Compared to conventional anti-adhesion hydrogels, these aggregates exhibit excellent dynamic mechanical properties, injectability, tissue adhesion, and inhibition of fibroblast and bacterial adhesion. The mechanical properties and tissue adhesion strength of the aggregates can be regulated by varying the heating time. This overcomes the key issues of conventional anti-adhesion materials: the mismatch between mechanical strength and soft tissue, and the conflict between tissue adhesion and anti-adhesion performance.

[0007] A second object of the present invention is to provide an injectable aggregate having both tissue adhesion and anti-adhesion properties.

[0008] A third object of the present invention is to provide applications of the injectable aggregates having both tissue adhesion and anti-adhesion properties.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A method for preparing an injectable aggregate having both tissue adhesion and anti-adhesion properties comprises the following steps:

[0011] (1) dissolving L-arginine (Arg) in a mixed solvent of water, dioxane, and triethylamine (TEA) to obtain an Arg solution;

[0012] (2) adding methacrylic anhydride (MA) dropwise to the Arg solution in step (1) and stirring uniformly to obtain a mixture reaction solution;

[0013] (3) stirring the reaction solution in step (2), recrystallizing the reaction solution with acetone, and vacuum drying the precipitate to obtain white M-Arg powder;

[0014] (4) dissolving the M-Arg powder, N-isopropylacrylamide (NIPAM), N,N'-methylenebisacrylamide (MBA), hexadecyltrimethylammonium bromide (CTAB), and 2,2'-azobisisobutylamidine dihydrochloride (V-50) in step (3) in water, heating and stirring, and dialyzing to obtain a TNG nanogel solution;

[0015] (5) adjusting the concentration of the TNGD nanogel solution in step (4), adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stirring to obtain a mixed solution;

[0016] (6) injecting dopamine (DA) solution into the mixed solution in step (5), stirring, and dialyzing to obtain a TNGD nanogel dispersion;

[0017] (7) Adjusting the concentration of the TNGD nanogel dispersion in step (6), heating at 40°C to 70°C for 10 to 60 minutes and absorbing the precipitated water, the resulting coacervate is the injectable aggregate having both tissue adhesion and anti-adhesion properties.

[0018] The aggregates were denoted as TAHx-y, where x is the concentration of TNGD nanogels and y is the heating time at 60°C (in minutes).

[0019] Furthermore, the water described in the present invention is all deionized water.

[0020] Furthermore, the concentration of L-arginine in the Arg solution in step (1) is 6±1 g / 100 mL; the proportions of the components in the mixed solvent are: deionized water 60±5 v / v%, dioxane 26±3 v / v%, and triethylamine 14±2 v / v%.

[0021] Furthermore, the ratio of L-arginine, water, dioxane and triethylamine in step (1) is 2g:20mL:8.5mL:4.5mL.

[0022] Furthermore, the concentration of methacrylic anhydride in the mixture reaction solution in step (2) is 8.4±1 v / v%.

[0023] Furthermore, based on the amount of L-arginine used in step (1) being 2 g, the amount of methacrylic anhydride added in step (2) is 3 mL.

[0024] Furthermore, the stirring conditions in step (2) are: speed 200-300 rpm, time 10-20 min.

[0025] Furthermore, the stirring conditions in step (3) are: a speed of 200 to 300 rpm, and a time of overnight (8 to 18 hours).

[0026] Furthermore, the amount of acetone used in step (3) is 3 to 5 times the volume of the mixture reaction solution.

[0027] Furthermore, in the TNG nanogel solution in step (4), the concentration of M-Arg is 0.1 to 0.5 g / 100 mL, preferably 0.5 g / 100 mL; the concentration of N-isopropylacrylamide is 1 to 5 g / 100 mL, preferably 2.5 g / 100 mL; the concentration of N,N'-methylenebisacrylamide is 0.01 to 0.05 g / 100 mL, preferably 0.025 g / 100 mL; the concentration of 2,2'-azobisisobutylamidine dihydrochloride is 0.01 to 0.1 g / 100 mL, preferably 0.05 g / 100 mL; and the concentration of hexadecyltrimethylammonium bromide is 0.25 to 0.5 g / 100 mL, preferably 0.3 g / 100 mL.

[0028] Furthermore, the stirring conditions in step (4) are: speed 200-300 rpm, time 7-9 h, temperature 65-75°C.

[0029] Furthermore, the molecular weight cut-off of the dialysis bag used for dialysis in step (4) is 8k to 14kDa.

[0030] Furthermore, the concentration of the TNG nanogel solution in step (5) is 1-5 g / 100 mL, preferably 3 g / 100 mL.

[0031] Furthermore, in the mixed solution in step (5), the concentration of EDC is 1-2 g / 100 mL, preferably 1.3 g / 100 mL; the concentration of NHS is 0.6-1.2 g / 100 mL, preferably 0.8 g / 100 mL.

[0032] Furthermore, the stirring conditions in step (5) are: nitrogen atmosphere, speed 200-300 rpm, time 0.5-2 h.

[0033] Furthermore, the concentration of the dopamine solution in step (6) is 0.1-0.2 g / mL, preferably 0.11 g / mL.

[0034] Furthermore, the amount of the dopamine solution in step (6) is calculated as follows: TNG nanogel solution: dopamine solution = 40-50 mL: 1-3 mL, preferably 45 mL: 2 mL.

[0035] Furthermore, the stirring conditions in step (6) are: speed 200-300 rpm, temperature 20-30° C., and time 20-30 h.

[0036] Furthermore, the molecular weight cut-off of the dialysis bag used for dialysis in step (6) is 8k to 14k Da.

[0037] Furthermore, the concentration of the TNGD nanogel in step (7) is 1 to 5 g / 100 mL, preferably 3 to 5 g / 100 mL.

[0038] Furthermore, the heating conditions in step (7) are: temperature 60° C., time 60 min.

[0039] An injectable aggregate with both tissue adhesion and anti-adhesion properties is obtained by the preparation method.

[0040] The use of the above-mentioned injectable aggregates having both tissue adhesion and anti-adhesion properties in the preparation of drugs having any one or more of the following effects: A. promoting wound healing, B. promoting tissue regeneration, C. preventing postoperative adhesion.

[0041] The aggregates of the present invention undergo a hydrophobic phase transition to form cohesions at temperatures between 40°C and 60°C. As the temperature increases, the aggregation-induced gelation time gradually decreases. As the heating time of the nanogel TNGD increases, the mechanical strength of the aggregates increases, and tissue adhesion is enhanced. Due to the presence of hydrophilic zwitterions in the components, the aggregates of the present invention can significantly inhibit fibroblast adhesion. The supramolecular aggregates are used to adhere to surgical sites as a physical barrier to prevent postoperative adhesions.

[0042] The present invention has the following advantages and effects compared to the prior art:

[0043] (1) The present invention overcomes the problem of the lack of tissue adhesion of existing anti-adhesion materials.

[0044] (2) The present invention overcomes the problem that large hydrogels are not conducive to filling complex gaps and have poor soft tissue adaptability.

[0045] (3) The present invention overcomes the problem that the polymer solution is easily lost and has a short functional lifespan.

[0046] (4) The supramolecular aggregates prepared by the present invention have good dynamic mechanical properties. Prolonging the heating time of the nanogel can further enhance the mechanical strength of the aggregates.

[0047] (5) The aggregates prepared by the present invention have both excellent injectability and self-healing properties, can adhere to the surface of complex soft tissues, and remain stable under dynamic deformation.

[0048] (6) The aggregates prepared by the present invention can effectively inhibit fibroblast adhesion and have great potential for preventing postoperative adhesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Actual photos of the coagulation of nanogels prepared in Example 1 and Comparative Example 1.

[0050] Figure 2 This is a photo of the adhesion of the TAH5-60 aggregates prepared in Example 1 to soft tissue.

[0051] Figure 3 This is a graph showing the results of measuring the anti-adhesion properties of the TAH5-60 aggregates prepared in Example 1 on fibroblasts.

[0052] Figure 4 This is a diagram showing the injectability of TAH3-60 aggregates prepared in Example 2.

[0053] Figure 5 This is the coagulation phase diagram of TNGD nanogels with different concentrations at different temperatures in Comparative Example 2.

[0054] Figure 6 This is a strain scanning curve diagram of the aggregate formed by heating for different times in Comparative Example 3.

[0055] Figure 7 This is a graph showing the results of measuring the interfacial adhesion strength of the aggregates formed by heating for different times in Comparative Example 3 to the small intestine.

[0056] Figure 8 This is a diagram showing the effect of the TAH5-60 aggregate prepared in Example 1 on preventing tissue adhesion after colon anastomosis in rats. DETAILED DESCRIPTION

[0057] The present invention will be further described in detail below with reference to specific embodiments and drawings, but the embodiments of the present invention are not limited thereto. The advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention.

[0058] Sources of the raw materials used in the following examples are as follows: L-arginine, methacrylic anhydride, N-isopropylacrylamide, N,N'-methylenebisacrylamide, cetyltrimethylammonium bromide, 2,2'-azobisisobutylamidine dihydrochloride, triethylamine, dioxane, acetone, dopamine hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide were all purchased from Aladdin.

[0059] Example 1

[0060] (1) 2 g of L-arginine (Arg) was dissolved in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution;

[0061] (2) adding 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stirring uniformly at a stirring speed of 200 rpm to obtain a mixture reaction solution;

[0062] (3) The reaction solution in step (2) was stirred overnight, the reaction solution was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white powder M-Arg;

[0063] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water;

[0064] (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution;

[0065] (6) The nanogel solution in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNG nanogel solution with a concentration of 3w / v%.

[0066] (7) 45 mL of the TNG nanogel solution in step (6) was added with 0.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS), and the mixture was stirred at 250 rpm for 1 h under a nitrogen atmosphere to obtain a mixed solution;

[0067] (8) injecting 2 mL of 0.11 g / mL dopamine solution into the mixed solution in step (7), stirring at 250 rpm at 25°C for 24 h to obtain a TNGD nanogel dispersion;

[0068] (9) The TNGD nanogel dispersion in step (8) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNGD nanogel with a concentration of 5w / v%.

[0069] (10) 2 mL of the TNGD nanogel prepared in step (9) was placed in a transparent vial, and the vial was heated at 60° C. for 60 min. The vial was then taken out to prepare an aggregate, which was designated as TAH5-60.

[0070] The TNGD nanogel dispersion in step (9) and the heated vial in step (10) were inverted and photographed to observe the coagulation. The results are as follows: Figure 1 As shown in the figure, it can be seen that the TNGD nanogel prepared in this example can form aggregates after heating.

[0071] Take the TAH5-60 prepared in this example, use a syringe to stick an appropriate amount of rhodamine-stained TAH5-60 to two pieces of tissue (including pig skin, pork, pig intestine), stretch, bend, and twist the tissue, observe the adhesion of the aggregates on the surface and take pictures. The results are as follows: Figure 2 As shown in the figure, TAH5-60 aggregates have good soft tissue adhesion properties and can adhere to various tissues such as pig skin, pork, and small intestine, and remain stable after dynamic deformation such as bending and torsion.

[0072] The TAH5-60 prepared in this example was injected into the bottom of a 24-well plate, and fibroblasts were seeded on the surface of the TAH5-60 and cultured for two days. Cells were directly cultured on the well plate without adding aggregates as a control. Live-dead staining was performed to observe the cell adhesion on the gel surface and fluorescence photos were taken. The results are shown in FIG. Figure 3 As shown in the figure, in the control group, cells cultured directly on the well plate adhered to the bottom of the well plate in large numbers, while only a small number of mouse fibroblasts adhered after being co-cultured with the aggregates for 2 days. This indicates that TAH5-60 aggregates have the potential to reduce fibroblast adhesion and prevent postoperative tissue adhesion.

[0073] The TAH5-60 prepared in this example was used in an animal experiment to study its effect in preventing postoperative adhesions. A 1 cm long incision was made longitudinally in the colon of rats using a scalpel. The wound was then sutured side-to-side with absorbable sutures to create a colon anastomosis model. TAH5-60 aggregates were applied to the anastomosis site. A control group with only the wound sutured served as the control group. Tissue adhesion at the surgical site was observed 7 and 14 days after surgery. The results are shown in Figure 2. Figure 8 As shown, rats treated with TAH5-60 showed no obvious tissue adhesion after surgery, while rats in the control group whose wounds were only sutured showed severe tissue adhesion, indicating that TAH5-60 aggregates have the effect of preventing postoperative tissue adhesion.

[0074] Example 2

[0075] (1) 2 g of L-arginine (Arg) was dissolved in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution;

[0076] (2) adding 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stirring uniformly at a stirring speed of 200 rpm to obtain a mixture reaction solution;

[0077] (3) The reaction solution in step (2) was stirred overnight, the reaction solution was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white powder M-Arg;

[0078] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water;

[0079] (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution;

[0080] (6) The nanogel solution in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNG nanogel solution with a concentration of 3w / v%.

[0081] (7) 45 mL of the TNG nanogel solution in step (6) was added with 0.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS), and the mixture was stirred at 250 rpm for 1 h under a nitrogen atmosphere to obtain a mixed solution;

[0082] (8) injecting 2 mL of 0.11 g / mL dopamine solution into the mixed solution in step (7), stirring at 250 rpm at 25°C for 24 h to obtain a TNGD nanogel dispersion;

[0083] (9) The TNGD nanogel dispersion in step (8) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNGD nanogel with a concentration of 3w / v%.

[0084] (10) 2 mL of the TNGD nanogel prepared in step (9) was placed in a transparent vial, and the vial was heated at 60° C. for 60 min. The vial was then taken out to prepare an aggregate, which was designated as TAH3-60.

[0085] Take the TAH3-60 prepared in this example, inject the TAH3-60 aggregate into molds of different shapes (star, tree) with a syringe, and inject different letters on a glass slide to observe its shape adaptability to irregular graphics. The results are as follows: Figure 4 As shown in the figure, it can be seen that TAH aggregates have good injectable properties, can fill various complex shapes, and have good shape adaptability.

[0086] Example 3

[0087] (1) 2 g of L-arginine (Arg) was dissolved in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution;

[0088] (2) adding 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stirring uniformly at a stirring speed of 200 rpm to obtain a mixture reaction solution;

[0089] (3) The reaction solution in step (2) was stirred overnight, the reaction solution was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white powder M-Arg;

[0090] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water;

[0091] (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution;

[0092] (6) The nanogel solution in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNG nanogel solution with a concentration of 3w / v%.

[0093] (7) 45 mL of the TNG nanogel solution in step (6) was added with 0.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS), and the mixture was stirred at 250 rpm for 1 h under a nitrogen atmosphere to obtain a mixed solution;

[0094] (8) injecting 2 mL of 0.11 g / mL dopamine solution into the mixed solution in step (7), stirring at 250 rpm at 25°C for 24 h to obtain a TNGD nanogel dispersion;

[0095] (9) The TNGD nanogel dispersion in step (8) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNGD nanogel with a concentration of 4w / v%.

[0096] (10) 2 mL of the TNGD nanogel prepared in step (9) was placed in a transparent vial, and the vial was heated at 60° C. for 60 min. The vial was then taken out to prepare an aggregate, which was designated as TAH4-60.

[0097] Comparative Example 1 (unmodified dopamine)

[0098] (1) 2 g of L-arginine (Arg) was dissolved in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg mixed solution;

[0099] (2) adding 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stirring uniformly at a stirring speed of 200 rpm to obtain a mixture reaction solution;

[0100] (3) The reaction solution in step (2) was stirred overnight, the reaction solution was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white powder M-Arg;

[0101] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water;

[0102] (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution;

[0103] (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and the nanogel solution was concentrated to obtain a TNG nanogel solution with a concentration of 5w / v%.

[0104] 2 mL of 5 w / v% TNG nanogel solution was taken into a transparent vial, and the vial was heated at 60°C for 60 min. The vial was taken out and inverted, and the coagulation was observed and photographed. Figure 1 .from Figure 1 It can be seen that the TNG nanogels without dopamine modification cannot form aggregates after heating.

[0105] Comparative Example 2 (Effects of Different TNGD Nanogel Concentrations and Heating Temperatures)

[0106] A method for preparing an aggregate having both injectability and adhesiveness comprises the following steps:

[0107] (1) 2 g of L-arginine (Arg) was dissolved in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution;

[0108] (2) adding 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stirring uniformly at a stirring speed of 200 rpm to obtain a mixture reaction solution;

[0109] (3) The reaction solution in step (2) was stirred overnight, the reaction solution was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white powder M-Arg;

[0110] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water;

[0111] (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution;

[0112] (6) The nanogel solution in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNG nanogel solution with a concentration of 3w / v%.

[0113] (7) 45 mL of the TNG nanogel solution in step (6) was added with 0.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS), and the mixture was stirred at 250 rpm for 1 h under a nitrogen atmosphere to obtain a mixed solution;

[0114] (8) injecting 2 mL of 0.11 g / mL dopamine solution into the mixed solution in step (7), stirring at 250 rpm at 25°C for 24 h to obtain a TNGD nanogel dispersion;

[0115] (9) The TNGD nanogel dispersion in step (8) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain nanogel TNGD with concentrations of 3w / v%, 4w / v%, and 5w / v%, respectively.

[0116] Take 2 mL of each TNGD nanogel in step (9) in a transparent vial and heat it at 40-70°C, setting a group every 5°C. Starting from the start time of heating, turn the bottle upside down every 20 seconds to observe the coagulation. Record the gelation time of the three groups of nanogels at different temperatures and draw a phase diagram. The results are as follows: Figure 5 As shown in the figure, it can be seen that with the increase of nanogel concentration, the gelation time is shorter at the same temperature; for nanogels of the same concentration, the higher the temperature, the shorter the gelation time.

[0117] Comparative Example 3 (Effect of Different Heating Times)

[0118] (1) 2 g of L-arginine (Arg) was dissolved in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution;

[0119] (2) adding 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stirring uniformly at a stirring speed of 200 rpm to obtain a mixture reaction solution;

[0120] (3) The reaction solution in step (2) was stirred overnight, the reaction solution was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white powder M-Arg;

[0121] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water;

[0122] (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution;

[0123] (6) The nanogel solution in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNG nanogel solution with a concentration of 3w / v%.

[0124] (7) 45 mL of the TNG nanogel solution in step (6) was added with 0.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS), and the mixture was stirred at 250 rpm for 1 h under a nitrogen atmosphere to obtain a mixed solution;

[0125] (8) injecting 2 mL of 0.11 g / mL dopamine solution into the mixed solution in step (7), stirring at 250 rpm at 25°C for 24 h to obtain a TNGD nanogel dispersion;

[0126] (9) The TNGD nanogel dispersion in step (8) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain a TNGD nanogel with a concentration of 5w / v%.

[0127] (10) 2 mL of the TNGD nanogel prepared in step (9) was placed in a transparent vial, and the vial was heated at 60°C for 20 min, 40 min, and 60 min, respectively. The vial was then taken out to prepare aggregates, which were designated as TAH5-20, TAH5-40, and TAH5-60.

[0128] The strain sweep of TAH5-20, TAH5-40 and TAH5-60 aggregates was carried out using a rheometer to test their storage modulus and loss modulus. Figure 6 As can be seen from the figure, for nanogel TNGD at the same concentration and temperature, the heating time will affect the storage modulus and mechanical strength of the aggregate. The longer the heating time, the higher the storage modulus and the stronger the mechanical strength of the aggregate.

[0129] The TAH5-20, TAH5-40, and TAH5-60 aggregates prepared in this example were adhered between two pieces of small intestine to test their adhesion strength. Figure 7 From the figure, it can be seen that the length of heating time will affect the adhesion strength of the aggregate to the tissue. As the heating time increases, the adhesion strength of the aggregate to the tissue gradually increases, and the maximum can exceed 10kPa.

[0130] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing an injectable aggregate having both tissue adhesion and anti-adhesion properties, characterized in that: The steps include: (1) dissolving L-arginine in a mixed solvent of water, dioxane and triethylamine to obtain an Arg solution; (2) adding methacrylic anhydride dropwise to the Arg solution in step (1) and stirring uniformly to obtain a mixture reaction solution; (3) stirring the reaction solution in step (2), recrystallizing the reaction solution with acetone, and vacuum drying the precipitate to obtain white M-Arg powder; (4) dissolving the M-Arg powder, N-isopropylacrylamide, N,N'-methylenebisacrylamide, hexadecyltrimethylammonium bromide, and 2,2'-azobisisobutylamidine dihydrochloride in step (3) in water, heating and stirring, and dialyzing to obtain a TNG nanogel solution; (5) adjusting the concentration of the TNG nanogel solution in step (4), adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stirring to obtain a mixed solution; (6) injecting dopamine solution into the mixed solution in step (5), stirring, and dialyzing to obtain a TNGD nanogel dispersion; (7) adjusting the concentration of the TNGD nanogel dispersion in step (6), heating at 40°C to 70°C for 10 to 60 minutes, and absorbing the precipitated water to obtain the injectable aggregate having both tissue adhesion and anti-adhesion properties.

2. The method for preparing the injectable aggregate having both tissue adhesion and anti-adhesion properties according to claim 1, characterized in that: The concentration of L-arginine in the Arg solution in step (1) is 6±1 g / 100 mL; the proportions of the components in the mixed solvent are: deionized water 60±5 v / v%, dioxane 26±3 v / v%, and triethylamine 14±2 v / v%; The concentration of methacrylic anhydride in the mixture reaction solution in step (2) is 8.4±1 v / v%; In the TNG nanogel solution in step (4), the concentration of M-Arg is 0.1 to 0.5 g / 100 mL; the concentration of N-isopropylacrylamide is 1 to 5 g / 100 mL; the concentration of N,N'-methylenebisacrylamide is 0.01 to 0.05 g / 100 mL; the concentration of 2,2'-azobisisobutylamidine dihydrochloride is 0.01 to 0.1 g / 100 mL; and the concentration of hexadecyltrimethylammonium bromide is 0.25 to 0.5 g / 100 mL.

3. The method for preparing the injectable aggregate having both tissue adhesion and anti-adhesion properties according to claim 1, characterized in that: The ratio of L-arginine, water, dioxane and triethylamine in step (1) is 2g:20mL:8.5mL:4.5mL; Based on the amount of L-arginine used in step (1) being 2 g, the amount of methacrylic anhydride added in step (2) is 3 mL; In the TNG nanogel solution in step (4), the concentration of M-Arg is 0.5 g / 100 mL; the concentration of N-isopropylacrylamide is 2.5 g / 100 mL; the concentration of N,N'-methylenebisacrylamide is 0.025 g / 100 mL; the concentration of 2,2'-azobisisobutylamidine dihydrochloride is 0.05 g / 100 mL; and the concentration of hexadecyltrimethylammonium bromide is 0.3 g / 100 mL.

4. The method for preparing the injectable aggregate having both tissue adhesion and anti-adhesion properties according to claim 1, 2 or 3, characterized in that: The stirring conditions in step (2) are: speed 200-300 rpm, time 10-20 min; The stirring conditions in step (3) are: speed 200-300 rpm, time 8-18 h; The amount of acetone used in step (3) is 3 to 5 times the volume of the mixture reaction solution; The stirring conditions in step (4) are: speed 200-300 rpm, time 7-9 h, temperature 65-75° C.; The molecular weight cut-off of the dialysis bag used in the dialysis in step (4) is 8k to 14kDa.

5. The method for preparing the injectable aggregate having both tissue adhesion and anti-adhesion properties according to claim 1, characterized in that: The concentration of the TNG nanogel solution in step (5) is 1-5 g / 100 mL; In the mixed solution in step (5), the concentration of EDC is 1-2 g / 100 mL; the concentration of NHS is 0.6-1.2 g / 100 mL; The amount of the dopamine solution in step (6) is calculated as follows: TNG nanogel solution: dopamine solution = 40-50 mL: 1-3 mL; The concentration of the TNGD nanogel in step (7) is 1-5 g / 100 mL.

6. The method for preparing the injectable aggregate having both tissue adhesion and anti-adhesion properties according to claim 1, characterized in that: The concentration of the TNG nanogel solution in step (5) is 3 g / 100 mL; In the mixed solution in step (5), the concentration of EDC is 1.3 g / 100 mL; the concentration of NHS is 0.8 g / 100 mL; The amount of the dopamine solution in step (6) is calculated as follows: TNG nanogel solution: dopamine solution = 45 mL: 2 mL; The concentration of the dopamine solution in step (6) is 0.1 to 0.2 g / mL; The concentration of the TNGD nanogel in step (7) is 3-5 g / 100 mL.

7. The method for preparing the injectable aggregate having both tissue adhesion and anti-adhesion properties according to claim 1, 5 or 6, characterized in that: The stirring conditions in step (5) are: nitrogen atmosphere, speed 200-300 rpm, time 0.5-2 h; The stirring conditions in step (6) are: speed 200-300 rpm, temperature 20-30° C., and time 20-30 h; The molecular weight cut-off of the dialysis bag used in the dialysis in step (6) is 8k-14k Da.

8. The method for preparing the injectable aggregate having both tissue adhesion and anti-adhesion properties according to claim 1, characterized in that: The heating conditions in step (7) are: temperature 60° C., time 60 min.

9. An injectable aggregate having both tissue adhesion and anti-adhesion properties, characterized in that: The method is obtained by the preparation method described in any one of claims 1 to 8.

10. Use of the injectable aggregate having both tissue adhesion and anti-adhesion properties as claimed in claim 9 in the preparation of a medicament having any one or more of the following effects: A. promoting wound healing, B. promoting tissue regeneration, C. preventing postoperative adhesion.

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