Preparation method, product and application of polymer coating

Abstract

The invention relates to the technical field of biological materials, in particular to a preparation method of a polymer coating. The preparation method comprises the following steps: mixing a dopamine solution and a hydrogen peroxide aqueous solution on the surface of a base material to be coated to generate the polydopamine coating. The invention provides a novel method for preparing a material for treating radioactive rectal injury, which can effectively improve the problems of tissue ischemia, oxygen deficit, microcirculation disturbance and the like caused by radioactive vascular endothelial injury of lesion intestinal canals, accelerate ulcer healing, promote tissue repair and improve the living quality of patients. Meanwhile, the polydopamine coating can remove various ROS (reactive oxygen species), including. OH,. O2 <-> and H2O2, so that the technical scheme can also be used for treating other inflammatory diseases, such as ulcerative colitis and the like. The technical scheme provided by the invention has a wide application prospect in the field of radioactive injury treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a method for preparing a polymer coating and a method for preparing the same. Background Art

[0002] Radiotherapy is a common method for treating certain types of cancer, especially gynecological, urological and gastrointestinal cancers. However, because the rectum is close to the treatment area, radiotherapy may cause structural damage and dysfunction of the intestinal epithelial barrier, leading to a series of complications, the most typical of which is radiation proctitis. Radiation rectal injury refers to rectal injury caused by radiotherapy, which is common in the treatment of cancers such as cervical cancer and prostate cancer. Mild radiation rectal injury can cause symptoms such as congestion, edema, and ulcers of the rectal mucosa, causing some patients to have changes in defecation habits; severe radiation rectal injury can cause massive rectal bleeding, intestinal perforation, intestinal obstruction, intestinal fistula and even complex multiple fistulas, which makes clinical diagnosis and treatment extremely difficult. Therefore, radiation rectal injury seriously affects the quality of life of patients. In addition, radiation rectal injury is also one of the main causes of radiation enteritis.

[0003] The current existing solutions mainly include drug therapy and surgical treatment. The drug treatment of radiation rectal injury includes a variety of drugs, mainly the following categories: 1. Anti-inflammatory drugs. Used to reduce the incidence and severity of diarrhea, and relieve symptoms such as diarrhea during the acute period of radiotherapy. 2. Antibiotics: Improve symptoms such as abdominal distension and diarrhea caused by intestinal flora ectopic and imbalance after radiotherapy. 3. Antioxidants: Used for adjuvant therapy, but the therapeutic effect is controversial. 4. Mucosal protectants: Used to improve local blood flow of ulcers, protect mucosa, and promote ulcer healing. 5. Compound enema preparations: Have significant short-term effects on mild to moderate bloody stools, but have poor effects on RLRI patients with ulcers. The prior art has also attempted to prevent and treat radiation intestinal injury with Chinese medicinal materials. For example, Chinese patent CN115252685A discloses the use of a Mongolian medicine Batri-7 in the prevention and treatment of radiation intestinal injury. However, there are some disadvantages in drug treatment of radiation-induced rectal injury, including controversial treatment effects, insufficient symptom control, long-term drug dependence, lack of standardized treatment regimens, side effects and tolerance issues, high costs, and drug accessibility.

[0004] Surgery is an important means of treating radiation-induced rectal injury, especially for patients with severe complications in the late stage. Surgery should follow the principles of "damage control" and "extended resection" to minimize the incidence of surgical complications and mortality, and improve the patient's long-term quality of life; especially for serious complications such as fistula or bleeding caused by chronic radiation-induced intestinal injury, surgical treatment is a necessary means.

[0005] The existing technologies for the treatment of radiation-induced rectal injury still have the following problems: 1. Existing drug treatments are not effective: Traditional drug treatments cannot completely eliminate the symptoms of rectal injury, and long-term use of antibiotics and other drugs may lead to problems such as bacterial resistance. 2. Existing surgical treatments are risky: Although surgical treatments can completely remove the damaged rectum, surgical treatments still have high surgical risks, high complication rates, and long recovery times. 3. Existing technologies are not effective in repairing the microenvironment of radiation-induced rectal injury: Existing technologies are not effective in repairing the microenvironment of radiation-induced rectal injury, and cannot effectively improve problems such as tissue ischemia, hypoxia, and microcirculatory disorders caused by radiation-induced vascular endothelial damage in the diseased intestine.

[0006] In summary, there is currently no effective and safe product on the market for rectal protection after radiotherapy; there is an urgent need to provide a product that can be effectively applied to radiation rectal injury to provide a barrier, lubrication, and repair effect for the affected area. Summary of the invention

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a method for preparing a polymer coating, the preparation steps comprising: mixing a dopamine solution and an aqueous hydrogen peroxide solution (commonly known as: hydrogen peroxide) on the surface of a substrate to be coated to generate a polydopamine coating.

[0008] In some preferred embodiments, the substrate to be coated is immersed in a dopamine solution, and an aqueous hydrogen peroxide solution is added dropwise to form a polydopamine coating on the surface of the substrate to be coated.

[0009] In some preferred embodiments, the method for preparing the dopamine solution comprises: dissolving dopamine monomers in an alkaline buffer to obtain a dopamine solution; the concentration of the dopamine monomers in the dopamine solution is 10-1000 mg / mL.

[0010] More preferably, the concentration of dopamine monomer in the dopamine solution is 20-200 mg / mL, which can be exemplified as 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, 160 mg / mL, 180 mg / mL, and 200 mg / mL.

[0011] In some preferred embodiments, the pH of the alkaline buffer is 7.5-9.0.

[0012] More preferably, the pH of the alkaline buffer is 8.0-8.6.

[0013] In some preferred embodiments, the alkaline buffer is a Tris (tris(hydroxymethyl)aminomethane) buffer; the preparation method of the Tris buffer is not particularly limited, as long as it can meet the pH requirement; for example, a mixture of PBS (phosphate buffered saline) and Tris can be used.

[0014] In some preferred embodiments, the dopamine solution contains catalase, and the concentration of the catalase is 0.5-4 mg / mL.

[0015] The catalase includes, but is not limited to, HRP (horseradish peroxidase), CAT (catalase), and the like.

[0016] In some preferred embodiments, the reaction temperature is 15-45° C., and the reaction time is 1-70 min.

[0017] More preferably, the reaction temperature is 17-40° C., and the reaction time is 2-60 min.

[0018] Most preferably, the reaction temperature is 37° C. and the reaction time is 10 min.

[0019] In some preferred embodiments, the concentration of hydrogen peroxide in the aqueous hydrogen peroxide solution is 0.01-50%.

[0020] More preferably, the concentration of hydrogen peroxide in the aqueous hydrogen peroxide solution is 1-10%, which can be exemplified as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%.

[0021] In some preferred embodiments, the thickness of the polydopamine coating is 10-500 nm. The coating thickness can be flexibly adjusted according to the reaction time and dopamine concentration during the preparation process.

[0022] In some preferred embodiments, the substrate to be coated is biological rectal tissue.

[0023] A second aspect of the present invention provides a polymer coating prepared by the preparation method described above.

[0024] A third aspect of the present invention provides an application of the polymer coating as described above, wherein the polymer coating is applied to prepare a material for radiation-induced intestinal damage.

[0025] On the one hand, the polydopamine coating can interact with the intestinal mucosa to enhance the repair ability of intestinal cells; on the other hand, it can alleviate intestinal damage caused by radiotherapy, inhibit oxidative damage to intestinal tissue after radiotherapy, improve intestinal barrier function, and prevent cell apoptosis and inflammatory response.

[0026] Furthermore, the polydopamine coating can also be loaded with functional components as required, such as antioxidants or anti-inflammatory drugs, to enhance the protective effect of the coating.

[0027] The present invention aims at the limitations of the current treatment of radiation rectal injury and provides an in-situ polymerized adhesive dopamine polymer coating, which can form a protective layer in the rectal injury area after radiotherapy, reduce inflammatory response, moisturize the intestine and promote tissue repair. Dopamine, catalase and hydrogen peroxide are selected to form a polydopamine barrier on the intestinal wall to protect and shield the intestinal damage site after radiotherapy. The coating provided by the present invention has low requirements for the use environment, and still has high reactivity even at temperatures far below body temperature. It can form a polydopamine coating in situ on the surface of the intestine such as the small intestine or rectum, and the pathological degree of the intestine has little effect on the formation of the coating. The polydopamine coating prepared by the present invention has universality, can form a polydopamine coating on different types of intestinal surfaces, is suitable for a variety of intestinal tissues, has a wide range of applications, and has huge market demand and application value.

[0028] Beneficial effects:

[0029] The present invention provides a preparation method, product and application of a polymer coating, which has the following advantages:

[0030] (1) The present invention mixes dopamine, catalase and hydrogen peroxide on the surface of a substrate to be coated to react and generate a polydopamine coating. The obtained polydopamine coating can effectively alleviate the symptoms of radiation rectal injury and will not cause problems such as bacterial resistance.

[0031] (2) The polydopamine coating of the present invention is generated by in situ polymerization at the affected area, which provides a mild and effective treatment plan for radiation rectal injury without the need for surgery, thus avoiding the risks and complications of surgery.

[0032] (3) The present invention uses catalase to catalyze the decomposition of hydrogen peroxide to produce oxygen and H 2 O 2 , H 2 O 2 It can induce white blood cells to gather at the wound surface, thereby recruiting immune cells and accelerating wound healing. It can further release oxygen through hydrogen peroxide and cooperate with the antioxidant effect of polydopamine to effectively improve tissue ischemia, hypoxia and microcirculation disorders caused by radiation-induced vascular endothelial damage in the diseased intestine, accelerate ulcer healing and promote tissue repair.

[0033] (4) The polydopamine coating of the present invention can play a barrier role. The polydopamine coating generated by in situ polymerization can serve as an effective intestinal mucosal barrier for the treatment of ulcerative enteritis, further protect the intestinal mucosa, effectively inhibit tissue damage and deterioration, and accelerate wound healing.

[0034] (5) The coating of the present invention has a flexible existence time. The existence time of the coating in the rectum can be flexibly adjusted (about 1-15 days) according to the specific conditions of the patient and the progression of the disease, and the treatment plan can be adaptively adjusted to achieve the best treatment effect.

[0035] (6) The reaction system for preparing polydopamine in the present invention is simple, the raw materials are easily available and safe and non-toxic, the therapeutic effect can be improved under a mild operating environment, and the price is cheaper than high-pressure oxygen therapy, and it is easy to promote and implement.

[0036] (7) The technical solution of the present invention can also be used to develop new antioxidants and cell protectants, etc., and can be well applied to multidisciplinary fields such as medical treatment, radiation injury treatment, biomaterials, and enzyme engineering. It is expected to have a positive impact in multiple fields and promote technological progress in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 .Schematic diagram of the reaction principle;

[0038] Figure 2 .Schematic diagram of the dopamine polymerization kinetics detection scheme;

[0039] Figure 3 .The extinction variation with time during the polymerization of dopamine solution (the dopamine concentrations from left to right are 25 mg / mL, 50 mg / mL and 100 mg / mL); Note: The dotted line indicates that the measured value exceeds the instrument range and is not displayed;

[0040] Figure 4 .Simulation diagram of dopamine solution polymerization process (detection wavelength 700nm);

[0041] Figure 5 . Fourier transform infrared spectra of dopamine and polydopamine;

[0042] Figure 6 .Dopamine polymerization on the surface of isolated small intestine tissue at different time points (left: 5min, right: 10min); the black part is polydopamine, the white part is intestinal tissue, and the scale is 500μm;

[0043] Figure 7 . Preparation of in situ polymerized polydopamine coating on the surface of isolated small intestine (A. clean intestine, B. ulcer model, C. dopamine solution added, D. reaction ended; reaction temperature: 17°C); the black part in the figure is polydopamine coating; the PBS soaked group is normal tissue; the 20% HAc and HAc treated groups represent small intestine tissues with different degrees of ulcers;

[0044] Figure 8. Preparation of in situ polymerized polydopamine coating on the surface of isolated small intestine (A. ulcer modeling, B. dopamine solution addition, C. reaction simulation, D. reaction completion; reaction temperature: 37°C); the black part in the figure is polydopamine coating; the PBS soaking group is normal tissue; the 20% HAc and HAc treatment groups represent small intestine tissues with different degrees of ulcers; Fig. 9 . Scraping experiment of in situ polymerized polydopamine coating on the surface of isolated small intestine (from left to right, before and after cotton swab scraping experiment, before and after metal spoon scraping experiment); the solution in the water bath in the figure is normal saline, and the black is polydopamine; the PBS soaking group is normal tissue; the 20% HAc and HAc treatment groups represent small intestine tissues with different degrees of ulcers;

[0045] Fig.10 .Preparation of in situ polydopamine coating on the rectal surface (from left to back: rectal segment, limiting, ulcer modeling, dripping of dopamine solution, body temperature simulation, reaction in progress, and reaction end); the black in the figure is polydopamine; the PBS soaking group is normal tissue; the 20% HAc and HAc treatment groups represent rectal tissues with different degrees of ulcers;

[0046] Fig.11 . Scraping test of in situ polymerized polydopamine coating on the rectal surface (from left to right, before and after the cotton swab scraping test, before and after the metal spoon scraping test); the solution in the water bath in the figure is normal saline, and the black is polydopamine; the PBS soaking group is normal tissue; the 20% HAc and HAc treatment groups represent rectal tissues with different degrees of ulcers;

[0047] Fig.12 . Photo of frozen section of rectal tissue; blue ruler is 1mm, red ruler is 200μm; black arrow indicates goblet cell, green arrow indicates polydopamine, red bracket indicates muscle layer;

[0048] Fig.13 .HE section photos of rectal tissue; the blue ruler in the figure is 1mm, and the red ruler is 200μm; the black arrow indicates goblet cells, the green arrow indicates polydopamine, and the red bracket indicates the muscle layer;

[0049] Fig.14 . Preparation of rectal in situ perfusion polydopamine coating (from left to right: fresh rectum, rectal segment, reaction solution injection, bottom closure, anatomical observation), the black part in the figure is polydopamine;

[0050] Fig.15 . Scratching stability test of polydopamine coating after in situ perfusion of the rectum. The black part in the figure is polydopamine, and the solution in the water bath is saline;

[0051] Fig.16. Preparation of in situ polydopamine coating on the inner wall of isolated small intestine (upper left is before treatment, upper right is after adding dopamine solution, lower right is after adding catalase, and lower left is after 10 minutes of reaction);

[0052] Fig.17 . In vitro polymerization reaction diagram of dopamine (the left picture shows a reaction time of 60 s, the middle picture shows a reaction time of 180 s, and the right picture shows a reaction time of 3600 s);

[0053] Fig.18 .Comparison of absorbance of in vitro polymerization reaction of dopamine (detection wavelength 405nm). DETAILED DESCRIPTION

[0054] Note:

[0055] 1) The isolated small intestinal tissue and rectal tissue in the specific embodiment of the present invention are both derived from large Yorkshire pigs.

[0056] 2) The catalase in the specific embodiment of the present invention is specifically catalase CAT.

[0057] 2) Unless otherwise specified, the solvents of the solutions involved in the present invention are all water; the concentrations of the solutions involved are all mass concentrations; and the raw materials used are all commercially available.

[0058] Preparation Example

[0059] Prepare Tris buffer: add 0.605 g of Tris to 45 mL of PBS and stir to dissolve; adjust the pH of the solution to 8.4-8.6 and adjust the volume to 50 ml. The following Tris buffer is from this preparation example.

[0060] Prepare dopamine solution: weigh 0.5 g dopamine hydrochloride, add it to 10 ml of the above Tris buffer, and stir until dissolved; weigh 10 mg catalase, dissolve it in the above mixture to obtain dopamine solution, which is reagent A;

[0061] Preparation of polydopamine: Prepare 3 wt% H 2 O 2 Aqueous solution, denoted as reagent B; reagent A and reagent B were mixed in a volume ratio of 1:1 and reacted at 17°C to verify the synthesis of polydopa; the experimental results are shown in Fig.17 , the absorbance value during the reaction is Fig.18 .

[0062] As can be seen from the figure, as the reaction time increases, the dopamine solution changes from colorless, light gray, brown to black, and solid particles appear in the solution. The absorbance value of the solution increases significantly with the increase of storage time.

[0063] Example 1

[0064] This example explores the in vitro polymerization kinetics of dopamine. The reaction principle diagram is shown in Figure 1 , see the schematic diagram of the scheme Figure 2 ; comprising the following steps:

[0065] S1. Prepare dopamine solution: add dopamine hydrochloride and catalase to Tris buffer and dissolve evenly to obtain dopamine mother solution; the concentration of dopamine in the dopamine mother solution is 100 mg / mL, and the concentration of catalase is 1.5 mg / mL.

[0066] S2. Mixing the reaction solution: dilute the mother solution to the target concentration according to the dopamine concentration of 25 mg / mL, 50 mg / mL, and 100 mg / mL, take 2 mL of dopamine solution of each concentration into a cuvette, add 0.05 mL of 3% hydrogen peroxide solution, and shake to mix; lay a circular stopper on the surface of the isolated small intestine, add the mixed reaction solution to the area inside the circular stopper, and react at 37°C; then remove the circular stopper (unless otherwise specified, the following embodiments also use similar operations).

[0067] S3. Detect the polymerization status of the reaction system at different time points.

[0068] Specifically, a UV-visible spectrophotometer was used to detect and record the absorbance of each reaction solution in the wavelength range of 200-700 nm; the acquisition frequency was once every 10 minutes until the absorbance of the reaction solution reached a stable state. The extinction of each reaction solution was calculated by Lambert-Beer's law, and the polymerization of reaction solutions of different concentrations at various time points was studied. The detection results during the polymerization process are shown in Figure 3-6 .

[0069] like Figure 5 As shown, at 1100-1250cm -1 In the range of 1550-1650cm, the peak intensity of the phenolic CO stretching vibration on PDA is weakened. This is because some phenolic hydroxyl groups are oxidized during the synthesis of PDA, resulting in a weakening of the peak intensity on PDA. -1 In the range of 2.5, the NH stretching vibration peak intensity on PDA weakens, which is due to the primary amino group (-NH 2 ) reacted with the aromatic ring, resulting in a decrease in the peak intensity on PDA. Figure 5 It is proved that dopamine in the reaction system of the present invention is polymerized to form polydopamine.

[0070] from Figure 6 As shown, 5 minutes after the polymerization reaction, a certain amount of black or brown substance with a wide coverage was formed on the surface of the isolated small intestinal tissue. After 10 minutes of the polymerization reaction, the surface of the small intestinal tissue was basically covered by the black or brown substance. Figure 6 It was shown that in vitro and at room temperature, dopamine solution could be gradually polymerized to form a black or dark brown polydopamine coating.

[0071] Example 2

[0072] In this example, small intestinal ulcer models with different disease severity were constructed. The isolated small intestinal tissue was treated with 20 wt% acetic acid aqueous solution (simulating ulcer, treatment time was 2 min), and then NaHCO 3 The polydopamine coating was then prepared on the surface of the isolated small intestine tissue to observe the stability of the coating; at the same time, the small intestine of the PBS-treated group was set as the normal group control.

[0073] The preparation method of the polydopamine coating is as follows: 0.5 mL of dopamine solution (dopamine concentration is 0.5 g / 10 mL, catalase concentration is 5 mg / mL, and the solvent is Tris buffer) is added dropwise to the surface of the treated isolated small intestine tissue, and then 0.1 mL of 3% hydrogen peroxide is added dropwise, and the reaction is carried out at 17 and 37 ° C for 10 minutes to obtain a polymer coating; the actual reaction diagram is shown in Figure 7 and Figure 8 , experimental test diagram see Fig. 9 .

[0074] As can be seen from the figure, a continuous polydopamine coating is generated on the surface of the small intestine after acid treatment, especially the coating obtained by the reaction at 37°C remains stable after multiple scrapings. The coating is tightly adhered to the small intestinal tissue and the coating network is highly stable.

[0075] Example 3

[0076] 0.5 mL of dopamine solution (dopamine concentration is 0.5 g / 10 mL, catalase concentration is 5 mg / mL, and the solvent is Tris buffer) was dripped onto the affected area of ​​rectal ulcers of different degrees (the ulcerated rectum was obtained from the rectum of normal pigs after being treated with acetic acid), and then 0.1 mL of 3% hydrogen peroxide was dripped, and the reaction was carried out at 37°C for 10 minutes to generate a polydopamine coating. See the actual reaction picture. Fig.10 , experimental test diagram see Fig.11 .

[0077] As can be seen from the figure, the obtained coating can still be stable after being scratched many times. Fig.12 , 13 .

[0078] Example 4

[0079] 0.5 mL of dopamine solution (dopamine concentration of 0.5 g / 10 mL, catalase concentration of 5 mg / mL, solvent is Tris buffer) was perfused into the rectal wall, and then 0.1 mL of 3% hydrogen peroxide was added dropwise, and reacted at 37°C for 10 minutes to generate a polydopamine coating. Experimental pictures are shown in Fig.14 , experimental test diagram see Fig.15 .

[0080] As can be seen from the figure, the obtained coating still exists stably after being scraped many times, indicating that the dopamine reaction system of the present invention does not only form a polydopamine coating due to deposition, but can also undergo in-situ polymerization at all contact sites with the intestinal wall to form a polydopamine coating.

[0081] Example 5

[0082] 0.5 mL of dopamine solution (dopamine concentration of 10 mg / mL, catalase concentration of 5 mg / mL, solvent is Tris buffer) was dripped onto the inner wall of the isolated small intestine, and then 0.1 mL of 3% hydrogen peroxide was dripped and reacted at 37°C for 10 minutes. No continuous polydopamine coating was found on the surface of the intestinal tissue. Experimental pictures are shown in Fig.16 .

[0083] As can be seen from the figure, the color of the small intestine did not change after contact with the dopamine solution. After the addition of hydrogen peroxide, the outer wall of the small intestine turned red, and the inner wall turned redder. After the reaction, no obvious continuous polydopamine coating was observed on the outer wall of the small intestine. This shows that low-concentration dopamine solutions cannot form a continuous polydopamine coating on the surface of the isolated small intestine to play an intestinal barrier and protective role.

Claims

1. A method for preparing a polymer coating, characterized in that: The preparation steps include: The dopamine solution and the hydrogen peroxide aqueous solution are mixed on the surface of the substrate to be coated to form a polydopamine coating.

2. The method for preparing a polymer coating according to claim 1, characterized in that: The method for preparing the dopamine solution comprises: dissolving dopamine monomers in an alkaline buffer solution to obtain a dopamine solution; the concentration of the dopamine monomers in the dopamine solution is 10-1000 mg / mL.

3. The method for preparing a polymer coating according to claim 2, characterized in that: The concentration of dopamine monomer in the dopamine solution is 20-200 mg / mL.

4. The method for preparing a polymer coating according to claim 1, characterized in that: The pH of the alkaline buffer solution is 7.5-9.

0.

5. The method for preparing a polymer coating according to claim 1, characterized in that: The dopamine solution contains catalase, and the concentration of the catalase is 0.5-4 mg / mL.

6. The method for preparing a polymer coating according to claim 5, characterized in that: The reaction temperature is 15-45°C, and the reaction time is 1-70 min.

7. The method for preparing a polymer coating according to claim 1, characterized in that: The thickness of the polydopamine coating is 10-500 nm.

8. The method for preparing a polymer coating according to claim 1, characterized in that: The substrate to be coated is biological rectal tissue.

9. A polymer coating prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the polymer coating according to claim 9, characterized in that: The polymer coating is used for preparing materials for radiation intestinal damage.

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