Polysaccharide-based gastrointestinal tract tissue marking hydrogel as well as preparation method and application thereof

Abstract

The invention discloses polysaccharide-based gastrointestinal tract tissue marking hydrogel as well as a preparation method and application thereof. The invention provides a preparation method of gastrointestinal tract tissue labeling hydrogel by utilizing the property that polysaccharide is subjected to Schiff base reaction with oxidized dextran after being grafted with polyaniline. Compared with the existing commercial marking liquid, the marking hydrogel disclosed by the invention has a better marking effect, and overcomes the defects that the conventional marking liquid is easy to coagulate and easy to leak during injection and the like. The polysaccharide-based gastrointestinal tract tissue marking hydrogel prepared by the invention has good biocompatibility, injectability, controllable degradability and excellent stability, and can be used for accurate positioning of gastrointestinal tract pathological tissues or specific parts under an endoscope and treatment of cancerous tissues.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials and relates to a polysaccharide-based hydrogel, specifically a gastrointestinal tissue marking hydrogel based on dextran and other natural polysaccharides, and a preparation method and application thereof. Background Art

[0002] With the continuous advancement of medical technology, non-invasive or minimally invasive endoscopy (gastroenteroscopy, laparoscopy) has become a key diagnostic and treatment tool, especially in the diagnosis and treatment of early lesions in the gastrointestinal tract or abdominal cavity. In addition, it can also assist surgeons in observing lesions that cannot be displayed by X-rays during endoscopic surgery, as well as regular follow-up observations of lesions. However, in later follow-up visits or endoscopic surgical interventions, due to changes in patient position or movement of internal organs and complete recovery of lesions, the endoscope may not be able to quickly and accurately locate the location of early lesions, thereby prolonging the examination time, bringing a bad experience to patients and reducing clinical work efficiency.

[0003] Endoscopic staining and marking technology refers to the use of dyes or dyes to mark gastrointestinal lesions under endoscopy (including endoscopic ultrasound) to guide subsequent surgical intervention or regular reexamination and other diagnosis and treatment methods. This is one of the most commonly used methods to quickly and accurately locate the location of lesions before endoscopic surgery.

[0004] The dyes used for this technology in the early stage include India ink, methylene blue, indocyanine green, etc. Due to the complex composition of India ink, it needs to be carefully diluted and sterilized when used, which limits its application. Compared with India ink, methylene blue and indocyanine green, which are safer, also have the disadvantages of short labeling time and are not suitable for patients with iodine allergy, which limits their application. At present, sterile carbide (SPOT) is the only FDA-approved marking liquid on the market. It is also the safest and best marking liquid in existence. However, its liquid physical properties may cause precipitation or leakage or diffusion from the tissue at the injection point, thereby reducing the utilization rate of the marking liquid. At the same time, its high cost hinders its widespread clinical use. Therefore, there is a clear need for an endoscopic marking dye that is both biosafe, durable, not easy to precipitate, not easy to leak, and cost-effective. Summary of the invention

[0005] In view of the problems existing in existing marking dyes, the present invention provides a polysaccharide-based gastrointestinal tissue marking hydrogel. This gastrointestinal tissue marking hydrogel based on dextran and other natural polysaccharides is intended to solve the problems of existing endoscopic marking dyes, such as high toxicity, short marking time, easy aggregation, and easy leakage.

[0006] The invention provides a preparation method of a polysaccharide-based gastrointestinal tissue marker hydrogel, comprising the following steps: step A: dissolving a polysaccharide in a first solvent, stirring the polysaccharide to completely dissolve the polysaccharide to form a polysaccharide solution, wherein the mass concentration of the polysaccharide after dissolution is 0.5%-5%, then adding aniline hydrochloride to the polysaccharide solution to form a first mixed solution, wherein the mass concentration of the aniline hydrochloride in the first mixed solution is 0.5%-5%, then adding a strong oxidant to the first mixed solution, wherein the mass concentration of the strong oxidant after dissolution is 0.05%-0.5%, then placing the first mixed solution with the strong oxidant under room temperature and stirring for 12-48 hours, adjusting the pH after the reaction, adding ethanol to precipitate a product, washing the product with a washing solution, dissolving the product in water, encapsulating the product in a dialysis bag, and dialyzing and purifying the product with deionized water for 2-6 days, and freeze-drying the purified product to obtain polysaccharide-polyaniline;

[0007] Step B: dissolving dextran in a second solvent, wherein the mass concentration of the dissolved dextran is 5%-10%, and then adding a strong oxidant to the dextran solution to form a second mixed solution, wherein the mass concentration of the strong oxidant in the second mixed solution is 10%-20%. The second mixed solution is placed in a heated water bath, reacted for 1-12 hours, and after the reaction is completed, the product is packaged in a dialysis bag, dialyzed and purified with deionized water for 2-6 days, and the purified product is freeze-dried to obtain oxidized dextran.

[0008] Step C: dissolving the polysaccharide-polyaniline in a third solvent to obtain a polysaccharide-polyaniline solution with a mass concentration of 5%-10%; dissolving the oxidized dextran in a third solvent to obtain an oxidized dextran solution with a mass concentration of 5%-10%; and uniformly mixing the polysaccharide-polyaniline solution and the oxidized dextran solution in a certain proportion to obtain a polysaccharide-based gastrointestinal tissue marker hydrogel.

[0009] Furthermore, in step A, the polysaccharide is any one of chitosan, carboxymethyl chitosan, hyaluronic acid and sodium alginate.

[0010] Furthermore, in step A, the first solvent is hydrochloric acid, and the concentration of the hydrochloric acid is 0.1-1.0M, specifically any one of 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, and 1.0M.

[0011] Furthermore, in step A, the strong oxidant is any one of ammonium persulfate, nitric acid, peracetic acid and hydrogen peroxide.

[0012] Furthermore, in step A, the pH value is 6.5-8, specifically 6.5, 7, 7.5 or 8, etc.

[0013] Furthermore, the washing liquid in step A is any one of ethanol, dimethyl sulfoxide, and N-methylpyrrolidone.

[0014] Furthermore, in step B, the molecular weight of dextran is any one of 40,000, 70,000, 100,000, 150,000, 250,000, and 500,000.

[0015] Furthermore, in step B, the second solvent is any one of deionized water, phosphate buffer solution and physiological saline, the pH of the phosphate buffer solution is 7.4, and the mass concentration of the physiological saline is 0.9%.

[0016] Furthermore, in step B, the strong oxidant is any one of sodium periodate, potassium permanganate, sodium hypochlorite and potassium dichromate.

[0017] Furthermore, the reaction temperature of the glucan and the strong oxidant in step B is 40-80°C, specifically any one of 40°C, 50°C, 60°C, 70°C, and 80°C.

[0018] Furthermore, in step C, the third solvent is any one of deionized water, phosphate buffer solution and physiological saline, the pH of the phosphate buffer solution is 7.4, and the mass concentration of the physiological saline is 0.9%.

[0019] Furthermore, in step C, the mixing ratio of the polysaccharide-polyaniline solution and the oxidized dextran solution is 1:1 to 10:1, specifically any one of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1.

[0020] The present invention firstly adds aniline hydrochloride and a strong oxidant to a polysaccharide such as chitosan solution to polymerize aniline while grafting polyaniline to the polysaccharide molecular chain through non-covalent bonds, thereby obtaining polysaccharide-polyaniline. Then, a strong oxidant is added to a dextran solution to oxidize the aldehyde groups on the dextran molecular chain to carboxyl groups to obtain oxidized dextran. Subsequently, the obtained polyaniline-polysaccharide solution is mixed with the oxidized dextran solution to obtain a polysaccharide-based gastrointestinal tissue marker hydrogel through covalent bonding.

[0021] The invention provides a method for preparing a polysaccharide-based gastrointestinal tissue marking hydrogel by utilizing the property of Schiff base reaction between polysaccharide-polyaniline and oxidized dextran.

[0022] The present invention provides a polysaccharide-based gastrointestinal tissue marking hydrogel. The preparation process of the present invention is simple, the product is easy to obtain, and the obtained polysaccharide-based gastrointestinal tissue marking hydrogel shows good biocompatibility in in vitro and in vivo simulation experiments. Different from the existing marking liquid, the present invention selects natural high molecular polysaccharide and non-toxic, extremely stable high molecular polymer for grafting, and then cross-links with natural high molecular polysaccharide through chemical bonds to form a hydrogel-like dye. It is worth mentioning that the hydrogel-like dye of the present invention has physical properties between liquid and solid, combines the injectability of liquid with the non-flowability of solid, solves the problem of easy precipitation and leakage of liquid marking liquid, and greatly improves the utilization rate of materials.

[0023] Compared with the prior art, the present invention has significant technical progress. The preparation process of the present invention is simple, the product is easily available, and it has excellent in vivo lesion marking ability, which can make up for the shortcomings of the existing marking materials such as low utilization efficiency and high toxicity. It is expected to be applied to the fields of safe and efficient accurate positioning of gastrointestinal lesions or specific parts, and has certain clinical application value.

[0024] The present invention provides the above application for accurate positioning of gastrointestinal lesions or specific parts. The polysaccharide-based gastrointestinal marker hydrogel prepared by the present invention has excellent stability and good biocompatibility, and can be used for safe and efficient accurate positioning of gastrointestinal lesions or specific parts.

[0025] The present invention also provides an application of ablating cancerous changes in the gastrointestinal tract through photothermal therapy. The polysaccharide-based gastrointestinal tract marking hydrogel prepared by the present invention has the application of safe and efficient accurate positioning of lesions or specific parts in the gastrointestinal tract. In addition, the present invention also has excellent photothermal performance, and can achieve the treatment of cancerous tissues through photothermal therapy while marking. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 a is a photo of CMCS-PANI prepared in Example 2.

[0027] Figure 1 b is a photograph of the CMCS-PANI / OD hydrogel prepared in Example 10.

[0028] Figure 1 c is a morphological photograph of the CMCS-PANI / OD hydrogel prepared in Example 15.

[0029] Figure 2 a is the FESEM image of CMCS-PANI prepared in Example 2.

[0030] Figure 2 b is the FESEM image of the CMCS / OD hydrogel prepared in Example 15.

[0031] Figure 2 c is the FESEM image of the CMCS-PANI / OD hydrogel prepared in Example 10.

[0032] Figure 3 This is a macroscopic image of the injectability of the CMCS-PANI / OD hydrogel prepared in Example 10.

[0033] Figure 4 a is the infrared spectra of carboxymethyl chitosan (and CMCS) and CMCS-PANI and CMCS-PANI / OD hydrogels prepared in Examples 2 and 9.

[0034] Figure 4 b is the thermogravimetric test spectra of aniline hydrochloride (i.e. ANI), carboxymethyl chitosan (i.e. CMCS) and CMCS-PANI prepared in Example 2.

[0035] Figure 5 These are the data results of the simulated in vitro degradation of the CMCS-PANI / OD hydrogel prepared in Example 10 in physiological saline, simulated gastric fluid, simulated intestinal fluid, deionized water and phosphate buffer solution (i.e., PBS).

[0036] Figure 6 a is the hemolysis test result of the CMCS-PANI / OD hydrogel prepared in Example 10.

[0037] Figure 6 b is the cell survival rate after co-culture of the CMCS-PANI / OD hydrogel prepared in Example 10 with two fibroblasts (HBE and Beas-2B).

[0038] Figure 7 Dead / Live staining results of CMCS-PANI / OD hydrogel prepared in Example 10 and fibroblasts.

[0039] Figure 8 This is a graph showing the results of labeling the CMCS-PANI / OD hydrogel prepared in Example 10 under the gastric mucosa of Kunming rats for 1, 30, 60, and 120 days.

[0040] Fig. 9 This is the H&E staining result of the biosafety of the CMCS-PANI / OD hydrogel prepared in Example 10 labeled under the gastric mucosa of Kunming rats for 0, 1, 30, 60, and 120 days.

[0041] Fig.10 For different materials at 1w / cm 2Thermal imaging images of deionized water, CMCS, and CMCS-PANI, OD, CMCS-PANI / OD hydrogel, and CMCS / OD hydrogel prepared in Examples 2, 5, 10, and 15 after irradiation with infrared laser of high power density for 5 min.

[0042] Fig.11 The thermal imaging images of the CMCS-PANI / OD hydrogel prepared in Example 10 irradiated with infrared laser at different power densities for 5 min, i.e., 0.5 W / cm 2 , 0.8W / cm 2 , 1W / cm 2 .

[0043] Fig.12 a is different materials at 1W / cm 2 Temperature variation data diagram of infrared laser irradiation with high power density for 5 min, i.e., deionized water, CMCS, and CMCS-PANI, OD, CMCS-PANI / OD hydrogel and CMCS / OD hydrogel prepared in Examples 2, 5, 10 and 15.

[0044] Fig.12 b is the temperature variation data of the CMCS-PANI / OD hydrogel prepared in Example 10 under infrared laser irradiation for 5 min at different power densities, i.e., 0.5 W / cm 2 , 0.8W / cm 2 , 1W / cm 2 .

[0045] Fig.12 c is the CMCS-PANI / OD hydrogel prepared in Example 10 at 1 W / cm 2 Graph of the infrared laser irradiation cycle as a function of power density.

[0046] Fig.12 d is the CMCS-PANI / OD hydrogel prepared in Example 10 at a power density of 1 W / cm 2 The photothermal efficiency results are shown in Figure 2. DETAILED DESCRIPTION

[0047] Example 1

[0048] Chitosan is dissolved in 0.2M hydrochloric acid and stirred to completely dissolve to form a solution, wherein the mass concentration of the dissolved chitosan is 5%, and then aniline hydrochloride is added to the chitosan solution to form a mixed solution, wherein the mass concentration of the aniline hydrochloride in the mixed solution is 2.5%, and then ammonium persulfate is added to the mixed solution, wherein the mass concentration of the dissolved ammonium persulfate is 0.25%, and the mixed solution with the added ammonium persulfate is stirred at room temperature for 12 hours. After the reaction is completed, the pH is adjusted to 7.5, and ethanol is added to precipitate the product, and the product is washed with dimethyl sulfoxide, and then the product is dissolved in water and encapsulated in a 14000Da dialysis bag, and dialyzed and purified with deionized water for 3 days, and the purified product is freeze-dried to obtain chitosan-polyaniline, i.e., CS-PANI.

[0049] Example 2

[0050] Dissolve carboxymethyl chitosan in 0.5M hydrochloric acid and stir to completely dissolve it to form a solution. The mass concentration of the dissolved carboxymethyl chitosan is 2.5%. Then add aniline hydrochloride to the carboxymethyl chitosan solution to form a mixed solution. The mass concentration of aniline hydrochloride in the mixed solution is 2.5%. Then add ammonium persulfate to the mixed solution. The mass concentration of the dissolved ammonium persulfate is 0.05%-0.5%. The mixed solution with ammonium persulfate is stirred at room temperature for 24 hours. After the reaction is completed, adjust the pH to 7, add ethanol to precipitate the product, wash the product with N-methylpyrrolidone, dissolve the product in water, encapsulate it in a 14000Da dialysis bag, and dialyze and purify it with deionized water for 3 days. The purified product is freeze-dried to obtain carboxymethyl chitosan-polyaniline, i.e., CMCS-PANI.

[0051] Example 3

[0052] Dissolve hyaluronic acid in 0.3M hydrochloric acid and stir to completely dissolve it to form a solution. The mass concentration of the dissolved hyaluronic acid is 1%. Then add aniline hydrochloride to the hyaluronic acid solution to form a mixed solution. The mass concentration of aniline hydrochloride in the mixed solution is 5%. Then add nitric acid to the mixed solution. The mass concentration of the dissolved nitric acid is 0.05%. The mixed solution with nitric acid is stirred at room temperature for 48 hours. After the reaction is completed, adjust the pH to 8, add ethanol to precipitate the product, wash the product with ethanol, dissolve the product in water and encapsulate it in a 14000Da dialysis bag, and dialyze and purify it with deionized water for 5 days. The purified product is freeze-dried to obtain hyaluronic acid-polyaniline, i.e., HA-PANI.

[0053] Example 4

[0054] Sodium alginate was dissolved in 0.7M hydrochloric acid and stirred to completely dissolve to form a solution, wherein the mass concentration of the dissolved sodium alginate was 1%. Aniline hydrochloride was then added to the sodium alginate solution to form a mixed solution, wherein the mass concentration of the aniline hydrochloride in the mixed solution was 5%. Peracetic acid was then added to the mixed solution, wherein the mass concentration of the dissolved peracetic acid was 0.5%. The mixed solution to which the peracetic acid was added was stirred at room temperature for 24 hours. After the reaction was completed, the pH was adjusted to 7.5, and ethanol was added to precipitate the product. The product was washed with N-methylpyrrolidone, dissolved in water and encapsulated in a 14000Da dialysis bag, and dialyzed and purified with deionized water for 3 days. The purified product was freeze-dried to obtain sodium alginate-polyaniline, i.e., SA-PANI.

[0055] Example 5

[0056] Dextran with a molecular weight of 100,000 was dissolved in deionized water, and the mass concentration of dextran after dissolution was 5%. Then sodium periodate was added to the dextran solution to form a mixed solution, and the mass concentration of sodium periodate in the mixed solution was 10%. The mixed solution was placed in a 60°C water bath and reacted for 2 hours. After the reaction, the product was packaged in a 14,000Da dialysis bag, dialyzed and purified with deionized water for 2 days, and the purified product was freeze-dried to obtain oxidized dextran, namely OD.

[0057] Example 6

[0058] Dextran with a molecular weight of 70,000 was dissolved in a phosphate buffer solution, and the mass concentration of dextran after dissolution was 10%. Potassium permanganate was then added to the dextran solution to form a mixed solution, and the mass concentration of potassium permanganate in the mixed solution was 15%. The mixed solution was placed in a 40°C water bath and reacted for 6 hours. After the reaction, the product was packaged in a 14,000Da dialysis bag, dialyzed and purified with deionized water for 3 days, and the purified product was freeze-dried to obtain oxidized dextran, namely OD-1.

[0059] Example 7

[0060] Dextran with a molecular weight of 150,000 was dissolved in a phosphate buffer solution, and the mass concentration of dextran after dissolution was 7%. Then sodium hypochlorite was added to the dextran solution to form a mixed solution, and the mass concentration of sodium hypochlorite in the mixed solution was 12%. The mixed solution was placed in a 50°C water bath and reacted for 12 hours. After the reaction, the product was packaged in a 14,000Da dialysis bag, dialyzed and purified with deionized water for 5 days, and the purified product was freeze-dried to obtain oxidized dextran, namely OD-2.

[0061] Example 8

[0062] Dextran with a molecular weight of 250,000 was dissolved in physiological saline, and the mass concentration of dextran after dissolution was 6%. Potassium dichromate was then added to the dextran solution to form a mixed solution, and the mass concentration of potassium dichromate in the mixed solution was 18%. The mixed solution was placed in a 60°C water bath and reacted for 12 hours. After the reaction, the product was packaged in a 14,000Da dialysis bag, dialyzed and purified with deionized water for 5 days, and the purified product was freeze-dried to obtain oxidized dextran, namely OD-3.

[0063] Example 9

[0064] Dextran with a molecular weight of 500,000 was dissolved in physiological saline, and the mass concentration of dextran after dissolution was 5%. Then sodium periodate was added to the dextran solution to form a mixed solution, and the mass concentration of sodium periodate in the mixed solution was 20%. The mixed solution was placed in a water bath at 80°C and reacted for 12 hours. After the reaction, the product was packaged in a 14000Da dialysis bag, dialyzed and purified with deionized water for 5 days, and the purified product was freeze-dried to obtain oxidized dextran, namely OD-4.

[0065] Example 10

[0066] The product prepared in Example 2 was dissolved in deionized water to obtain a CMCS-PANI solution with a mass concentration of 5%. The product prepared in Example 5 was dissolved in a solvent to obtain an OD solution with a mass concentration of 5%. The above two solutions were mixed in a ratio of 2:1 to obtain a gastrointestinal tissue labeling hydrogel, i.e., CMCS-PANI / OD hydrogel.

[0067] Embodiment 11

[0068] The product prepared in Example 2 was dissolved in deionized water to obtain a CMCS-PANI solution with a mass concentration of 5%. The product prepared in Example 5 was dissolved in a solvent to obtain an OD solution with a mass concentration of 6%. The above two solutions were mixed in a ratio of 3:1 to obtain a gastrointestinal tissue labeling hydrogel, i.e., CMCS-PANI / OD-1 hydrogel.

[0069] Example 12

[0070] The product prepared in Example 2 was dissolved in deionized water to obtain a CMCS-PANI solution with a mass concentration of 5%. The product prepared in Example 5 was dissolved in a solvent to obtain an OD solution with a mass concentration of 8%. The above two solutions were mixed in a ratio of 6:1 to obtain a gastrointestinal tissue labeling hydrogel, i.e., CMCS-PANI / OD-2 hydrogel.

[0071] Example 13

[0072] The product prepared in Example 2 was dissolved in deionized water to obtain a CMCS-PANI solution with a mass concentration of 5%. The product prepared in Example 5 was dissolved in a solvent to obtain an OD solution with a mass concentration of 10%. The above two solutions were mixed in a ratio of 10:1 to obtain a gastrointestinal tissue labeling hydrogel, i.e., CMCS-PANI / OD-3 hydrogel.

[0073] Embodiment 14

[0074] The product prepared in Example 2 was dissolved in deionized water to obtain a CMCS-PANI solution with a mass concentration of 10%. The product prepared in Example 5 was dissolved in a solvent to obtain an OD solution with a mass concentration of 5%. The above two solutions were mixed in a ratio of 1:1 to obtain a gastrointestinal tissue labeling hydrogel, i.e., CMCS-PANI / OD-4 hydrogel.

[0075] Embodiment 15

[0076] Carboxymethyl chitosan was dissolved in a solvent to obtain a CMCS solution with a mass concentration of 5%. The product prepared in Example 2 was dissolved in a solvent to obtain an OD solution with a mass concentration of 5%. The above two solutions were mixed in a ratio of 2:1 to obtain a CMCS / OD hydrogel.

[0077] Example 16

[0078] The product of Example 2 was dissolved in deionized water to obtain a CMCS-PANI solution with a concentration of 5% (w / v). The CMCS-PANI solution and the CMCS-PANI / OD hydrogel prepared in Example 9 were respectively placed in 20 mL glass bottles, and the glass bottles were tilted to take photos.

[0079] Depend on Figure 1 a and Figure 1 b It can be seen that CMCS-PANI is a liquid, while CMCS-PANI / OD is a hydrogel between liquid and solid. Figure 1 As can be seen from the macroscopic morphology of c, the gastrointestinal tissue labeling hydrogel (i.e., CMCS-PANI / OD hydrogel) prepared by the present invention has excellent plasticity.

[0080] Embodiment 17

[0081] After the CMCS-PANI, CMCS-PANI / OD and CMCS / OD prepared in Examples 2, 9 and 13 were dried, FESEM images were taken.

[0082] according to Figure 2 a, it can be seen that CMCS-PANI is a non-network material. Figure 2 b and Figure 2CMCS / OD and CMCS-PANI / OD in c have a hydrogel network structure. Figure 2 In c, filamentous substances can be observed connected to the network structure, indicating that polyaniline (PANI) was successfully grafted onto carboxymethyl chitosan (CMCS).

[0083] Embodiment 18

[0084] To explore the injectability of the hydrogel, the CMCS-PANI / OD hydrogel prepared in Example 9 was loaded into a syringe and injected. Figure 3 ad) The present invention has excellent injectability and can achieve in vivo gastroscopic injection.

[0085] Embodiment 19

[0086] Dried carboxymethyl chitosan (CMCS) and CMCS-PANI and CMCS-PANI / OD prepared in Examples 2 and 9 were subjected to infrared characterization tests. According to the infrared characterization results ( Figure 4 a) It can be seen that CMCS-PANI grafted with PANI has a wavelength of 1000 cm -1 There is a strong peak on the left and right, which is the stretching vibration peak of the benzene ring in PANI, which once again verifies that PANI is successfully grafted onto CMCS.

[0087] Embodiment 20

[0088] Dried aniline hydrochloride (ie, ANI), carboxymethyl chitosan (ie, CMCS), and CMCS-PANI prepared in Example 2 were subjected to thermogravimetric testing.

[0089] According to the thermogravimetric test results ( Figure 4 b) It can be calculated that the grafting rate of PANI in CMCS-PANI is about 12%.

[0090] Embodiment 21

[0091] To investigate the in vitro degradation ability of the hydrogel, the freeze-dried CMCS-PANI / OD hydrogel was immersed in 20 mL of physiological saline, simulated gastric fluid, simulated intestinal fluid, deionized water, and phosphate buffer solution (PBS) (n=3). The hydrogel samples were taken out at fixed time intervals, washed with deionized water to remove impurities, and then freeze-dried and weighed to draw the corresponding in vitro degradation curve.

[0092] According to the degradation test results ( Figure 5) It can be seen that the CMCS-PANI / OD hydrogel is almost not degraded in physiological saline and PBS, degraded by about 50% in simulated intestinal fluid and deionized water, and degraded by about 80% in simulated gastric fluid within 2 hours. According to the degradation experimental results, it can be concluded that the CMCS-PANI / OD hydrogel has controllable degradability. When the labeled hydrogel is injected into the stomach or intestine, it is rapidly degraded within 2 hours under the action of gastric juice or intestinal juice. However, it is almost not degraded in PBS and physiological saline, so as to achieve the stability of long-term labeling of CMCS-PANI / OD hydrogel in vivo.

[0093] Embodiment 22

[0094] In order to explore the blood compatibility of gastrointestinal tissue-labeled hydrogel. 2mL of whole blood was centrifuged (5000rpm, 5 minutes) and rinsed three times with phosphate buffered solution (i.e., PBS) to obtain red blood cells. The obtained red blood cells were stored in 50mL of phosphate buffered solution for further use. In the hemolysis test, 0.5mL of the above mouse red blood cells were placed in a 5.0mL centrifuge tube and incubated in a 37°C water bath for 2 hours with 2.5mL PBS solution (negative control), 2.5mL deionized water (positive control) and different concentrations of the tissue-labeled hydrogel prepared in Example 3 (5, 10, 15mg / mL, in 2.5mL PBS solution). The absorbance of the supernatant at 541nm was collected, and the hemolysis rate of the red blood cells was calculated.

[0095] like Figure 6 As shown in a, the calculated hemolysis rates of the tissue-labeled hydrogels were all less than 5%. From the supernatant photos, it can be seen that the supernatant of red blood cells incubated with CMCS-PANI / OD hydrogel and PBS solution is transparent. However, the blood treated with deionized water showed a distinct red color due to positive hemolysis. The results show that CMCS-PANI / OD hydrogel has good blood compatibility.

[0096] Embodiment 23

[0097] In order to evaluate the in vitro cell compatibility of gastrointestinal tissue marker hydrogel as a biomedical material, the CMCS-PANI / OD hydrogel in Example 9 was measured by CCK-8 test kit and Dead / Live cell staining to determine the influence of cell morphology and cell viability. The hydrogel was soaked in a serum-containing culture medium and incubated for a period of time to obtain different concentrations of leaching solution (i.e. 0, 5, 10, 15 mg / mL). Cells were seeded in 96-well plates at a certain density and cultured with hydrogel leaching solution. After 24 h, PBS solution was used to gently wash the cell culture plate 3 times and quantitatively measure cell viability. After cell staining, an inverted fluorescence microscope was used to observe cell morphology and assess cell viability.

[0098] Depend on Figure 6 b It can be seen that both cells co-cultured with the hydrogel extract have good cell survival rates. Figure 7 It can be seen that the cells treated by the hydrogel extract have normal morphology, and most cells are stained green (live cells), while red cells (dead cells) are not counted. In summary, the above results show that CMCS-PANI / OD hydrogel has good cell compatibility.

[0099] Embodiment 24

[0100] The CMCS-PANI / OD hydrogel obtained in Example 9 was injected into the subgastric mucosa of Kunming rats. The subgastric mucosa of Kunming rats was observed on days 0, 1, 30, 60, and 120. The heart, liver, spleen, lung, and kidney were obtained for H&E staining observation.

[0101] like Figure 8 As shown in the figure, after 1, 30, 60, and 120 days of labeling, the hydrogel can still be clearly observed, which confirms that the CMCS-PANI / OD tissue labeling hydrogel has excellent stability. Fig. 9 As shown, CMCS-PANI / OD hydrogel did not have any effect on the important organs of mice after 1, 30, 60, and 120 days of labeling in vivo, indicating that CMCS-PANI / OD hydrogel has good in vivo safety.

[0102] Embodiment 25

[0103] The photothermal conversion performance of CMCS-PANI / OD hydrogel was evaluated using near-infrared light at 808 nm. 2 The infrared laser was used to irradiate different materials (deionized water, CMCS, and CMCS-PANI, OD, CMCS-PANI / OD, and CMCS / OD prepared in Examples 1, 2, 3, and 4) to explore whether the CMCS-PANI / OD hydrogel has photothermal conversion performance. 2 , 0.5W / cm 2 and 0.8W / cm 2 ) infrared light was used to irradiate CMCS-PANI / OD hydrogel to explore the effect of infrared laser power density on the photothermal conversion rate of hydrogel. The temperature change image within 5 minutes was recorded using a FLIR E60 near-infrared thermal imager. When studying the photothermal stability of CMCS-PANI / OD hydrogel, we used an 808nm infrared laser (power density: W / cm 2) was used to irradiate the CMCS-PANI / OD hydrogel. The laser was turned on for 5 minutes, then turned off for 5 minutes, and this step was repeated 10 times. In the study of photothermal conversion efficiency, an 808 nm infrared laser (power density: 1 W / cm 2 ) irradiated 1 mg / mL CMCS-PANI / OD hydrogel for 10 min, and then the photothermal conversion efficiency of CMCS-PANI / OD hydrogel was calculated using the relevant formula.

[0104] like Fig.10 and Fig.12 As shown in a, CMCS-PANI and CMCS-PANI / OD grafted with PANI have photothermal conversion capabilities, which is due to the ability of PANI to convert light energy into thermal energy. Fig.11 and Fig.12 As shown in (b), the photothermal conversion capacity of CMCS-PANI / OD hydrogel increases with the increase of power density. Fig.12 As shown in Figure c, under 5 cycles of laser switching, the temperature difference of CMCS-PANI / OD hydrogel is always maintained at about 50°C, proving that the tissue labeling hydrogel has good photothermal stability. In addition, photothermal conversion efficiency is another important criterion for measuring photothermal materials, and a higher photothermal conversion efficiency is the key to cancerous tissue treatment. Therefore, the photothermal conversion efficiency of CMCS-PANI / OD hydrogel is calculated to be 74.5% ( Fig.12 d), which proves that CMCS-PANI / OD hydrogel has excellent photothermal conversion ability and has broad application prospects in the photothermal treatment platform of cancerous tissues.

[0105] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all belong to the protection scope of the invention.

Claims

1. A method for preparing a polysaccharide-based gastrointestinal tissue marker hydrogel, characterized in that: include, Step A: dissolving a polysaccharide in a first solvent, stirring to completely dissolve the polysaccharide to form a polysaccharide solution, wherein the mass concentration of the polysaccharide after dissolution is 0.5%-5%, then adding aniline hydrochloride to the polysaccharide solution to form a first mixed solution, wherein the mass concentration of aniline hydrochloride in the first mixed solution is 0.5%-5%; then adding a strong oxidant to the first mixed solution, wherein the mass concentration of the strong oxidant after dissolution is 0.05%-0.5%; then placing the first mixed solution with the strong oxidant under room temperature and stirring for 12-48 hours, adjusting the pH after the reaction, adding ethanol to precipitate the product, washing the product with a washing solution, dissolving the product in water and encapsulating it in a dialysis bag, and dialysis and purification with deionized water for 2-6 days, and freeze-drying the purified product to obtain polysaccharide-polyaniline; Step B: dissolving dextran in a second solvent, wherein the mass concentration of the dissolved dextran is 5%-10%, and then adding a strong oxidant to the dextran solution to form a second mixed solution, wherein the mass concentration of the strong oxidant in the second mixed solution is 10%-20%. The second mixed solution is placed in a heated water bath, reacted for 1-12 hours, and after the reaction is completed, the product is packaged in a dialysis bag, dialyzed and purified with deionized water for 2-6 days, and the purified product is freeze-dried to obtain oxidized dextran; Step C: dissolving the polysaccharide-polyaniline in a third solvent to obtain a polysaccharide-polyaniline solution with a mass concentration of 5%-10%; dissolving the oxidized dextran in a third solvent to obtain an oxidized dextran solution with a mass concentration of 5%-10%; and uniformly mixing the polysaccharide-polyaniline solution and the oxidized dextran solution in a certain proportion to obtain a polysaccharide-based gastrointestinal tissue marker hydrogel.

2. The method for preparing a polysaccharide-based gastrointestinal tissue marker hydrogel according to claim 1, characterized in that: The polysaccharide in step A is any one of chitosan, carboxymethyl chitosan, hyaluronic acid and sodium alginate.

3. The method for preparing a polysaccharide-based gastrointestinal tissue marker hydrogel according to claim 1, characterized in that: In step A, the first solvent is hydrochloric acid, and the concentration of the hydrochloric acid is 0.1-1.0M.

4. The method for preparing a polysaccharide-based gastrointestinal tissue marker hydrogel according to claim 1, characterized in that: In step A, the strong oxidant is any one of ammonium persulfate, nitric acid, peracetic acid, and hydrogen peroxide; The pH value in step A is 6.5-8; The washing liquid in step A is any one of ethanol, dimethyl sulfoxide and N-methylpyrrolidone.

5. The method for preparing a polysaccharide-based gastrointestinal tissue marker hydrogel according to claim 1, characterized in that: In step B, the molecular weight of the dextran is any one of 40,000, 70,000, 100,000, 150,000, 250,000, and 500,000.

6. The method for preparing a polysaccharide-based gastrointestinal tissue marker hydrogel according to claim 1, characterized in that: The second solvent in step B is any one of deionized water, phosphate buffer solution and normal saline. The third solvent in step C is any one of deionized water, phosphate buffer solution and normal saline. The pH of the phosphate buffer solution is 7.4, and the mass concentration of the normal saline is 0.9%.

7. The method for preparing a polysaccharide-based gastrointestinal tissue marker hydrogel according to claim 1, characterized in that: In step B, the strong oxidant is any one of sodium periodate, potassium permanganate, sodium hypochlorite and potassium dichromate; The reaction temperature of dextran and strong oxidant is 40-80°C.

8. The method for preparing a polysaccharide-based gastrointestinal tissue marker hydrogel according to claim 1, characterized in that: In step C, the mixing ratio of the polysaccharide-polyaniline and the oxidized dextran solution is 1:1 to 10:

1.

9. The polysaccharide-based gastrointestinal tissue marker hydrogel obtained according to the preparation method according to any one of claims 1 to 8.

10. Use of the polysaccharide-based gastrointestinal tissue marker hydrogel according to claim 9 in the preparation of tissue marker materials.

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