Intestinal micro-needle anastomosis stent with antibacterial function and preparation method and application thereof

Abstract

The application discloses an intestinal micro-needle anastomosis stent with an antibacterial function and a preparation method and application thereof, which can effectively physically isolate an anastomotic stoma from intestinal contents, provides a relatively clean local environment for healing of the anastomotic stoma, and further reduces tension at the anastomotic stoma by fixing the micro-needle anastomosis stent at the anastomotic stoma, so that anastomotic leakage caused by excessive tension is avoided; a micro-needle layer on a surface of the stent can realize in-situ long-acting slow release of berberine hydrochloride (BBH) on a superficial layer of the anastomotic stoma, so that drug utilization of the BBH is improved, and occurrence of initial inflammation of the anastomotic stoma is effectively reduced, and healing of the anastomotic stoma is better regulated. Animal experiment results prove that implantation of the micro-needle anastomosis stent can effectively reduce inflammatory cell infiltration of local tissues, reduce anastomotic arrangement, and regulate smooth healing of the anastomotic stoma.

Description

Intestinal microneedle anastomosis scaffold with antibacterial function, its preparation method and application Technical Field

[0001] This invention relates to the field of flexible anastomotic stent technology for the digestive tract, specifically to an intestinal microneedle anastomotic stent with antibacterial function, its preparation method, and its application. Background Technology

[0002] Despite continuous improvements in surgical techniques, anastomotic leakage (AL) remains an unresolved postoperative complication in colorectal cancer surgery, with an incidence rate as high as 30%. AL is a serious clinical syndrome caused by poor anastomotic healing, leading to leakage of intestinal contents into the abdominal cavity. In severe cases, it can progress to abdominal infection, organ infection, and sepsis, ultimately resulting in patient death. This also exacerbates doctor-patient conflicts and causes adverse social impacts.

[0003] Intestinal anastomotic healing typically occurs in three phases: the inflammatory phase, the fibrotic proliferative phase, and the remodeling phase. Factors influencing anastomotic healing are complex, with high local tension, insufficient blood supply, and postoperative infection being the main causes of difficulty in healing. Intestinal anastomotic healing is a complex cell-mediated process aimed at restoring intestinal wall continuity. The early stages of anastomotic healing are most susceptible to various stimuli, potentially due to early anastomotic incomplete closure. Effectively preventing anastomotic leakage is a critical scientific issue that urgently needs to be addressed. Studies have reported that implanting rigid or flexible stents at the anastomosis site to effectively physically isolate the digestive tract anastomosis from intestinal contents creates a favorable local healing environment, which is an effective measure to reduce the incidence of anastomotic leakage. However, how to reduce anastomotic inflammation and promote anastomotic healing through local drug administration remains crucial for ensuring successful postoperative anastomotic healing.

[0004] During the inflammatory phase, an ideal flexible scaffold must exhibit effective antibacterial and anti-inflammatory properties. However, previous studies have shown that drug delivery on flexible scaffolds is only done epidermally. Due to the complex physiological environment of the gastrointestinal tract, the utilization rate of drug delivery on scaffolds is greatly reduced. Therefore, it is essential to develop a biodegradable flexible anastomotic scaffold that can deliver drugs in situ. Summary of the Invention

[0005] To address the technical deficiencies of existing technologies, this invention provides an intestinal microneedle anastomosis scaffold with antibacterial function, its preparation method, and its application. On the one hand, it isolates the wound and contents, providing a relatively clean healing environment for the intestinal anastomosis and reducing bacterial infection in the early stages of healing. On the other hand, the release of BBH from the drug-loaded microneedles plays an antibacterial and anti-inflammatory role in the early stages of inflammation.

[0006] The technical solution adopted in this invention is: an intestinal microneedle anastomosis scaffold with antibacterial function. The intestinal microneedle anastomosis scaffold is composed of a flexible scaffold base layer and a drug-loaded microneedle outer layer organically combined. The flexible scaffold base layer is obtained by blending PTMC-b-PEG-b-PTMC triblock copolymer with silk fibroin (SF). The drug-loaded microneedles are obtained by mixing tyramine-modified hyaluronic acid (HA-Tyr) and SF, adding berberine hydrochloride (BBH), and then casting. The organic combination of the flexible scaffold base layer and the drug-loaded microneedles is achieved by using the SF contained in both, and cross-linking the dityrosine on the SF with ultraviolet light to modify the microneedles on the outer surface of the scaffold, thus obtaining an anastomosis scaffold with an antibacterial and anti-inflammatory microneedle layer.

[0007] In the blending of PTMC-b-PEG-b-PTMC triblock copolymer and silk fibroin (SF), the mass ratio of PTMC-b-PEG-b-PTMC triblock copolymer to silk fibroin (SF) is 70 / 30.

[0008] The PEG content in the PTMC-b-PEG-b-PTMC triblock copolymer is 0.3 wt%.

[0009] The degree of substitution of the tyramine-modified hyaluronic acid (HA-Tyr) is 31%.

[0010] The mass ratio of SF to tyramine-modified hyaluronic acid (HA-Tyr) in the drug-loaded microneedles is 80 / 20.

[0011] The microneedle taper of the drug-loaded microneedle is 30-35°.

[0012] The distance between the microneedle tips in the drug-loaded microneedles is 1500 μm.

[0013] The content of berberine hydrochloride (BBH) in the drug-loaded microneedles is 750-1000 μg / mL.

[0014] A method for preparing an intestinal microneedle anastomosis scaffold with antibacterial function includes the following steps:

[0015] (1) Synthesis of PTMC-b-PEG-b-PTMC: In an anhydrous and oxygen-free glove box, a certain proportion of PEG and TMC monomers were weighed, and 0.05wt% of catalyst Sn(Oct)2 was added. A magnetic flask was placed in the glove box and the mouth of the reaction flask was sealed with vacuum silicone grease. Finally, the mouth of the reaction flask was sealed with a sealing film. The sealed reaction flask was taken out of the glove box and placed in an oil bath at 130~150 ℃ for 36~48 h. After the reaction was completed, the reaction flask was taken out and cooled to room temperature. Then, trichloroethane solvent was added to dissolve the reaction product. After complete dissolution, the solution was taken out and added dropwise to n-hexane to precipitate the product. This was repeated 3 times. Finally, the purified polymer product was dried to obtain PTMC-b-PEG-b-PTMC.

[0016] (2) Electrospinning of PTMC-b-PEG-b-PTMC with silk fibroin (SF): Weigh a certain amount of PTMC-b-PEG-b-PTMC and add an appropriate proportion of SF. Mix the two and dissolve them in HFIP. Place the mixture on a shaker at 37 ℃ to fully dissolve the sample and prepare a uniform electrospinning solution with a concentration of 4%. Load the spinning solution into an electrospinning syringe, attach a 20 G metal needle to the syringe, and use a single nozzle to perform electrospinning to form a flexible scaffold base layer, which is then dried.

[0017] (3) Synthesis of HA-Tyr: Hyaluronic acid (HA) was weighed to prepare an aqueous solution with a concentration of 1 wt%. Then, a certain amount of EDC and NHS were added sequentially under stirring to activate the carboxyl group of HA. Tyramine hydrochloride (Tyr) was then dissolved in the activated aqueous solution of HA and left to stand overnight at room temperature. After the reaction was completed, the unreacted Tyr, EDC and NHS were dialyzed and filtered through a dialysis bag with a molecular weight cutoff of 3500 Da. The dialysis solution was then freeze-dried to obtain the product HA-Tyr.

[0018] (4) Preparation of SF / HA-Tyr microneedles: Add tris(2,2-bipyridine)ruthenium(II) hexahydrate Ru(II)bpy3 to deionized water. 2+Let this be solution A; add sodium persulfate (SPS) to deionized water, let this be solution B; mix equal amounts of solution A and solution B, let this be solution C; then, weigh out a certain amount of SF, HA-Tyr and berberine hydrochloride (BBH) and dissolve them in solution C to prepare an 8% (w / v) prepolymer solution; cast the prepolymer solution into a 10×10 array of PDMS microneedles and evacuate it in a vacuum degassing device with a vacuum degree of 0.1 MPa; then place the mold in a constant temperature and humidity chamber at a temperature of 25±2℃ and a humidity of 65±5% to dry and shape it. During this period, the prepolymer solution is added repeatedly to replenish the liquid, and the same conditions of evacuation and drying are repeated 3 to 4 times. After blue light curing, it is placed in a desiccator containing methanol for methanol vapor treatment.

[0019] (5) Preparation of microneedle anastomosis scaffold: When preparing microneedles, after repeated heating and concentration, the microneedles are directly treated with methanol vapor for 8 hours. The prepared flexible scaffold base layer is then attached to the surface of a microneedle mold containing SF / HA-Tyr solution. The microneedles are then irradiated with blue light for 20-30 minutes. Through the cross-linking effect of dityrosine in SF, which is contained in both microneedles, the microneedle anastomosis scaffold is finally prepared.

[0020] Application of an intestinal microneedle anastomosis scaffold in the preparation of materials for preventing intestinal anastomotic leakage and promoting anastomotic healing.

[0021] The beneficial effects of this invention are as follows: This invention provides an intestinal microneedle anastomosis scaffold with antibacterial function, its preparation method, and its application. It can effectively physically isolate the anastomosis from intestinal contents, providing a relatively clean local environment for anastomotic healing. By fixing the microneedle anastomosis scaffold at the anastomosis site, the tension at the anastomosis site can be further reduced, preventing anastomotic leakage caused by excessive tension. The microneedle layer on the scaffold surface can achieve in-situ long-acting sustained release of berberine hydrochloride (BBH) in the superficial layer of the anastomosis, which not only improves the drug utilization rate of BBH but also effectively reduces the occurrence of initial inflammation at the anastomosis site, regulating better anastomotic healing. Animal experimental results confirm that the implantation of the microneedle anastomosis scaffold can effectively reduce the infiltration of inflammatory cells in local tissues, reduce anastomotic sludge, and regulate smooth anastomotic healing. Figure description:

[0022] Figure 1 shows the synthesis of PTMC-b-PEG-b-PTMC: (A) Schematic diagram of the preparation of PTMC-b-PEG-b-PTMC; (B) Molecular weight and (C) Contact angle of PTMC-b-PEG-b-PTMC with different PEG addition amounts (0.05 wt%, 0.3 wt%, 0.6 wt%, 0.9 wt%, 1.2 wt%, 1.5 wt%).

[0023] Figure 2 shows the infrared spectra of PEG20000, PTMC, and PTMC-b-PEG-b-PTMC.

[0024] Figure 3 shows the NMR spectrum of PTMC-b-PEG-b-PTMC.

[0025] Figure 4 shows the chemical properties of PTMC-b-PEG-b-PTMC / SF: (A) Infrared spectra of PTMC-b-PEG-b-PTMC, SF, and PTMC-b-PEG-b-PTMC / SF; (B) Infrared spectra of the PTMC-b-PEG-b-PTMC / SF scaffold before and after methanol treatment.

[0026] Figure 5 shows the mechanical properties of the PTMC-b-PEG-b-PTMC / SF scaffold: (A) elongation at break in rat intestines of PTMC-b-PEG-b-PTMC / SF scaffolds with different ratios; (B) Young's modulus of PTMC-b-PEG-b-PTMC / SF scaffolds with different ratios; (C) PTMC-b-PEG-b-PTMC / SF scaffolds. 8020 and PTMC-b-PEG-b-PTMC / SF 5050 A stretched image of the actual object.

[0027] Figure 6 shows SEM images and diameter distributions of PTMC-b-PEG-b-PTMC / SF fiber membranes with different proportions.

[0028] Figure 7 shows the degradation rates of PTMC-b-PEG-b-PTMC / SF fiber membranes with different ratios.

[0029] Figure 8 shows the SEM images of the local degradation of PTMC-b-PEG-b-PTMC / SF scaffolds with different ratios at different time points.

[0030] Figure 9 shows the intestinal insertion experiments of SF microneedles and HA-Tyr / SF: (A) Local morphology of pure SF microneedles; (B) Pure SF microneedles inserted into the intestine; (C) Pure SF microneedles after insertion into the intestine; (D) Local morphology of HA-Tyr / SF microneedles; (E) HA-Tyr / SF microneedles inserted into the intestine; (F) HA-Tyr / SF microneedles after insertion into the intestine.

[0031] Figure 10 shows the synthesis scheme and NMR characterization of HA-Tyr.

[0032] Figure 11 shows the swelling and optical images of SF / HA-Tyr microneedles with different ratios. (A) Overall morphology of SF / HA-Tyr microneedles; (B) Local morphology of SF / HA-Tyr microneedles; (C) SF / HA-Tyr microneedles after swelling; (D) Swelling rate of SF / HA-Tyr microneedles with different ratios.

[0033] Figure 12 shows the mechanical properties of SF / HA-Tyr microneedles with different ratios: (A) SF 100 Compression curves and compression images of microneedles; (B) SF / HA-Tyr 9010 Compression curves and compression images of microneedles; (C) SF / HA-Tyr 8020 Compression curves and compression images of microneedles; (D) SF / HA-Tyr 7030 Compression curves and compression images of microneedles; (E) SF / HA-Tyr 6040 Compression curves and compression images of microneedles; (F) SF / HA-Tyr 5050 Compression curves and compressed images of microneedles.

[0034] Figure 13 shows the compressive force of a single needle in microneedles with different ratios of SF / HA-Tyr (100 / 0, 90 / 10, 80 / 20, 70 / 30, 60 / 40, 50 / 50).

[0035] Figure 14 shows SF / HA-Tyr with different tapers. 80 / 20 Microneedle insertion into the intestine: (A) Optical images of microneedles with different tapers (20°, 25°, 30°, 35°, 40°); (B) Compression curves of microneedles with different tapers (20°, 25°, 30°, 35°, 40°) and anastomosing scaffolds.

[0036] Figure 15 shows the SF / HA-Tyr needles with different tip pitches. 80 / 20 Microneedle insertion into the intestine: (A) Optical images of microneedles with different tip distances (1000μm, 1250μm, 1500μm, 1750μm, 2000μm); (B) Compression curves of microneedles anastomosing scaffolds with different tip distances (1000μm, 1250μm, 1500μm, 1750μm, 2000μm).

[0037] Figure 16 shows the in vitro antibacterial experiments of microneedle scaffolds with different BBH contents (500 μg / mL, 750 μg / mL, 1000 μg / mL): (A) Plate coating of microneedle scaffolds with different BBH contents; (B) Live and dead staining images of microneedle scaffolds with different BBH contents; (C) SEM images of microneedle scaffolds with different BBH contents after treatment; (D) Bacterial survival rate of microneedle scaffolds with different BBH contents after co-incubation with S. aureus; (E) Bacterial survival rate of microneedle scaffolds with different BBH contents after co-incubation with E. coli.

[0038] Figure 17 shows the hemolysis rate of microneedle scaffolds after blood compatibility testing and centrifugation: positive control, negative control, and different BBH addition amounts (500 μg / mL, 750 μg / mL, 1000 μg / mL).

[0039] Figure 18 shows the cell compatibility test: the cell viability of microneedle scaffolds with different BBH additions (500 μg / mL, 750 μg / mL, 1000 μg / mL) after co-culturing with L929 cells for 24 h.

[0040] Figure 19 shows the drug release experiment: (A) Standard curve of BBH; (B) Release curve of microneedles with 750 μg / mL BBH added in PBS environment.

[0041] Figure 20 shows PEG-PTMC / SF 7030 / BBH 750 @SF / HA-Tyr 8020 Compression curves and compression optical images of the support.

[0042] Figure 21 shows PEG-PTMC / SF 7030 / BBH 750 @SF / HA-Tyr 8020 Comparison of colonic mechanical properties between scaffold and SD rat.

[0043] Figure 22 is a schematic diagram of stent implantation. The target site is exposed; the tissue is cut and feces are removed; CG-1, CG-2, and EG group stents are implanted; manual suturing and stent suturing are performed; a schematic diagram of intestinal healing after 14 days.

[0044] Figure 23 shows the intestinal healing of the EG, BG, CG-1, and CG-2 groups on postoperative day 14. Figure 24 shows the H&E staining results of different groups: EG, BG, CG-1, and CG-2 groups on postoperative days 3, 7, and 14.

[0045] Figure 25 shows the Masson staining results of different groups: EG group, BG group, CG-1 group, and CG-2 group on the 3rd, 7th, and 14th days after surgery. Detailed Implementation

[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Example 1

[0048] Synthesis of PTMC-b-PEG-b-PTMC

[0049] A triblock PTMC-b-PEG-b-PTMC copolymer was synthesized using ring-opening polymerization. Details are as follows:

[0050] (1) In an anhydrous and oxygen-free glove box, weigh a certain proportion of PEG (0.3wt%, 0.6wt%, 0.9wt%, 1.2wt%, 1.5wt%) and TMC monomer and add them to a completely dry round-bottom flask. Then add 0.05wt% of catalyst Sn(Oct)2 (dissolved in anhydrous toluene, 20mg / mL), place a magnetic flask in it and seal the mouth of the reaction flask with vacuum silicone grease. Finally, seal the mouth of the reaction flask with a sealing film to ensure that no oxygen or moisture enters.

[0051] (2) Take the round-bottom flask containing the sample out of the glove box and place it in an oil bath at 130~150 ℃ for 36~48 h.

[0052] (3) After the reaction was completed, the reaction flask was removed from the oil bath and placed in liquid nitrogen to cool to room temperature. Trichloroethane solvent was then added several times until completely dissolved. The polymer solution was then purified and precipitated with three times the amount of n-hexane, and this process was repeated three times. Finally, the purified polymer was dried in a vacuum drying oven for 48 h to obtain PTMC-b-PEG-b-PTMC for later use. The molecular weight and contact angle of the polymers synthesized in different proportions were tested to determine the appropriate proportion.

[0053] Exploration of the blending ratio of PTMC-b-PEG-b-PTMC and SF

[0054] (1) Weigh a certain amount of the prepared PTMC-b-PEG-b-PTMC and add different amounts of purchased SF to make PTMC-b-PEG-b-PTMC / SF form a certain ratio (90 / 10, 80 / 20, 70 / 30, 60 / 40, 50 / 50). Dissolve the above-mentioned PTMC-b-PEG-b-PTMC and SF mixtures in HFIP to prepare a spinning solution with a concentration of 4%. Place the solution in a shaker at 37°C to fully dissolve the sample and obtain a uniform electrospinning solution.

[0055] (2) The spinning solution was loaded into a 2.5 mL syringe, and a 20 G metal needle was attached to the front of the syringe. Electrospinning was performed using a traditional single nozzle. The specific spinning conditions are shown in Table 3. Finally, the blended support was placed in a desiccator containing methanol for 8 h of treatment.

[0056] Table 3 Electrospinning process parameters

[0057]

[0058] Synthesis and characterization results of PTMC-b-PEG-b-PTMC

[0059] Figure 1A shows the synthesis mechanism of PTMC-b-PEG-b-PTMC. In the presence of polyethylene glycol as the initiator and Sn(Oct)2 as the catalyst, TMC underwent ring-opening polymerization to synthesize the PTMC-b-PEG-b-PTMC triblock copolymer. Block copolymers with different molecular weights exhibit significant differences in degradation rate and mechanical properties, and different PEG addition amounts have a significant impact on the molecular weight of the block copolymer. For the product to be implanted into the intestine, it needs to have a suitable degradation rate and excellent mechanical properties. Therefore, this experiment focuses on investigating the effects of TMC monomers and different amounts of PEG in the raw materials on the molecular weight of the block copolymer PTMC-b-PEG-b-PTMC and its contact angle. In Figures 1B and 1C, as the amount of PEG added increases, the molecular weight and contact angle of PTMC-b-PEG-b-PTMC decrease. In order for PTMC-b-PEG-b-PTMC to better integrate with SF, another material of the scaffold, in the spinning solution during electrospinning, a ratio (0.3~1.5 wt%) similar to the contact angle of SF is selected. Furthermore, because the spun scaffold becomes transparent quickly as the molecular weight of PTMC-b-PEG-b-PTMC decreases, a PEG addition of 0.3 wt% and a molecular weight of 114 are ultimately selected for PTMC-b-PEG-b-PTMC.

[0060] The infrared spectra of PTMC, PEG, and the block copolymer PTMC-b-PEG-b-PTMC are shown in Figure 2. The copolymer PTMC-b-PEG-b-PTMC and PTMC show similar infrared spectra at 1735 cm⁻¹. -1 and 1218cm -1 The characteristic absorption peaks overlap at these locations, corresponding to the characteristic peaks of the carbonate carbonyl group and the ether bond, respectively. The red indicator band represents the characteristic absorption peak of CO, and the values ​​for PTMC-b-PEG-b-PTMC, PTMC, and PEG at this location are 1103 cm⁻¹. -1 1095 cm -1 and 1091 cm -1 .

[0061] PTMC-b-PEG-b-PTMC 1 As shown in Figure 3, the H-NMR peaks at chemical shifts of 4.25 ppm (a), 2.06 ppm (b), and 3.68 ppm (c) correspond to the methylene protons next to oxygen in PTMC, other methylene protons in PTMC, and methylene protons in the PEG backbone, respectively.

[0062] FTIR of PTMC-b-PEG-b-PTMC / SF anastomotic stent

[0063] In this invention, the infrared spectra of PTMC-b-PEG-b-PTMC (abbreviated as PEG-PTMC), SF, and PTMC-b-PEG-b-PTMC / SF (abbreviated as PEG-PTMC / SF) nanofiber membranes were tested (Figure 4A). For the PEG-PTMC fiber membrane, an absorption peak of the carbonate carbonyl group was observed at 1735 cm⁻¹. -1 At this point, the -O- absorption peak is at 1218 cm⁻¹. -1 Tensile vibration at 1520-1540 cm⁻¹. For SF₂ films, this occurs at 1520-1540 cm⁻¹. -1 (Amide I, carbonyl stretching) and 1650 cm -1 A characteristic peak exists at (amide II; NH bending vibration). For the PEG-PTMC / SF composite film, all characteristic peaks belonging to PTMC-b-PEG-b-PTMC and SF can be found, and the peak positions do not change significantly. Therefore, it can be inferred that there is almost no interaction between PTMC-b-PEG-b-PTMC and SF molecules in the PEG-PTMC / SF nanofiber layer, their respective chemical properties remain unchanged, and their respective material properties are preserved. Figure 4B shows the infrared spectra of the PEG-PTMC / SF fiber film before and after methanol treatment. The infrared spectrum before methanol treatment is 1650 cm⁻¹. -1 1540cm -1 The characteristic peaks of amide I and amide II in SF were observed. After methanol treatment, the characteristic peaks of amide I and amide II shifted to 1632 cm⁻¹. -1 1535cm -1 The movement indicates that the conformation of SF changed after methanol treatment, transforming into a β-sheet structure.

[0064] Mechanical properties of PTMC-b-PEG-b-PTMC / SF anastomotic stent

[0065] PEG-PTMC / SF nanofiber membranes should possess suitable mechanical properties (Figures 5A and 5B). It can be observed that as the proportion of SF increases, the tensile elongation of the PEG-PTMC / SF scaffold gradually decreases, the Young's modulus gradually increases, and the scaffold gradually stiffens. However, when the PEG-PTMC / SF ratio is 50 / 50, the tensile elongation increases. In the tensile test, the 50 / 50 fiber membrane shows a continuous fracture (Figure 5C), unlike the brittle fracture of other ratios (Figure 5C). This is presumably because the amount of SF added has reached a saturation point. When the PEG-PTMC / SF ratio is 70 / 30 or 60 / 40, its Young's modulus is between 17-43 MPa, and its elongation at break reaches 70%-132%. This gives PEG-PTMC / SF good flexibility and a certain degree of rigidity, meeting the toughness required by rat intestines and enhancing the support of PTMC, which is much greater than the elongation at break (36%-54%) and Young's modulus (1-3 MPa) of rat intestines. It can be seen that the elongation at break and Young's modulus of the scaffold before and after fumigation are not significantly different, indicating that methanol treatment did not affect the mechanical properties of the scaffold.

[0066] SEM images of PTMC-b-PEG-b-PTMC / SF anastomotic stents

[0067] The microstructure of the anastomotic stent can be observed by SEM. As shown in Figure 6, SEM images and fiber diameter diagrams of PEG-PTMC / SF anastomotic stents with different ratios can be seen. The 90 / 10 electrospun fiber membrane has an average diameter of 2.2 μm, with a fiber distribution range of 0.05-3.5 μm, of which fibers with a diameter of 1.5-3.0 μm account for 74% of the total fiber count. The 80 / 20 electrospun fiber membrane has an average diameter of 1.2 μm, with a fiber distribution range of 0.05-3.0 μm, of which fibers with diameters of 0-0.5 μm and 1.5-2.0 μm account for 22% and 44% of the total fiber count, respectively. The 70 / 30 electrospun fiber membrane has an average diameter of 1.2 μm, with a fiber distribution range of 0.05-2.0 μm, of which fibers with diameters of 0-0.5 μm and 1.5-2.0 μm account for 34% and 54% of the total fiber count, respectively. The 60 / 40 electrospun fiber membrane has an average diameter of 0.2 μm, with a fiber distribution range of 0.05-1.4 μm, of which fibers with a diameter of 0-0.4 μm account for 74% of the total fiber count. Fibers with diameters of 0.8 μm and 0.8-1.2 μm account for 30% and 42% of the total fiber count, respectively; the average diameter of the 50 / 50 electrospun fiber membrane is 1.2 μm, with fiber distribution ranging from 0.05 to 2.0 μm, of which fibers with diameters between 0.5 and 1.5 μm account for 64% of the total fiber count. It can be observed that both materials exhibit a relatively uniform fiber structure under electrospinning, and the fiber diameter decreases with increasing SF content.

[0068] In vitro degradation of PTMC-b-PEG-b-PTMC / SF anastomotic scaffold

[0069] Based on the above experiments, this invention found that PEG-PTMC / SF nanofiber layers with a ratio between 70 / 30 and 60 / 40 exhibit better tensile properties, and therefore further explored their in vitro degradation. The in vitro weight loss rate was calculated (Figure 7), revealing that: under the same time conditions, the in vitro weight loss rates of PEG-PTMC scaffolds and PEG-PTMC / SF scaffolds with a ratio of 90 / 10 were not significantly different, ranging from 0-10%, indicating slow degradation; the degradation rate of PEG-PTMC / SF scaffolds with a ratio between 80 / 20 and 50 / 50 increased with increasing SF ratio within 5 days. During the 6-21 day period, the weight loss rates of 60 / 40 and 50 / 50 ratio scaffolds showed significant fluctuations, while the 70 / 30 ratio scaffolds exhibited a more stable degradation process, with a weight loss rate between 40-50% over 21 days. Because the intestinal environment is complex, and this experiment involved in vitro degradation, the scaffold degradation rate was accelerated. It is speculated that a 70 / 30 PEG-PTMC / SF ratio is more suitable. This stage corresponds to the end of the proliferative phase of intestinal healing.

[0070] Scanning electron microscopy (SEM) allows for better observation of the microscopic morphological changes of the fibers during the degradation process. By observing SEM images of PEG-PTMC / SF fiber layers with different ratios (Figure 8), this invention reveals that the PEG-PTMC fiber layers are prone to dissolution during in vitro degradation, and the dissolution rate is rapid. As the SF ratio increases, the dissolution and degradation of the PEG-PTMC / SF fiber layers gradually slows down. When the ratio of PEG-PTMC / SF fiber layers is 70 / 30, the randomly oriented fibers maintain a good morphology for approximately 21 days as the degradation time increases, further demonstrating that a 70 / 30 ratio PEG-PTMC / SF fiber layer better meets the expectations of this invention.

[0071] Summary of PTMC-b-PEG-b-PTMC / SF Anastomotic Stent

[0072] This invention utilizes the biocompatible triblock copolymer PEG-PTMC and SF to prepare anastomotic scaffolds. By analyzing molecular weight and contact angle, the optimal PEG content in PTMC-b-PEG-b-PTMC was determined to be 0.3 wt%, at which point the molecular weight of the triblock copolymer is 100~120 × 10⁻⁶. 4 By using FTIR, this invention clarified that the chemical composition and properties of the PEG-PTMC and SF blend remained unchanged. Furthermore, through mechanical tensile testing and degradation performance analysis, the optimal ratio of the PEG-PTMC / SF scaffold was determined to be PEG-PTMC / SF. 70 / 30 .

[0073] Example 2

[0074] Synthesis of HA-Tyr

[0075] HA (1 g) was dissolved in 100 mL of PBS buffer (pH=6.8) to prepare a 1 wt% HA aqueous solution. Then, EDC (2.5 mM) and NHS (2.5 mM) were added sequentially to the HA solution, and the reaction was carried out for 30 min to activate the carboxyl group of HA. Subsequently, Tyr (2-3 mM) was dissolved in the activated HA solution and reacted overnight at room temperature. After the reaction, unreacted Tyr, EDC, and NHS were purified by dialyzing using a dialysis bag with a molecular weight cutoff of 3500 Da. The dialyzed solution was pre-frozen overnight at -80 ℃ and then freeze-dried to obtain the final product HA-Tyr. The sample was stored in a desiccator for later use.

[0076] Preparation of SF / HA-Tyr microneedles

[0077] (1) Solution preparation. Add Ru(II)bpy3 to deionized water. 2+(1 mM), denoted as solution A, then add SPS (20~40 mM) to deionized water, denoted as solution B, mix equal amounts of solution A and solution B, denoted as solution C, and dissolve different proportions of SF / HA-Tyr (100 / 0, 90 / 10, 80 / 20, 70 / 30, 60 / 40, 50 / 50) in solution C to prepare an 8% (w / v) prepolymer solution.

[0078] (2) Casting and centrifugal drying. 200 μL of prepolymer solution was filled into a 10×10 array of PDMS microneedles and vacuumed three times in a vacuum degassing device with a vacuum degree of 0.1 MPa for 5~10 min each time. Then the mold was placed in a constant temperature and humidity chamber at 25±2℃ and 65±5% to dry.

[0079] (3) Heating concentration. Add 100 μL of prepolymer solution to replenish the liquid. After vacuuming under the same conditions as above, place the mold in a constant temperature desiccator at 25±2 ℃ and 65±5% humidity for 1~3 h. Repeat the heating concentration 3~4 times.

[0080] (4) In blue light (460nm, 30w / cm) 2 Cured under light for 20 minutes.

[0081] (5) Drying and demolding. Place the mold containing the microneedles in a constant temperature desiccator at 25±2 ℃ and 65±5% humidity for 24 h and then demold.

[0082] (6) Methanol vapor treatment. The microneedles were placed in a dryer containing methanol for methanol vapor treatment for 8 hours.

[0083] Exploration of SF Microneedles

[0084] Figures 9A-C show that pure SF microneedles completely dissolve and deform within 1-3 minutes after insertion into the rat intestine, which is not conducive to sustained drug release in the intestine. Subsequently, this invention prepared microneedles by blending SF with HA-Tyr. Tyrosine in tyramine can be cross-linked with tyrosine in SF through dityrosine under visible light. After insertion into the intestine, the microneedles exhibit swelling but not dissolution characteristics within 1-3 minutes (Figures 9D-F), enabling drug delivery to the target intestine and sustained release through the exchange of substances with the fluid in the tissue.

[0085] NMR spectrum and grafting ratio of HA-Tyr

[0086] Tyr was successfully synthesized by coupling it to the HA backbone via an EDC / NHS amidation reaction between the carboxyl group of HA and the amino group of Tyr (Figure 10A). The number of Tyr molecules introduced into each repeating unit of the HA polymer was determined by... 1H-NMR spectroscopy confirmed the grafting of tyramine. As shown in Figure 10B, the absorption peaks at 6.9 ppm and 7.2 ppm (a and b) are proton absorption peaks on the benzene ring of tyramine, and the NMR peak at 1.96 ppm (c) is a proton absorption peak on the hyaluronic acid side chain, indicating that tyramine was successfully grafted onto the hyaluronic acid side chain. The degree of substitution of tyramine was calculated using the following formula:

[0087]

[0088] Where DS represents the degree of substitution, and I Tyr and I HA These are the integrated areas of the proton absorption peaks of the tyramine benzene ring and the hyaluronic acid side chain, respectively.

[0089] The degree of substitution was calculated to be 31%.

[0090] Swelling properties of SF / HA-Tyr microneedles

[0091] HA (hyaluronic acid) is a natural component of the extracellular matrix of animals and microorganisms (including human tissues) and is used in many applications, from injections to hydrogels and creams. It has been shown to stimulate fibroblast proliferation and adhesion at wound sites, promoting healing. When HA is combined with the good mechanical properties of SF (sulfuric acid), SF / HA-Tyr microneedles are ideal materials for microneedle sustained release. Swellable microneedles made from SF / HA-Tyr crosslinked hydrogels swell without dissolving after absorbing water, which can promote the controlled release of pre-loaded drugs. The swelling process can be qualitatively and quantitatively examined in vitro. Typically, the swelling capacity is measured by immersing the microneedles in phosphate-buffered saline. As shown in Figures 11A-C, SF / HA-Tyr microneedles can be seen to transform into hydrogel microneedles after absorbing water. As shown in Figure 11D, the average swelling rates of SF / HA-Tyr microneedles with different ratios were 244.7%, 249.2%, 355.1%, 717.2%, 855.8%, and 540.9%, respectively. With increasing HA content, the swelling rate of SF / HA-Tyr microneedles increased until the SF / HA-Tyr ratio reached 50 / 50, at which point the swelling rate began to decrease, suggesting that the HA addition had reached a saturation point. Considering the potential harm to the intestines from excessively swollen microneedles, and in conjunction with the sustained-release drug, a more moderate swelling rate, namely an SF / HA-Tyr microneedle material ratio of 80 / 20, was selected for subsequent experiments.

[0092] Compression performance of SF / HA-Tyr microneedles

[0093] Compression experiments demonstrated the compressibility of SF / HA-Tyr microneedles with different ratios. SF / HA-Tyr microneedles with varying ratios (100 / 0, 90 / 10, 80 / 20, 70 / 30, 60 / 40, 50 / 50) were prepared. The microneedle mold data was a needle length of 1800 μm, a tip-to-needle distance of 1200 μm, and an array of 10×10 regular square pyramids. As shown in Figures 12A-F, the force-displacement curves of the SF / HA-Tyr microneedles can be further divided into four stages. In stage 1 (light blue area), the force is quite low because no microneedles are truly compressed; the slight increase in force corresponds to warping of the sample. Then, in stage 2 (light green area), the SF / HA-Tyr microneedles flatten, and the compressive force is applied directly to the microneedle body. The compressive force increases rapidly and then suddenly decreases because the needle tip breaks; its peak is selected as the point of fracture force (marked as point F in the figure). In stage 3 (light yellow area), the microneedle body is slowly crushed into small pieces, and the compressive force increases again. In the final stage, stage 4 (light gray area), the fragments of the microneedle body are tightly compacted, resulting in an almost linear force-displacement curve. As can be seen from Figures 12A-F, as the SF content decreases, the mechanical properties of the microneedle also weaken accordingly. This is because SF has high mechanical strength to support effective microneedle insertion.

[0094] Summary of SF / HA-Tyr Microneedles

[0095] Because pure SF microneedles are highly soluble, this invention uses SF / HA-Tyr, which combines biocompatibility and mechanical strength, to prepare the microneedles. The degree of tyramine substitution was calculated to be 31% using NMR spectroscopy. Furthermore, compression and swelling tests determined the optimal material ratio for SF / HA-Tyr microneedles to be SF / HA-Tyr. 80 / 20 .

[0096] Example 3

[0097] Synthesis of microneedle scaffolds

[0098] The scaffold was prepared according to Example 1, and the microneedles were prepared according to Example 2. The difference was that, during the synthesis of the microneedles, after heating and concentration, the microneedles were directly treated with methanol for 8 hours, and then the scaffold was attached to the surface of the microneedle mold under blue light (460nm, 30w / cm). 2 Illuminate for 20-30 minutes.

[0099] Compression performance of microneedle anastomotic stents with different tapers

[0100] As shown in Figure 14, the present invention has customized designs indicating different taper (20) 0 25 0 30 0 35 0 40 0Microneedle molds were prepared and different microneedle scaffolds were fabricated to observe the puncture performance of microneedle anastomosis scaffolds with different tapers for rat intestines, and to select an appropriate microneedle taper. Microneedle molds with different tapers (20) were prepared. 0 25 0 30 0 35 0 40 0 Figure 14A shows optical images of microneedles with different tapers, and Figure 14B shows the puncture force of microneedles with different tapers on the rat intestine. The forces at the puncture points are 0.1024 N, 0.1264 N, 0.129 N, 0.1944 N, and 0.3715 N, respectively. Analysis of the compression curves shows that as the taper increases, the force required for the microneedle to penetrate the intestine increases with the taper, under the same displacement. It can also be seen that at 35°... 0 The puncture force of the microneedle stent is greater than that of 30. 0 The puncture force is relatively large, but 30 0 With 25 0 The puncture force of microneedle stents is basically the same, so 30 was chosen based on drug loading and puncture damage. 0 We will use microneedle scaffolds for subsequent experiments.

[0101] Compression performance of microneedle anastomotic stents with different tip pitches

[0102] Based on the above puncture experiments with different taper angles, the present invention is performed at 35°. 0 Based on the existing microneedle molds, we customized microneedle molds with different tip distances (1000μm, 1250μm, 1500μm, 1750μm, 2000μm) and prepared different microneedle anastomosis scaffolds to observe the puncture performance of microneedle anastomosis scaffolds with different tip distances for rat intestines and to select the appropriate microneedle tip distance. Figure 15A shows optical images of microneedles with different tip pitches, and Figure 15B shows the puncture force of microneedle scaffolds with different tip pitches into the rat intestine. The forces at the puncture points are 0.4173 N, 0.2761 N, 0.1713 N, 0.1592 N, and 0.1043 N, respectively. According to the compression curve analysis, as the tip pitch increases, the force required for the microneedle to penetrate the intestine decreases under the same displacement. It can also be seen that the puncture force of the microneedle scaffold with a tip pitch of 1250 μm is relatively larger than that of the 1500 μm scaffold, but the puncture forces of the 1500 μm and 1750 μm microneedle scaffolds are basically similar. Therefore, considering the drug loading and puncture damage, the 1500 μm microneedle scaffold was selected for subsequent experiments.

[0103] Biocompatibility of drug-eluting microneedle anastomotic scaffolds

[0104] After determining the components of each part of the microneedle scaffold (PEG-PTMC / SF)7030 SF / HA-Tyr 8020 The microneedle mold has a taper of 30. 0 (The microneedle mold tip distance is 1500μm). At this point, the drug loading of BBH needs to be considered, which is determined by the antibacterial properties, cell compatibility, and hemolysis rate of drug-loaded microneedle scaffolds with different drug contents.

[0105] Evaluation of the antibacterial properties of microneedle anastomotic scaffolds with different BBH contents

[0106] To investigate PEG-PTMC / SF 7030 / BBH@SF / HA-Tyr 8020 To assess the antibacterial properties, drug-loaded microneedle scaffolds with different BBH contents (500 μg / mL, 750 μg / mL, and 1000 μg / mL) were prepared. The antibacterial effect of the drug-loaded microneedle scaffolds was verified by dilution plating (Figure 16A), bacterial viability / death staining (Figure 16B), and scanning electron microscopy (Figure 16C). The bactericidal rate of the material was determined by the dilution plating method. National standards stipulate that only products capable of killing >50% of bacteria can be labeled as having antibacterial effects; and only products capable of killing >90% of bacteria can be labeled as having bactericidal effects. As shown in Figures 16A and 16E, the survival rates of *S. aureus* corresponding to 500 μg / mL, 750 μg / mL, and 1000 μg / mL were 21%, 3.67%, and 0%, respectively; and the survival rates of *E. coli* corresponding to 500 μg / mL, 750 μg / mL, and 1000 μg / mL were 23.67%, 7.67%, and 0%, respectively. Therefore, the microneedle scaffolds with 750 μg / mL and 1000 μg / mL met the bactericidal rate standard. The trends of *S. aureus* and *E. coli* under both live and dead bacterial staining were the same; therefore, only *S. aureus* was analyzed here. Figure 16B shows that the 750 μg / mL and 1000 μg / mL microneedle scaffolds were almost entirely stained red. To further understand the bactericidal mechanism of BBH, this invention also used SEM to observe the morphological changes of *S. aureus* and *E. coli* (Figure 16C). Untreated bacteria had smooth surfaces, while after treatment with the microneedle scaffold, both bacteria exhibited obvious cell wall shrinkage, which visually demonstrates the destructive effect of the microneedle scaffold on bacteria.

[0107] Blood compatibility evaluation

[0108] Blood compatibility is an important characteristic of implantable materials because most materials come into direct contact with blood during implantation. The most common indicator of blood compatibility is hemolysis rate. This invention uses in vitro hemolysis to evaluate the blood compatibility of drug-eluting microneedle anastomotic stents. Using physiological saline as a negative control and deionized water as a positive control, the hemolysis rate of microneedle anastomotic stents with different BBH addition levels (500 μg / mL, 750 μg / mL, and 1000 μg / mL) was measured. Images of the centrifuged supernatant of all microneedle anastomotic stents and the control group, along with the final hemolysis rate, are shown in Figure 17. This invention found that the hemolysis rates of the 500 μg / mL, 750 μg / mL, and 1000 μg / mL microneedle anastomotic stents were 0.6%, 0.33%, and 0.32%, respectively, all less than 5%. Generally, a hemolysis rate below 5% indicates that hemolysis does not occur upon contact with blood. Therefore, the microneedle anastomotic stent prepared in this invention exhibits good blood compatibility and is highly suitable for implantation.

[0109] Cell compatibility assessment

[0110] Cell compatibility refers to the ability of a material to establish a good interaction with cells. Good cell compatibility means that the material surface will not cause cell damage or inflammatory response. The cytotoxicity of implanted materials is a crucial factor affecting their long-term stability in vivo. The in vitro compatibility of drug-loaded microneedle anastomotic scaffolds was evaluated using the CCK-8 assay, as shown in Figure 18. The results showed that after co-culturing microneedle anastomotic scaffolds with different BBH additions (500 μg / mL, 750 μg / mL, and 1000 μg / mL) with L929 cells for 24 h, the cell viability of the 500 μg / mL and 750 μg / mL drug-loaded microneedle anastomotic scaffolds was above 75%, indicating that the drug-loaded microneedle anastomotic scaffolds have good cell compatibility. Based on the antibacterial experiment, this invention ultimately selected a drug loading of 750 μg / mL for subsequent research.

[0111] Drug-releasing microneedle anastomotic stent

[0112] Based on the above research, this invention yields the final microneedle anastomosis stent. For the flexible stent portion, a 70 / 30 ratio of PEG-PTMC / SF was selected. 7030 For the scaffold; for the microneedle portion, BBH with a concentration of 750 μg / mL and an SF / HA-Tyr material ratio of 80 / 20 was selected. 750 @SF / HA-Tyr 8020 Microneedles; a taper of 30 was selected. 0 Microneedles with a tip-to-tip distance of 1500 μm were used for subsequent experiments.

[0113] Figure 19A shows the standard curve of absorbance of BBH at 345 nm measured with standard solution versus concentration. After function fitting, R... 2 The value was 0.99925, greater than 0.999, confirming the reliability of the standard curve. Furthermore, the release of BBH at a dosage of 750 μg / mL was investigated in PBS at 37 °C, as shown in Figure 19B. At 0.5 h, the drug exhibited a burst release, reaching 58%, followed by a slow release at 4 h, reaching 93% at 12 h. The early stages of intestinal anastomosis healing are most susceptible to various stimuli, and intestinal healing is in an inflammatory phase during this period. BBH, being an antibacterial and anti-inflammatory drug, provides adequate treatment during the inflammatory phase with its release within the first 12 h.

[0114] Compression performance of drug-loaded microneedle anastomotic stents

[0115] PEG-PTMC / SF 7030 / BBH 750 @SF / HA-Tyr 8020 The support was placed on the compression stage of a universal testing machine, and the mechanical properties of the microneedles were tested by uniform compression at a test speed of 0.1 mm / s. As shown in Figure 20, the breaking force of the microneedles was 0.5 N / needle, and the minimum force for effective skin penetration was 45 mN, which fully meets the mechanical properties required for insertion into the intestine.

[0116] Mechanical properties of microneedle anastomosis scaffolds and intestinal tissue

[0117] During implantation, the stent is subjected to external forces exerted by the surgeon. After implantation, the stent must provide support while also allowing intestinal contents to pass through smoothly. Therefore, the overall mechanical properties of the stent are crucial for its support of the intestine. As shown in Figure 21, a comparison of PEG-PTMC / SF... 7030 / BBH 750 @SF / HA-Tyr 8020 The mechanical properties of the stent and the colon of SD rats were compared. The present invention found that the overall intestinal stent had a tensile elongation rate of about 100%, while the rat colon had a tensile elongation rate of only about 40%. The overall mechanical properties of the stent were significantly better than those of the rat intestine, which made the stent more flexible than the rat colon. This provides strong evidence for stent implantation.

[0118] Summary of drug-eluting microneedle anastomotic stents

[0119] Through microneedle insertion experiments with different tapers and needle tip distances into the intestine, this invention clarifies that a taper of 30° is optimal. 0The microneedle scaffold with a needle tip distance of 1500 μm exhibited optimal drug loading and minimal damage to the intestine. Antibacterial, hemolytic, and cellular experiments confirmed that an optimal BBH addition level of 750 μg / mL was achieved, demonstrating both a certain bactericidal rate and excellent biocompatibility, thus ensuring successful implantation. Mechanical property testing demonstrated that the integral anastomotic scaffold outperformed the rat intestine, facilitating tension protection at the anastomosis site. Compression tests determined the single needle breaking force of the integral microneedle scaffold to be 0.5 N, and the in vitro release of BBH in PBS (37 ℃, 547 rpm) reached 93% after 12 h.

[0120] Example 4: Animal Experiment with Drug-Loaded Microneedle Anastomotic Scaffold

[0121] Animal implantation experiments

[0122] To further evaluate the efficacy of drug-loaded microneedle anastomotic stents in preventing anastomotic leakage, this invention employed a colorectal anastomotic leakage model in SD rats for in vivo implantation experiments. As shown in Figure 22, during model construction, the target site was surgically cut open, and the intestinal contents were emptied. Four groups were established: a blank group (traditional manual suturing group, BG), a control group-1 (implanted PTMC-b-PEG-b-PTMC / SF stent, CG-1), a control group-2 (implanted BBH-loaded PTMC-b-PEG-b-PTMC / SF stent, CG-2), and an experimental group (implanted BBH-loaded microneedle anastomotic stent, EG). The blank group was sutured using conventional surgical methods, while the control and experimental groups had stents implanted according to the schematic diagram of this invention.

[0123] Tissue physical observation

[0124] The intestinal recovery of SD rats in each group was observed and photographed on day 14 after stent placement (Figure 23). It was observed that, under the same suture method and number of stitches, the wound healing in the BG group was irregular. At day 14, some intestinal contents adhered to the sutures at the anastomosis site, leading to unclean wounds and susceptibility to infection, resulting in some areas failing to heal properly. In contrast, the intestinal wounds of rats in the CG-1 and CG-2 groups showed multiple folds at the healing site, with the wound in the CG-2 group being smoother and more even than that in the CG-1 group, demonstrating the effectiveness of BBH. The intestinal wounds of rats in the EG group were regular and healed systematically, with no visible wound traces at day 14, indicating good recovery. This may be because the microneedle anastomosis stent isolates intestinal contents and feces during intestinal healing, while the release of BBH from the microneedles has anti-inflammatory and antibacterial effects in the early stages of intestinal healing, ensuring successful healing around day 14. The observation that the intestinal anastomosis healed on day 14 further demonstrates the advantages of the drug-loaded microneedle anastomosis scaffold of the present invention in intestinal healing.

[0125] H&E staining

[0126] HE (Hematologic Analysis) is the gold standard for histological examination. This invention further validates the healing of intestinal anastomoses using HE pathological sections (Figure 24). The results of observing four groups of HE pathological sections are as follows: While the intestinal tissue in the BG group showed a general healing trend, further observation revealed inflammatory cell infiltration throughout the entire intestinal anastomosis healing process. The continuous damage from inflammatory cells significantly hindered wound healing, leading to excessive tissue proliferation. Although the mucosa showed a gradual healing trend, excessive tissue proliferation in the later stages indicated a tendency for scarring, with significant mucosal loss still observed at 14 days. The intestinal tissue recovery in the two CG groups was slightly stronger than in the control group. Inflammatory cell infiltration was still present. At 14 days, the CG-1 group showed slight mucosal defects, while the CG-2 group's mucosa was nearly intact under the influence of medication at 14 days. In contrast, the EG group, as observed by this invention, showed significantly less inflammatory cell infiltration at day 3 compared to the other three groups. Furthermore, the inflammatory infiltration began to decrease around day 7, with the anastomosis gradually healing, the damaged muscle layer gradually repairing, and the extent of mucosal damage decreasing, leading to gradual mucosal coverage. By day 7, mucosal apposition was nearly complete, and the degree of inflammatory infiltration was significantly reduced. Initial healing was achieved by day 14. This is primarily attributed to the excellent antibacterial and anti-inflammatory properties of the drug-loaded microneedle anastomosis scaffold. The protection of the anastomosis site and the release of drugs from the microneedles resulted in significantly lower inflammatory cell infiltration compared to the other three groups, and also led to significantly better mucosal recovery.

[0127] Masson staining

[0128] Collagen deposition is also an important indicator of the anastomotic healing process, and Masson staining is an authoritative and classic technique for staining collagen fibers. The same pattern as H&E staining can be observed in Masson staining images (Figure 25). Therefore, this invention also shows that collagen fiber formation in the BG group is significantly delayed, with only a small amount of collagen forming around day 7, leading to a further prolongation of the healing cycle. In the CG-1 group, collagen fiber formation is minimal in the early stages, resulting in a slow healing rate and excessive collagen fiber deposition by day 14, leading to slow healing. Collagen fiber proliferation gradually increases in the CG-2 and EG groups, returning to normal levels after two weeks, but the EG group shows better results than the CG-2 group. This again demonstrates that the drug-loaded microneedle scaffold of this invention protects the anastomotic site and provides favorable conditions for collagen formation in the tissue.

[0129] In summary, this invention constructs an implantable electrospun nanofiber anastomosis scaffold with PTMC-b-PEG-b-PTMC / SF as the main scaffold and combined with BBH-loaded SF / HA-Tyr microneedles as the antibacterial functional layer, for the prevention of anastomotic leakage after intestinal anastomosis. This intestinal drug-loaded microneedle anastomosis scaffold possesses both the mechanical flexibility to conform to intestinal tissue and the ability to isolate the wound from the influence of feces and intestinal contents. Simultaneously, it contains BBH-loaded hydrogel microneedles, which can release drugs sustainably in the early stages of inflammation to achieve antibacterial function. Therefore, this drug-loaded microneedle anastomosis scaffold not only plays an antibacterial role in the early inflammatory phase of anastomotic healing but also promotes tissue healing in the early stages of healing. Through the special morphology and electrospinning of the PTMC-b-PEG-b-PTMC / SF composite material, the scaffold material can maintain a certain cavity structure while degrading in vivo, without overall shrinkage affecting normal anastomotic healing. At the same time, the SF / HA-Tyr hydrogel microneedles can swell after insertion into the intestine to release drugs and promote wound healing. Furthermore, the drug-loaded microneedle anastomotic scaffold demonstrated a good ability to promote anastomotic healing in a simulated anastomotic leakage model in SD rats. These results provide a new approach for reducing the occurrence of anastomotic leakage after intestinal anastomosis using biodegradable drug-loaded microneedle anastomotic scaffolds.

[0130] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0131] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. An intestinal microneedle anastomosis scaffold with antibacterial function, characterized in that, The intestinal microneedle anastomosis scaffold is organically composed of a flexible scaffold base layer and a drug-loaded microneedle outer layer. The flexible scaffold base layer is obtained by blending PTMC-b-PEG-b-PTMC triblock copolymer with silk fibroin (SF). The drug-loaded microneedles are obtained by mixing tyramine-modified hyaluronic acid (HA-Tyr) and SF, adding berberine hydrochloride (BBH), and then casting. The flexible scaffold base layer and the drug-loaded microneedles are organically bonded through the SF contained in both, using the dityrosine on the SF to crosslink with blue light, thus modifying the microneedles on the outer surface of the scaffold, resulting in an anastomosis scaffold with an antibacterial and anti-inflammatory microneedle layer.

2. The intestinal microneedle anastomosis scaffold with antibacterial function according to claim 1, characterized in that, In the blending of PTMC-b-PEG-b-PTMC triblock copolymer and silk fibroin (SF), the mass ratio of PTMC-b-PEG-b-PTMC triblock copolymer to silk fibroin (SF) is 70 / 30.

3. The intestinal microneedle anastomosis scaffold with antibacterial function according to claim 1, characterized in that, The PEG content in the PTMC-b-PEG-b-PTMC triblock copolymer is 0.3 wt%.

4. The intestinal microneedle anastomosis scaffold with antibacterial function according to claim 2, characterized in that, The degree of substitution of the tyramine-modified hyaluronic acid (HA-Tyr) is 31%.

5. The intestinal microneedle anastomosis scaffold with antibacterial function according to claim 1, characterized in that, The mass ratio of SF to tyramine-modified hyaluronic acid (HA-Tyr) in the drug-loaded microneedles is 80 / 20.

6. The intestinal microneedle anastomosis scaffold with antibacterial function according to claim 1, characterized in that, The microneedle taper of the drug-loaded microneedle is 30-35°.

7. The intestinal microneedle anastomosis scaffold with antibacterial function according to claim 1, characterized in that, The distance between the microneedle tips in the drug-loaded microneedles is 1500 μm.

8. The intestinal microneedle anastomosis scaffold with antibacterial function according to claim 1, characterized in that, The content of berberine hydrochloride (BBH) in the drug-loaded microneedles is 750-1000 μg / mL.

9. A method for preparing an intestinal microneedle anastomosis scaffold with antibacterial function as described in claim 1, characterized in that, Includes the following steps: (1) Synthesis of PTMC-b-PEG-b-PTMC: In an anhydrous and oxygen-free environment, PEG and TMC monomers were weighed, and 0.05 wt% of catalyst Sn(Oct)2 was added. A magnetic flask was placed in the flask and the mouth of the flask was sealed with vacuum silicone grease. Finally, the mouth of the flask was sealed with a sealing film. The sealed flask was taken out of the glove box and placed in an oil bath at 130~150 ℃ for reaction for 36~48 minutes. h, after the reaction is completed, take out the reaction bottle and cool it to room temperature, then add trichloroethane solvent to dissolve the reaction product. After complete dissolution, take the solution and add it dropwise to n-hexane to precipitate. Repeat 3 times. Finally, dry the purified polymer product to obtain PTMC-b-PEG-b-PTMC; (2) Electrospinning of PTMC-b-PEG-b-PTMC with silk fibroin (SF): Add SF to PTMC-b-PEG-b-PTMC, mix the two and dissolve them in HFIP, so that the sample is fully dissolved and a uniform spinning solution with a concentration of 4% is prepared. The spinning solution is loaded into the electrospinning syringe, the syringe is connected to a 20 G metal needle, and electrospinning is performed using a single nozzle to obtain a flexible scaffold base layer, and then dried; (3) Synthesis of HA-Tyr: Hyaluronic acid (HA) is prepared to obtain a concentration of 1 wt% HA aqueous solution, then, under stirring, EDC and NHS were added to activate the carboxyl group of HA. After that, tyramine hydrochloride (Tyr) was dissolved in the activated HA aqueous solution and left at room temperature overnight. After the reaction was completed, the unreacted Tyr, EDC and NHS were dialyzed and filtered using a dialysis bag with a molecular weight cutoff of 3500 Da. The dialysis solution was freeze-dried to obtain the product HA-Tyr; (4) Preparation of SF / HA-Tyr microneedles: Add tris(2,2-bipyridine)ruthenium(II) hexahydrate Ru(II)bpy3 to deionized water. 2+ Let this be denoted as solution A; Sodium persulfate (SPS) was added to deionized water and labeled as solution B; equal amounts of solution A and solution B were mixed and labeled as solution C; then, SF, HA-Tyr and berberine hydrochloride (BBH) were dissolved in solution C to prepare an 8% w / v prepolymer solution; the prepolymer solution was cast into a 10×10 array of PDMS microneedles and vacuumed in a vacuum degassing device with a vacuum degree of 0.1 MPa; then the mold was placed in a constant temperature and humidity chamber with a temperature of 25±2℃ and a humidity of 65±5% to dry and form. During this period, the prepolymer solution was added repeatedly to replenish the liquid, and the same conditions of vacuuming and drying were repeated 3~4 times. After blue light curing, it was placed in a desiccator containing methanol for methanol vapor treatment; (5) Preparation of microneedle anastomosis scaffold: when preparing microneedles, after repeated heating and concentration, methanol vapor treatment was directly applied for 8 h, then attach the prepared flexible scaffold base layer to the surface of a microneedle mold in which SF / HA-Tyr solution is cast, and irradiate with blue light for 20~30 min. Through the cross-linking effect of dityrosine in SF, which is contained in both, the microneedle anastomotic scaffold is finally prepared.

10. The use of the intestinal microneedle anastomosis scaffold of claim 1 in the preparation of materials for preventing intestinal anastomotic leakage and promoting anastomotic healing.

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