Multifunctional electrospun material, application thereof and artificial blood vessel and preparation method thereof

Abstract

This invention provides a multifunctional electrospun material and its application, as well as an artificial blood vessel and its preparation method. The electrospun material includes an electrospun material substrate and deferoxamine. This electrospun material possesses anti-inflammatory, antioxidant, antibacterial, and anticoagulant functions. When applied to the preparation of artificial blood vessels, it can improve the regenerative capacity of blood vessels, especially by protecting vascular cells through inhibiting inflammation and hyperglycemia-induced oxidative stress in diabetic and elderly patients, thereby promoting angiogenesis. The loaded deferoxamine can achieve long-term, slow release according to the environment of the transplantation site. The released deferoxamine can prevent acute thrombosis by inhibiting platelet adhesion and activation, and can also protect the microenvironment of the transplantation site by resisting oxidative stress and inflammation levels, promoting cell infiltration and regeneration, and protecting regenerated cells.

Description

Technical Field

[0001] This invention belongs to the field of artificial blood vessel technology, and particularly relates to a multifunctional electrospun material and its application, as well as artificial blood vessels and their preparation methods. Background Technology

[0002] Vascular transplantation is an important way to treat cardiovascular diseases, and vascular diseases in the context of diabetes and the elderly have higher requirements for vascular grafts due to their special physiological environment.

[0003] Autologous bypass grafting remains the gold standard for treating coronary artery disease; however, its application is limited by the availability of donor grafts and lesions at the donor site. Therefore, artificial grafts are often used for transplantation. While large-diameter artificial grafts have achieved success in clinical applications, current products lack regenerative capacity and do not possess anti-inflammatory, antibacterial, anticoagulant, or antioxidant properties, failing to achieve vascular regeneration and protection. Consequently, small-diameter (inner diameter < 6 mm) artificial grafts often suffer from problems such as thrombosis (acute thrombosis) and intimal hyperplasia, resulting in a low success rate within 5 years post-procedure, making it difficult to meet clinical needs. Studies show that inflammation and oxidative stress play a role in aging, diabetes, cardiovascular disease, and other diabetic complications. Increasing evidence suggests that ferroptosis, a newly discovered non-apoptotic regulated cell death, plays a central role in CVD and may contribute to or exacerbate the progression of cardiovascular disease, potentially representing a therapeutic target for cardiovascular disease.

[0004] Current understanding suggests that small-diameter artificial blood vessels implanted in diabetic and elderly patients must simultaneously possess anti-inflammatory, antioxidant, and anticoagulant effects. Surface modification of artificial blood vessels is considered one of the best ways to achieve these multiple functions; however, achieving this requires loading various active substances onto the surface of the artificial blood vessel. This process involves multiple steps, significantly increasing preparation time and cost. Furthermore, these multiple processing steps may lead to the inactivation of active substances. Due to these limitations, there are currently no reports in the literature or technological inventions regarding artificial blood vessels that simultaneously possess all of these functions. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a multifunctional electrospun material and its application, and an artificial blood vessel and its preparation method. The electrospun material contains deferoxamine, which can inhibit platelet adhesion and activation and reduce thrombus formation.

[0006] This invention provides a multifunctional electrospun material, comprising an electrospun material substrate and deferoxamine.

[0007] Preferably, deferoxamine is loaded onto the electrospun material substrate in a covalent bond manner;

[0008] Covalent bonds exhibit ROS responsiveness.

[0009] Preferably, the concentration of deferoxamine in the multifunctional electrospun material is 0.04~0.16 mmol / L.

[0010] Preferably, the electrospun material substrate is selected from one or more of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly(3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly-L-lactide-caprolactone, and poly(p-dioxane-hexanone).

[0011] This invention provides an application of the multifunctional electrospun material described above in the preparation of artificial blood vessels, vascular patches, valved blood vessels, heart valves, or skin dressings.

[0012] This invention provides a multifunctional electrospun artificial blood vessel, the material of which is the multifunctional electrospun material described in the above technical solution.

[0013] This invention provides a method for preparing the multifunctional electrospun artificial blood vessel described above, comprising the following steps:

[0014] The electrospinning material substrate is dissolved in an organic solvent to obtain a spinning solution;

[0015] The spinning solution is electrospun to obtain an electrospun artificial blood vessel;

[0016] The electrospun artificial blood vessel was immersed in a functional solution containing deferoxamine to obtain a multifunctional electrospun artificial blood vessel.

[0017] Preferably, the organic solvent is a mixture of chloroform and methanol in a volume ratio of 5:0.9~1.1.

[0018] Preferably, the concentration of deferoxamine powder in the spinning solution is 0.04~0.16 mmol / L;

[0019] The mass ratio of the matrix material to the deferoxamine powder is 1:(0.896×10⁻⁶). -4 ~3.584×10 -4 ).

[0020] Preferably, the parameters of the electrospinning are:

[0021] The flow rate of the spinning solution is 7~9 mL / h, the high voltage DC voltage is 10~12 kV, and the receiving distance is 10~15 cm.

[0022] This invention provides a multifunctional electrospun material, comprising an electrospun material substrate and deferoxamine. This electrospun material possesses anti-inflammatory, antioxidant, antibacterial, and anticoagulant functions; when applied to the fabrication of artificial blood vessels, it can enhance the regenerative capacity of blood vessels, particularly by protecting vascular cells through inhibiting inflammation and hyperglycemia-induced oxidative stress in diabetic and elderly patients, thereby promoting vascular regeneration. Attached Figure Description

[0023] Figure 1 The relative fluorescence intensities of the embodiments and comparative examples of the present invention are shown below.

[0024] Figure 2 This is a diagram showing the proportion of iNOS+ cells in the embodiments and comparative examples of the present invention;

[0025] Figure 3 This is a diagram showing the proportion of CD206+ cells in the embodiments and comparative examples of the present invention;

[0026] Figure 4 OD of cell proliferation after CCK-8 experiments in the embodiments and comparative examples of this invention 450 picture;

[0027] Figure 5 This is a diagram showing the diameter of the inhibition zone in the embodiments and comparative examples of the present invention;

[0028] Figure 6 OD for embodiments and comparative examples of the present invention 490 picture;

[0029] Figure 7 OD of platelet activation number in the embodiments and comparative examples of the present invention 450 picture. Detailed Implementation

[0030] This invention provides a multifunctional electrospun material, comprising an electrospun material substrate and deferoxamine.

[0031] In this invention, deferoxamine is covalently loaded onto an artificial membrane material. The covalent bonds of the deferoxamine loading exhibit ROS responsiveness. Specifically, if the artificial membrane material is an artificial blood vessel, then deferoxamine is covalently loaded onto the artificial blood vessel.

[0032] In this invention, the electrospun material substrate is selected from one or more of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly(3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly-L-lactide-caprolactone, and polydioxane, preferably polycaprolactone.

[0033] This invention can prepare the above-mentioned multifunctional electrospun artificial membrane material into artificial blood vessels, vascular patches, skin dressings, valved blood vessels, or heart valves. Preferably, in the embodiments of this invention, the above-mentioned multifunctional electrospun artificial membrane material can be prepared into artificial blood vessels with anti-inflammatory, antibacterial, antioxidant, and anticoagulant functions.

[0034] In this invention, the concentration of deferoxamine in the artificial membrane material is 0.04~0.16 mmol / L. In this invention, based on mass parts, the artificial blood vessel matrix material is 1 part, and the deferoxamine is 0.896 × 10⁻⁶ mmol / L. -4 ~3.584×10 -4 share.

[0035] Ferroptosis is a newly discovered cell death pathway, characterized by non-apoptotic cell death induced by reactive oxygen species (ROS) or iron-dependent mechanisms. Diabetic and elderly patients are often in a state of chronic inflammation and elevated levels of reactive oxygen species. Desferrioxamine (DFO), used in this invention, is an iron chelator that prevents oxidative damage by inhibiting the formation of iron-induced hydroxyl radicals, stabilizing hypoxia-inducible factor-1α (HIF1-α), upregulating vascular endothelial growth factor and angiopoietin-2, and enhancing angiogenesis.

[0036] This invention loads deferoxamine onto artificial blood vessels via ROS-responsive bonds (TK), enabling the slow, on-demand release of DFO in a high-level ROS environment, and giving the artificial blood vessels anti-inflammatory, antioxidant, antibacterial, and anticoagulant functions.

[0037] This invention provides an application of the multifunctional electrospun artificial membrane material described above in the preparation of artificial blood vessels, vascular patches, valved blood vessels, heart valves, or skin dressings.

[0038] Artificial blood vessels can reduce thrombus formation by releasing deferoxamine to inhibit platelet adhesion and activation.

[0039] This invention provides a multifunctional electrospun artificial blood vessel, the material of which is the multifunctional electrospun material described in the above technical solution.

[0040] This invention provides a method for preparing the multifunctional electrospun artificial blood vessel described above, comprising the following steps:

[0041] The electrospinning material substrate is dissolved in an organic solvent to obtain a spinning solution;

[0042] The spinning solution is electrospun to obtain an electrospun artificial blood vessel;

[0043] The electrospun artificial blood vessel was immersed in a functional solution containing deferoxamine to obtain a multifunctional electrospun artificial blood vessel.

[0044] This invention dissolves an electrospinning material substrate in an organic solvent to obtain a spinning solution. The electrospinning material substrate is preferably one or more of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly(3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly-L-lactide-caprolactone, and poly(p-dioxane-hexanone), with polycaprolactone being more preferred.

[0045] The organic solvent used in this invention is a mixture of chloroform and methanol, more preferably a mixture of chloroform and methanol in a volume ratio of 5:0.9~1.1; in a specific embodiment, the organic solvent is a mixture of chloroform and methanol in a volume ratio of 5:1.

[0046] In this invention, the concentration of the matrix material in the matrix solution is 0.04~0.16 mmol / L; the mass ratio of the matrix material to the deferoxamine powder is 1:(0.896×10⁻⁶). -4 ~3.584×10 -4 ).

[0047] In a specific embodiment, the concentration of polycaprolactone in the spinning solution is 0.25 g / mL. The concentration of the DFO solution is 0.04 mol / L, 0.08 mol / L, or 0.16 mol / L.

[0048] After obtaining the spinning solution, the present invention electrospins the spinning solution to obtain an electrospun artificial blood vessel.

[0049] In this invention, the spinning solution is preferably transferred to a 10mL syringe, and the syringe is inverted to remove air bubbles from the spinning solution. After the air bubbles are removed, a 21G needle is installed and fixed, and then the syringe is placed into the spinning device for electrospinning.

[0050] In this invention, the parameters of the electrospinning are:

[0051] The flow rate of the spinning solution is 7~9 mL / h, the high voltage DC voltage is 10~12 kV, and the receiving distance is 10~15 cm. In a specific embodiment, the flow rate of the spinning solution is 7 mL / h; the high voltage DC voltage is 11 kV; and the receiving distance is 10 cm.

[0052] If the product to be prepared is an electrospun membrane, the receiver for electrospinning is a grounded metal roller with a diameter of 15~20cm, the roller speed is 250~300rpm, and the spinning time is 20~25min.

[0053] If the product being prepared is an artificial blood vessel, the receiver for electrospinning is a grounded stainless steel rod with a diameter of 2 mm. The rotation speed of the stainless steel rod is 120~180 rpm, and the spinning time is 4~6 min. In a specific embodiment, the rotation speed of the stainless steel rod is 150 rpm, and the spinning time is 6 min.

[0054] After electrospinning, the present invention preferably soaks the electrospinned product in an alkaline solution to increase its hydrophilicity; the alkaline solution is preferably a 0.5 mol / L NaOH solution, and the soaking time is preferably 25-35 min.

[0055] After alkali treatment, the artificial blood vessels were washed three times with PBS, then immersed in MES (pH 5.6, 0.05 mol / L) for 25-35 minutes. They were then reacted in a mixed solution containing EDC, NHS, and amino-polyethylene glycol-ketithyl thioglycol-carboxyl groups (NH2-PEG-TK-COOH). If the electrospun material substrate was polycaprolactone (PCL), the carboxyl groups on the PCL surface would be exposed after alkali treatment, and these carboxyl groups would react with the amino groups of NH2-PEG-TK-COOH. In a specific embodiment, the mixed solution was 0.2% EDC + 0.12% NHS + 500 µM NH2-PEG-TK-COOH. The reaction temperature was 35-40°C, and the reaction time was 3.5-4.5 h; in a specific embodiment, the reaction temperature was 37°C, and the reaction time was 4 h. After the reaction was complete, the vessels were washed 3-6 times with PBS.

[0056] After washing, the electrospun artificial blood vessel is immersed in a functional solution containing deferoxamine to obtain a multifunctional electrospun artificial blood vessel. The concentration of deferoxamine in the functional solution is 0.04~0.16 mol / L; in specific embodiments, the concentrations of deferoxamine in the functional solution are 0.04 mol / L, 0.08 mol / L, and 0.16 mol / L. The immersion time is 55~65 min.

[0057] This invention achieves multiple functions by loading deferoxamine, greatly saving time and economic costs; the loading method is simple and does not affect the activity of deferoxamine.

[0058] This invention uses electrospun materials to fabricate artificial blood vessels, which can inhibit platelet adhesion and activation by releasing deferoxamine, thereby reducing thrombus formation. The deferoxamine released by the artificial blood vessels can resist oxidative stress and inflammation induced by high glucose and free fatty acid levels in diabetic and elderly patients, thus protecting endothelial cells and promoting long-term vascular patency and regeneration. The loaded deferoxamine can achieve long-term slow release according to the environment of the transplantation site. The released deferoxamine can prevent acute thrombus formation by inhibiting platelet adhesion and activation, and can also change the microenvironment of the transplantation site by resisting oxidative stress and inflammation, promoting cell infiltration and regeneration, and protecting regenerated cells.

[0059] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a multifunctional electrospun material and its application, as well as an electrospun artificial blood vessel and its preparation method. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] A chloroform-methanol solution with a volume ratio of 5:1 was added to a glass vial, followed by polycaprolactone (PCL) granules at a concentration of 0.25 g / mL. The vial was then tightened and sealed with sealing film. The vial was then placed on a magnetic stirrer and stirred continuously at room temperature in the dark for 12 hours. The final PCL concentration in the mixed solution was 0.25 g / mL. Electrospinning was then performed using the above solution to prepare electrospun PCL artificial blood vessels and electrospun membranes loaded with deferoxamine. Specific operations and parameters were as follows: The well-stirred spinning solution was transferred to a 10 mL syringe, and the syringe was inverted to remove air bubbles. After the air bubbles were removed, a 21G needle was installed and fixed, and the device was placed in the spinning apparatus for electrospinning. The solution flow rate was set to 7 mL / h, the high-voltage DC voltage to 11 kV, the electrospun membrane receiving distance to 10 cm, the receiver being a grounded stainless steel rod with a diameter of 15 cm, the rotation speed of the stainless steel rod to 250 rpm, and the spinning time to 25 min. The receiving distance of the artificial blood vessel was set to 10cm, the receiver was a grounded stainless steel rod with a diameter of 2mm, the rotation speed of the stainless steel rod was 150rpm, and the spinning time was 6min; the electrospun artificial blood vessel was prepared according to the above settings.

[0062] Electrospun artificial blood vessels were treated with alkali by soaking them in 0.5M NaOH for 30 minutes to increase their hydrophilicity.

[0063] After alkali treatment, the artificial blood vessels were washed three times with PBS, then immersed in MES (pH 5.6, 0.05 mol / L) for 0.5 h. Subsequently, the alkali-treated artificial blood vessels were placed in a mixed solution containing EDC, NHS, and amino-polyethylene glycol-ketithyl thioglycol-carboxyl group (NH2-PEG-TK-COOH) (0.2% EDC + 0.12% NHS + 500 µM NH2-PEG-TK-COOH) at 37°C for 4 h. After the reaction, the vessels were washed three times with PBS, immersed in 0.04 mol / L DFO solution for 1 h, washed five times with PBS after the reaction, and finally dried under nitrogen for later use.

[0064] Example 2

[0065] Add a chloroform-methanol solution (5:1 volume ratio) to a glass vial, then add polycaprolactone (PCL) granules at a rate of 0.25 g / mL. Tighten the cap and seal the vial with sealing film. Place the vial on a magnetic stirrer and stir continuously at room temperature in the dark for 12 hours. The final PCL concentration in the mixed solution is 0.25 g / mL. Electrospinning is then performed using the above solution to prepare electrospun PCL artificial blood vessels and electrospun membranes loaded with deferoxamine. Specific operations and parameters are as follows: Transfer the well-stirred spinning solution to a 10 mL syringe and invert the syringe to remove air bubbles. After removing the air bubbles, install and fix a 21G needle, and place the vial into the spinning apparatus for electrospinning. The solution flow rate is set to 7 mL / h, the high-voltage DC voltage is set to 11 kV, the electrospun membrane receiving distance is set to 10 cm, the receiver is a grounded stainless steel rod with a diameter of 15 cm, the rotation speed of the stainless steel rod is 250 rpm, and the spinning time is 25 min. The receiving distance of the artificial blood vessel was set to 10cm, the receiver was a grounded stainless steel rod with a diameter of 2mm, the rotation speed of the stainless steel rod was 150rpm, and the spinning time was 6min; the electrospun artificial blood vessel was prepared according to the above settings.

[0066] Electrospun artificial blood vessels were treated with alkali by soaking them in 0.5M NaOH for 30 minutes to increase their hydrophilicity.

[0067] After alkali treatment, the artificial blood vessels were washed three times with PBS, then immersed in MES (pH 5.6, 0.05 mol / L) for 0.5 h. Subsequently, the alkali-treated artificial blood vessels were placed in a mixed solution containing EDC, NHS, and amino-polyethylene glycol-ketithyl thioglycol-carboxyl group (NH2-PEG-TK-COOH) (0.2% EDC + 0.12% NHS + 500 µM NH2-PEG-TK-COOH) at 37°C for 4 h. After the reaction, the vessels were washed three times with PBS, immersed in 0.08 mol / L DFO solution for 1 h, washed five times with PBS after the reaction, and finally dried under nitrogen for later use.

[0068] Example 3

[0069] A chloroform-methanol solution with a volume ratio of 5:1 was added to a glass vial, followed by polycaprolactone (PCL) granules at a concentration of 0.25 g / mL. The vial was then tightened and sealed with sealing film. The vial was then placed on a magnetic stirrer and stirred continuously at room temperature in the dark for 12 hours. The final PCL concentration in the mixed solution was 0.25 g / mL. Electrospinning was then performed using the above solution to prepare electrospun PCL artificial blood vessels and electrospun membranes loaded with deferoxamine. Specific operations and parameters were as follows: The well-stirred spinning solution was transferred to a 10 mL syringe, and the syringe was inverted to remove air bubbles. After the air bubbles were removed, a 21G needle was installed and fixed, and the device was placed in the spinning apparatus for electrospinning. The solution flow rate was set to 7 mL / h, the high-voltage DC voltage to 11 kV, the electrospun membrane receiving distance to 10 cm, the receiver being a grounded stainless steel rod with a diameter of 15 cm, the rotation speed of the stainless steel rod to 250 rpm, and the spinning time to 25 min. The receiving distance of the artificial blood vessel was set to 10cm, the receiver was a grounded stainless steel rod with a diameter of 2mm, the rotation speed of the stainless steel rod was 150rpm, and the spinning time was 6min; the electrospun artificial blood vessel was prepared according to the above settings.

[0070] Electrospun artificial blood vessels were treated with alkali by soaking them in 0.5M NaOH for 30 minutes to increase their hydrophilicity.

[0071] After alkali treatment, the artificial blood vessels were washed three times with PBS, then immersed in MES (pH 5.6, 0.05 mol / L) for 0.5 h. Subsequently, the alkali-treated artificial blood vessels were placed in a mixed solution containing EDC, NHS, and amino-polyethylene glycol-ketithyl thioglycol-carboxyl group (NH2-PEG-TK-COOH) (0.2% EDC + 0.12% NHS + 500 µM NH2-PEG-TK-COOH) at 37°C for 4 h. After the reaction, the vessels were washed three times with PBS, immersed in 0.16 mol / L DFO solution for 1 h, washed five times with PBS after the reaction, and finally dried under nitrogen for later use.

[0072] Comparative Example 1

[0073] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, and polycaprolactone (PCL) granules were added at a rate of 0.25 g / mL. The vial was then tightened and sealed with sealing film. The vial was then placed on a magnetic stirrer and stirred continuously at room temperature in the dark for 12 hours. The concentration of PCL in the above mixed solution was 0.25 g / mL. Electrospinning was then performed using the above solution to prepare electrospun PCL artificial blood vessels and electrospun membranes. The specific operation and parameters are as follows: The well-stirred spinning solution was transferred to a 10 mL syringe, and the syringe was inverted to remove air bubbles from the spinning solution. After the air bubbles were removed, a 21G needle was installed and fixed, and the device was placed in the spinning apparatus for electrospinning. The flow rate of the solution was set to 7 mL / h, the high-voltage DC voltage was set to 11 kV, the receiving distance of the electrospun membrane was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 15 cm, the rotation speed of the stainless steel rod was 250 rpm, and the spinning time was 25 min. The receiving distance for the artificial blood vessel was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 2 mm, the rotation speed of the stainless steel rod was 150 rpm, and the spinning time was 6 min; the electrospun artificial blood vessel was prepared according to the above settings. After sterilization by cobalt-60 irradiation, it was ready for use.

[0074] Comparative Example 2

[0075] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, and polycaprolactone (PCL) granules were added at a rate of 0.25 g / mL. The vial was then tightened and sealed with sealing film. The vial was then placed on a magnetic stirrer and stirred continuously at room temperature in the dark for 12 hours. The concentration of PCL in the above mixed solution was 0.25 g / mL. Electrospinning was then performed using the above solution to prepare electrospun PCL artificial blood vessels and electrospun membranes. The specific operation and parameters are as follows: The well-stirred spinning solution was transferred to a 10 mL syringe, and the syringe was inverted to remove air bubbles from the spinning solution. After the air bubbles were removed, a 21G needle was installed and fixed, and the device was placed in the spinning apparatus for electrospinning. The flow rate of the solution was set to 7 mL / h, the high-voltage DC voltage was set to 11 kV, the receiving distance of the electrospun membrane was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 15 cm, the rotation speed of the stainless steel rod was 250 rpm, and the spinning time was 25 min. The receiving distance of the artificial blood vessel was set to 10cm, the receiver was a grounded stainless steel rod with a diameter of 2mm, the rotation speed of the stainless steel rod was 150rpm, and the spinning time was 6min; the electrospun artificial blood vessel was prepared according to the above settings.

[0076] The electrospun artificial blood vessels were soaked in 0.5M NaOH for 30 minutes to treat them with alkali, which increased their hydrophilicity. They were then sterilized by cobalt-60 irradiation and put into use.

[0077] Comparative Example 3

[0078] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, and polycaprolactone (PCL) granules were added at a rate of 0.25 g / mL. The vial was then tightened and sealed with sealing film. The vial was then placed on a magnetic stirrer and stirred continuously at room temperature in the dark for 12 hours. The concentration of PCL in the above mixed solution was 0.25 g / mL. Electrospinning was then performed using the above solution to prepare electrospun PCL artificial blood vessels and electrospun membranes. The specific operation and parameters are as follows: The well-stirred spinning solution was transferred to a 10 mL syringe, and the syringe was inverted to remove air bubbles from the spinning solution. After the air bubbles were removed, a 21G needle was installed and fixed, and the device was placed in the spinning apparatus for electrospinning. The flow rate of the solution was set to 7 mL / h, the high-voltage DC voltage was set to 11 kV, the receiving distance of the electrospun membrane was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 15 cm, the rotation speed of the stainless steel rod was 250 rpm, and the spinning time was 25 min. The receiving distance of the artificial blood vessel was set to 10cm, the receiver was a grounded stainless steel rod with a diameter of 2mm, the rotation speed of the stainless steel rod was 150rpm, and the spinning time was 6min; the electrospun artificial blood vessel was prepared according to the above settings.

[0079] After alkali treatment, the artificial blood vessels were washed three times with PBS, then immersed in MES (pH 5.6, 0.05 mol / L) for 0.5 h. Subsequently, the alkali-treated artificial blood vessels were placed in a mixed solution containing EDC and NHS (0.2% EDC + 0.12% NHS) at 37°C for 4 h. After completion, they were washed five times with PBS and finally dried under nitrogen for later use.

[0080] This invention characterizes and tests the microstructure, tensile strength, suture strength, burst pressure, release, antioxidant stress, anti-inflammatory, antibacterial, and anticoagulant functions of the artificial blood vessels prepared in Examples 1-3 and Comparative Examples 1-3. Simultaneously, a portion of the blood vessels were selected for in vivo implantation evaluation in diabetic rat models and aged rat models. The specific methods are as follows:

[0081] 1) Microstructure characterization:

[0082] The artificial blood vessels prepared in Examples 1-3 and Comparative Examples 1-3 were placed in liquid nitrogen, and the blood vessels were broken into transverse and longitudinal sections using a blade treated with liquid nitrogen. After fixation, they were sputtered with gold, photographed using a Phenom desktop scanning electron microscope (SEM), and the diameter of the blood vessels and the diameter of the fibers were measured using ImageJ.

[0083] 2) Tensile force characterization

[0084] The axial tensile force, radial tensile force, and suture strength of the artificial blood vessels prepared in Examples 1-3 and Comparative Examples 1-3 were tested using an Instron-3345 tensile testing machine.

[0085] The axial mechanical testing method is as follows: Fix the upper and lower ends of a 2.5cm long blood vessel to the upper and lower clamps of the tensile testing machine, ensuring that the distance between the upper and lower clamps is controlled at 1cm. Then input the various parameters of the sample, adjust the tensile speed to 20mm / min, set the tensile force in the software to zero, and start the tensile testing machine until the sample completely breaks. Obtain the Young's modulus, stress, and maximum elongation at break of the sample based on the stress-strain curve.

[0086] The radial mechanics test method is as follows: a sample with a length of about 5 mm is fixed to a tensile testing machine with a steel ring, the parameters of the sample are input, the tensile speed is adjusted to 10 mm / min, the tensile force in the software is zeroed, and the tensile testing machine is started until the sample is completely broken. The Young's modulus, stress and maximum elongation at break of the sample are obtained according to the stress-strain curve.

[0087] 3) Characterization of suture strength

[0088] The suture strength at the flat end (0°) of the artificial blood vessels prepared in Examples 1-3 and Comparative Examples 1-3 was tested according to ISO 7198:2016 A.5.7 standard. The test method is as follows: the blood vessel was cut into a small segment with a length of about 1 cm. 6-0 medical suture was passed through the sample tube wall about 2 mm from the horizontal end of the blood vessel. The suture was knotted to form a closed loop. The knotted suture was clamped with the air pump clamp of the tensile testing machine. The other end of the blood vessel was also clamped with the air pump clamp of the tensile testing machine. The tensile testing machine was balanced and zeroed. The tensile test was performed at a tensile speed of 8.00 mm / min until the 6-0 suture penetrated the end of the sample.

[0089] 4) Characterization of burst pressure

[0090] The artificial blood vessels prepared in Examples 1-3 and Comparative Examples 1-3 were tested according to section 8.3.3.3 of YY0500-2004 / ISO7198, which describes the pressure rupture strength – balloon method. The specific operation method is as follows: Vaseline lubricant was evenly applied to the balloon, and the balloon was carefully placed into the artificial blood vessel sample. One end of the blood vessel was directly connected to the catheter of the sensor, and the other end was sealed. CO2 gas was introduced at a rate of kPa / s until the blood vessel ruptured. The pressure change was recorded by a paperless pressure recorder on the other side of the sensor. The maximum pressure value was retrieved, and the rupture pressure of the blood vessel was calculated according to the conversion standard of kPa to mmHg.

[0091] 5) Antioxidant stress characteristics testing

[0092] Human umbilical vein endothelial cells (HUVECs) were cultured in ECM at a glucose concentration of 25 mmol / L for 24 hours to obtain high glucose-induced HUVECs. Electrospun membranes prepared in Examples 1-3 and Comparative Examples 1-3 were cut into 24-well plates and placed in 24-well plates, with 1 × 10⁻⁶ membranes per well. 4 High-glucose-induced HUVECs were added to the membrane. After 24 hours, the oxidative stress level of the cells was detected using the DCFH-DA probe. In this experiment, the fluorescence signal detected in Comparative Example 1 was defined as 100%.

[0093] 6) Detection of anti-inflammatory properties

[0094] RAW 264.7 macrophages were cultured in DMEM with a glucose concentration of 25 mmol / L for 24 hours to obtain high glucose-induced macrophages. Membranes from Examples 1-3 and Comparative Examples 1-3 were cut to 6-well plate size and placed in 6-well plates, with 3 × 10⁶ cells per well. 5 High-glucose-induced macrophages were added to the membrane and cultured in high-glucose medium for 3 days. Flow cytometry was then used to analyze the expression of iNOS (pro-inflammatory phenotype) and CD206 (anti-inflammatory phenotype) on the cell surface. iNOS is a pro-inflammatory phenotype marker, and CD206 is an anti-inflammatory phenotype marker; a higher percentage of CD206 indicates a better anti-inflammatory effect.

[0095] 7) Cell proliferation detection

[0096] Human umbilical vein endothelial cells (HUVECs) were cultured in ECM at a glucose concentration of 25 mmol / L for 24 hours to obtain high glucose-induced HUVECs. Membranes from Examples 1-3 and Comparative Examples 1-3 were cut to the size of 48-well plates and placed in 48-well plates, with 8 × 10⁸ cells per well. 3 High-glucose-induced HUVECs were added to the membrane, and after culturing for 3 days, the absorbance of the supernatant at 450 nm was detected using CCK-8 assay. The higher the absorbance, the better the cell proliferation.

[0097] 8) Antibacterial property testing

[0098] Weigh out 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar powder sequentially. Place them in a glass beaker, add 900 mL of distilled water, and stir thoroughly on a magnetic stirrer until dissolved and mixed. Then, bring the volume to 1 L. Transfer the solution to an Erlenmeyer flask, seal it, and sterilize it in an autoclave. When the temperature drops to approximately 60°C, pour the LB medium from the Erlenmeyer flask into a petri dish using a laminar flow hood. After solidification, add 0.2 mL of Staphylococcus aureus suspension to the petri dish and spread it evenly using a sterile triangular spreader. Place the membranes (6 mm in diameter) from Examples 1-3 and Comparative Examples 1-3 into the plates using sterile forceps and incubate at 37°C for 24 h. Finally, observe and measure the diameter of the inhibition zone. A larger inhibition zone diameter indicates stronger antibacterial ability.

[0099] 9) Anticoagulation property testing

[0100] Evaluation of platelet adhesion and activation: The membranes from Examples 1-3 and Comparative Examples 1-3 were cut into 48-well plates. 500 μL of platelet-rich plasma (PRP) was added to each well, and the plates were incubated at 37°C for 2 hours. Then, the adhered and activated platelets were quantified using a lactate dehydrogenase (LDH) kit and a soluble P-selectin ELISA kit, respectively. The absorbance at 490 nm was measured using a multi-mode microplate reader to represent platelet adhesion data; a higher absorbance value indicated more adhered platelets. The absorbance at 450 nm was measured using the same multi-mode microplate reader to represent platelet activation data; a higher absorbance value indicated more activated platelets.

[0101] 10) Evaluation of implantation

[0102] In vivo implantation in diabetic rats: SD rats were induced to develop diabetes by intraperitoneal injection of streptozotocin (STZ). Vascular materials prepared in Examples 1-3 and Comparative Examples 1-3 were cut into 1.1 cm pieces and implanted into the abdominal aorta of rats using interrupted end-to-end sutures with 9-0 sutures. One month after transplantation, the patency of the vessels was assessed using Vevo 2100 Doppler ultrasound. Subsequently, the vascular tissue was harvested and longitudinally sectioned. The presence of thrombus formation on the vascular lumen was observed under a stereomicroscope. After fixation with 2.5% glutaraldehyde and graded alcohol dehydration, the presence of endothelial coverage and adhesion to the coagulation matrix on the vascular lumen was observed using scanning electron microscopy. One sample was placed in OCT tissue embedding medium, flash-frozen in liquid nitrogen, and analyzed as a frozen section. Immunofluorescence staining with CD31 antibody was performed, and the endothelial coverage rate was calculated as: vascular endothelial coverage rate = (length of CD31-positive vascular lumen on longitudinal section / total length of vascular lumen on longitudinal section) × 100%. Inflammatory cells in blood vessels were assessed using CD206 and iNOS antibodies. Five regions were randomly selected from each sample for 40x magnification imaging, and CD206 levels were statistically analyzed.+ Cells and iNOS + The proportion of cells.

[0103] In vivo implantation in aged rats: SD rats were raised to 18 months of age. After anesthesia, the vascular materials prepared in Examples 1-3 and Comparative Examples 1-3 were cut into 1.1 cm pieces and implanted into the abdominal aorta of rats using interrupted end-to-end sutures with 9-0 sutures. One month after transplantation, the patency of the vessels was tested using Vevo 2100 Doppler ultrasound. Afterwards, the vascular tissue was harvested and longitudinally sectioned. The presence of thrombus formation on the vascular lumen was observed under a stereomicroscope. The tissue was then fixed with 2.5% glutaraldehyde and dehydrated using a gradient alcohol treatment. The presence of endothelial coverage and coagulation matrix adhesion on the vascular lumen was observed using scanning electron microscopy. One sample was placed in OCT tissue embedding medium, flash-frozen in liquid nitrogen, and analyzed as a frozen section. Immunofluorescence staining with CD31 antibody was performed, and the endothelial coverage rate was calculated as: vascular endothelial coverage rate = (length of CD31-positive vascular lumen on longitudinal section / total length of vascular lumen on longitudinal section) × 100%. Inflammatory cells in blood vessels were assessed using CD206 and iNOS antibodies. Five regions were randomly selected from each sample for 40x magnification imaging, and CD206 levels were statistically analyzed. + Cells and iNOS + The proportion of cells.

[0104] 11) Data Analysis

[0105] The data were analyzed using GraphPad Prism 9.0.0 software, and all data were expressed as mean ± standard error.

[0106] Table 1. Microscopic and mechanical characterization of blood vessels (parallel experiments, n=3, mean ± standard error)

[0107]

[0108] The results in Table 1 show that, compared with Comparative Example 1, Examples 1-3 did not show significant changes in diameter, fiber diameter, pore size, radial maximum stress, radial Young's modulus, radial elongation at break, axial maximum stress, axial Young's modulus, axial elongation at break, suture strength, and burst pressure. This indicates that the introduction of DFO does not change the microstructure and mechanical properties of blood vessels.

[0109] Table 2. Test results for antioxidant stress, anti-inflammatory, antibacterial, and anticoagulant properties (parallel experiments, n=3, average value ± standard error).

[0110]

[0111] The results in Table 2 show that the oxidative stress level of Comparative Example 1 was defined as 100%. The values ​​of Comparative Examples 2 and 3 were comparable to those of Comparative Example 1, indicating that the material did not change the oxidative stress level. After the introduction of DFO, the oxidative stress level of the examples was significantly reduced compared to their comparative examples, and the higher the DFO concentration, the lower the value. Figure 1 This indicates that the introduction of DFO can increase the antioxidant stress capacity of blood vessels.

[0112] The iNOS detection results in Table 2 show the iNOS levels in comparative examples 1-3. + The number of inflammatory cells was around 49%, and after the introduction of DFO, the corresponding iNOS... + The number of inflammatory cells decreased in all cases, and statistical data showed that these changes were significantly different. Figure 2 The detection results for CD206 showed that CD206 in Comparative Examples 1-3... + The number of anti-inflammatory cells was only 12%, while the corresponding CD206 in Examples 1-3 + The number of anti-inflammatory cells increased in all cases, and statistical data showed that all of these changes were statistically significant. Figure 3 The results above show that the introduction of DFO can increase the anti-inflammatory capacity of blood vessels.

[0113] Cell proliferation detection data showed that OD ratios of 1-3 were... 450 The value was around 0.55, while the values ​​for Examples 1-3 decreased, especially the value for Example 3, which showed a significant change. Figure 4 This indicates that high concentrations of DFO inhibit cell proliferation, while low concentrations of DFO have no significant effect on cell proliferation.

[0114] The results of the inhibition zone diameter test showed that the diameter of the inhibition zones in Comparative Examples 1-3 was about 6 mm, which is the same as the size of the sample, indicating that Comparative Examples 1-3 had no antibacterial or bacteriostatic ability. However, the inhibition zones in Examples 1-3 all showed an increase. Statistical data showed that the above changes were all significantly different. Figure 5 This indicates that the presence of DFO increases the antibacterial ability of blood vessels.

[0115] The platelet adhesion test results in Table 2 show that there was no difference in platelet adhesion in Comparative Examples 1-3. However, after the introduction of DFO, the number of platelets adhering in the corresponding examples decreased. Statistical data show that the above changes are significant. Figure 6 The data on platelet activation showed that platelet activation was observed in all three comparative examples (1-3) with no significant difference, while the number of activated platelets decreased in all three corresponding examples (1-3). Statistical data showed that all of these changes were significantly different. Figure 7 The results above show that the introduction of DFO reduces platelet adhesion and activation, and increases the anticoagulant properties of blood vessels.

[0116] Table 3. Evaluation of vascular implantation in diabetic rats (parallel experiments, n=3, mean ± standard error)

[0117]

[0118] Results of vascular implantation in diabetic rats showed that, one month after implantation, all vascularization methods in Comparative Examples 1-3 resulted in thrombosis and blockage. However, the patency rates in Examples 1-3 were significantly improved, with Examples 2-3 achieving 100% patency, indicating that the introduction of DFO significantly improves vascular patency. Endothelial coverage results showed that, one month after implantation in diabetic rats, the endothelial coverage of Comparative Examples 1-3 was approximately 18%, while the endothelial coverage rates of the corresponding examples all increased, especially in Example 2, which reached approximately 50%, significantly higher than the other examples. In vivo staining results showed that Comparative Examples 1-3 contained a relatively high amount of iNOS. + Inflammatory cells, with only a small number of CD206 + Anti-inflammatory cells, and the presence of DFO reduced the number of inflammatory cells and increased the number of anti-inflammatory cells in the examples, which is consistent with the results of in vitro experiments. Figure 2 and Figure 3 The results above show that the introduction of DFO can significantly improve vascular patency, increase the anti-inflammatory properties of blood vessels, and promote vascular endothelialization, especially when the final concentration of DFO is 0.08 mol / L, it has a better effect on promoting endothelialization.

[0119] Table 4. Evaluation of vascular implantation in aged rats (parallel experiments, n=3, mean ± standard error).

[0120]

[0121] One month after vascular implantation in aged rats, Comparative Example 1 was completely blocked due to thrombosis, while Comparative Examples 2 and 3 showed only 1 / 3 patency, with visible thrombi present in the patent vessels. Examples 1-3, however, showed improved patency rates, especially Examples 2-3 which achieved 100% patency, indicating that the introduction of DFO significantly improves vascular patency. Endothelial coverage results showed that one month after implantation in aged rats, Comparative Example 2 had approximately 12% endothelial coverage, while the corresponding examples all showed increased endothelial coverage, particularly Example 2, which showed a significant increase. In vivo staining results showed that Comparative Examples 1 and 2 contained relatively high levels of iNOS. + Inflammatory cells, with only a small number of CD206+ Anti-inflammatory cells were observed, but in the example, the number of inflammatory cells decreased while the number of anti-inflammatory cells increased, which is consistent with the in vitro experimental results. Figure 2 and Figure 3 The results above show that the introduction of DFO can significantly improve vascular patency, increase the anti-inflammatory properties of blood vessels, and promote vascular endothelialization, especially when the final concentration of DFO is 0.08 mol / L, it has a better effect on promoting endothelialization.

[0122] As can be seen from the above embodiments, the present invention provides a multifunctional electrospun material, comprising an electrospun material substrate and deferoxamine. This electrospun material possesses anti-inflammatory, antioxidant, antibacterial, and anticoagulant functions; when applied to artificial blood vessel fabrication, it can improve the regenerative capacity of blood vessels, especially by protecting vascular cells through inhibiting inflammation and hyperglycemia-induced oxidative stress in diabetic and elderly patients, thereby promoting vascular regeneration. The loaded deferoxamine can achieve long-term, slow release according to the environment of the transplantation site. The released deferoxamine can prevent acute thrombosis by inhibiting platelet adhesion and activation, and can also protect the microenvironment of the transplantation site by resisting oxidative stress and inflammation levels, promoting cell infiltration and regeneration, and protecting regenerated cells.

[0123] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multifunctional electrospun material, characterized in that, Including electrospun material substrates and deferoxamine; Deferroamine is covalently loaded onto an electrospun material substrate; Covalent bonds exhibit ROS responsiveness; The mass ratio of the electrospun material substrate to deferoxamine is 1:(0.896×10⁻⁶). -4 ~3.584×10 -4 ); The multifunctional electrospun material, when applied to the fabrication of artificial blood vessels, can enhance the regenerative capacity of blood vessels and protect vascular cells by inhibiting inflammation and hyperglycemia-induced oxidative stress in diabetic and elderly patients, thereby promoting vascular regeneration. Specifically, the following steps are included: The electrospinning material substrate is dissolved in an organic solvent to obtain a spinning solution; The spinning solution was electrospun, and the electrospun product was soaked in an alkaline solution to obtain an alkaline-treated electrospun artificial blood vessel. The alkaline-treated electrospun artificial blood vessel was washed three times with PBS, and then soaked in MES at pH 5.6 and a concentration of 0.05 mol / L for 25-35 min. It was then placed in a mixed solution containing EDC, NHS, and amino-polyethylene glycol-ketethioyl thioglycol-carboxyl NH2-PEG-TK-COOH for reaction. The reacted electrospun artificial blood vessel was then soaked in a functional solution containing deferoxamine.

2. The multifunctional electrospun material according to claim 1, characterized in that, Deferroamine exists as a functional solution containing deferroamine; The concentration of deferoxamine in the functional solution containing deferoxamine is 0.04~0.16 mol / L.

3. The multifunctional electrospun material according to claim 1, characterized in that, The base material of the electrospun material is selected from one or more of polycaprolactone, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly(3-hydroxybutyrate-co-4-hydroxybutyrate), polylactic acid, poly-L-lactide-caprolactone, and poly(p-dioxane-hexanone).

4. The use of the multifunctional electrospun material according to any one of claims 1 to 3 in the preparation of artificial blood vessels, vascular patches, valved blood vessels, heart valves or skin dressings.

5. A multifunctional electrospun artificial blood vessel, characterized in that, The material is the multifunctional electrospun material as described in claim 1.

6. A method for preparing the multifunctional electrospun artificial blood vessel according to claim 5, comprising the following steps: The electrospinning material substrate is dissolved in an organic solvent to obtain a spinning solution; The spinning solution was electrospun, and the electrospun product was soaked in an alkaline solution to obtain an alkaline-treated electrospun artificial blood vessel. The alkaline-treated electrospun artificial blood vessel was washed three times with PBS, and then soaked in MES at pH 5.6 and a concentration of 0.05 mol / L for 25-35 min. It was then placed in a mixed solution containing EDC, NHS, and amino-polyethylene glycol-ketithiolide-carboxyl NH2-PEG-TK-COOH for reaction. The reacted electrospun artificial blood vessel was then soaked in a functional solution containing deferoxamine to obtain a multifunctional electrospun artificial blood vessel.

7. The preparation method according to claim 6, characterized in that, The organic solvent is a mixture of chloroform and methanol in a volume ratio of 5:0.9~1.

1.

8. The preparation method according to claim 6, characterized in that, The concentration of deferoxamine powder in the functional solution containing deferoxamine is 0.04~0.16 mol / L; The mass ratio of the electrospun material substrate to the deferoxamine powder is 1:(0.896×10⁻⁶). -4 ~3.584×10 -4 ).

9. The preparation method according to claim 6, characterized in that, The parameters of the electrospinning are as follows: The flow rate of the spinning solution is 7~9 mL / h, the high voltage DC voltage is 10~12 kV, and the receiving distance is 10~15 cm.

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