Preparation method of expanded polytetrafluoroethylene small-caliber artificial blood vessel

Abstract

The invention discloses a preparation method of an expanded polytetrafluoroethylene (ePTFE) small-caliber artificial blood vessel based on directly grafted heparin of polydopamine nanoparticles. The method comprises the following steps: performing plasma treatment on a cleaned ePTFE small-caliber artificial blood vessel, grafting PDA-NPs on the treated artificial blood vessel, activating heparin by using EDCHCl and NHS, and reacting the ePTFE / PDA / HEP small-caliber artificial blood vessel grafted with the PDA-NPs with an activated heparin solution to obtain the heparin-grafted ePTFE / PDA / HEP small-caliber artificial blood vessel. And finally, washing and freeze-drying to obtain the artificial blood vessel. By utilizing the synergistic effect of plasma treatment and PDA-NPs modification, the loading capacity and stability of heparin are remarkably improved, the prepared artificial blood vessel has excellent anti-platelet adhesion performance, extremely low hemolysis rate and good biocompatibility, no toxic coupling agent needs to be added in the whole process, the safety is high, the process is stable, and the preparation method is suitable for industrial production. The method has a wide application prospect in the field of clinical transformation of small-caliber artificial blood vessels.

Description

Technical Field

[0001] This invention relates to the field of artificial blood vessel technology, specifically to a method for preparing small-diameter expanded polytetrafluoroethylene (ePTFE) artificial blood vessels based on heparin directly grafted onto polydopamine nanoparticles. Background Technology

[0002] Current main treatments for cardiovascular diseases include antithrombotic drug therapy, vascular stenting, and vascular replacement surgery, including bypass surgery. Vascular replacement surgery is an effective clinical treatment for severe cardiovascular diseases, and artificial blood vessels, as the core medical implant material for this type of surgery, have been widely used in surgical treatments such as coronary artery bypass grafting. Their performance directly determines the surgical efficacy and the patient's postoperative quality of life. As a medical material implanted in the human body, artificial blood vessels must meet stringent performance indicators, including good biocompatibility, excellent mechanical properties that match human blood vessels, durable antithrombotic properties, and compliance with physiological circulation. ePTFE, due to its excellent mechanical strength, stable biocompatibility, excellent bioinertness, and fatigue resistance, has become a clinically recognized safe implantable material system. It has been widely used clinically with no significant adverse reactions, demonstrating long-term in vivo stability, and is one of the preferred materials for artificial blood vessels.

[0003] However, the surface of ePTFE materials exhibits strong inertness and high hydrophobicity, which can easily lead to protein adsorption and platelet adhesion and aggregation after implantation in the human body, resulting in complications such as thrombosis and postoperative restenosis, severely limiting its clinical application in areas such as small-diameter artificial blood vessels. To improve its surface properties, surface modification techniques are usually required to enhance the material's hydrophilicity and cell compatibility. Currently common modification methods include chemical treatment, plasma treatment, and layer-by-layer self-assembly, but these methods each have their shortcomings in terms of modification stability, environmental friendliness, and feasibility for large-scale production. Some processes require the introduction of coupling agents to enhance the bonding between the modified layer and the substrate, but these chemical reagents may pose potential risks of cytotoxicity or immune reactions, further increasing the complexity of clinical applications and making it difficult to simultaneously meet the dual clinical requirements for modification efficacy and biosafety.

[0004] Existing research indicates that single surface modification techniques often have inherent limitations. For example, while plasma treatment can rapidly introduce active groups, its effects are difficult to sustain; and while simple polydopamine coatings enhance hydrophilicity, their function is singular and cannot synergistically achieve the dual goals of antithrombosis and endothelialization. Therefore, employing two or more modification techniques in a synergistic manner to complement each other's advantages has become an important direction for improving the performance of ePTFE. Among them, the combined strategy of plasma treatment with polydopamine grafting and heparin loading is considered to synergistically improve the hydrophilicity and biocompatibility of ePTFE. However, this combined technique still faces problems such as poor process integration, low stability of the modified layer, and insufficient precision in regulating heparin loading and release behavior, resulting in modified ePTFE materials still not fully meeting clinical implantation requirements. In summary, developing a rationally designed, stable, and non-toxic ePTFE combined surface modification technique to systematically address its challenges in hydrophobicity, biocompatibility, and long-term safety is of great significance for promoting the clinical translation of ePTFE artificial blood vessels. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and propose a method for preparing expanded polytetrafluoroethylene (ePTFE) small-diameter artificial blood vessels based on direct grafting of heparin onto polydopamine nanoparticles. This method provides a simplified, mild, biocompatible, and high-performance modification method that is beneficial for clinical application.

[0006] To achieve the above-mentioned technical objectives, the present invention provides a method for preparing a small-diameter artificial blood vessel made of expanded polytetrafluoroethylene (ePTFE), comprising the following steps:

[0007] Step 1: After cleaning the ePTFE small-diameter artificial blood vessel, cut it into several samples with a length of 1~10 cm, and then perform plasma treatment on the cleaned ePTFE small-diameter artificial blood vessel.

[0008] Step 2: Graft polydopamine nanoparticles (PDA-NPs) onto the treated ePTFE small-diameter artificial blood vessel obtained in Step 1 to prepare an ePTFE / PDA small-diameter artificial blood vessel.

[0009] Step 3: Activate heparin (HEP) with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS), and then react the ePTFE / PDA small-diameter artificial blood vessel obtained in step 2 with the activated HEP solution to obtain heparin-grafted ePTFE / PDA / HEP small-diameter artificial blood vessel;

[0010] Step 4: Use deionized water to thoroughly wash the ePTFE / PDA / HEP small-diameter artificial blood vessel obtained in step 3, and then freeze-dry it to obtain the expanded polytetrafluoroethylene small-diameter artificial blood vessel.

[0011] In some technical solutions of the present invention, the plasma processing gas in step 1 is oxygen or air.

[0012] In some technical solutions of this invention, the plasma treatment power in step 1 is 80-120 W, the pressure is 20-40 Pa, and the treatment time is 4-8 min. More preferably, the treatment time is 5-7 min. Plasma treatment can provide sufficient contact sites for the PDA load, thereby increasing the grafting amount and binding stability of heparin. As the plasma treatment time increases, hydrophilic groups can be introduced onto the material surface to increase the binding affinity with PDA. However, if the treatment time exceeds 6 min, the surface hydrophilic groups may begin to decompose, thus affecting the binding ability with PDA. If the treatment time is too long, exceeding 8 min, excessive etching occurs on the blood vessel surface, causing surface structure collapse, ultimately making it difficult to form a stable binding force with PDA, and thus making it difficult to graft sufficient heparin.

[0013] In some technical solutions of the present invention, the grafting of polydopamine nanoparticles in step 2 includes at least the following steps:

[0014] Step 2.1: Prepare a tris(hydroxymethyl)aminomethane (Tris) solution, adjust the Tris solution system with dilute hydrochloric acid, add dopamine hydrochloride to the Tris solution and stir at room temperature, then centrifuge to obtain the precipitate.

[0015] Step 2.2: Place the precipitate in a vacuum drying oven and dry it under vacuum at room temperature to obtain polydopamine nanoparticles (PDA-NPs).

[0016] Step 2.3: Prepare a PDA-NPs solution using phosphate buffered saline (PBS), and then react the plasma-treated ePTFE small-diameter artificial blood vessel with the PDA-NPs solution to form a PDA coating on its surface, thus obtaining the ePTFE / PDA small-diameter artificial blood vessel.

[0017] In some preferred embodiments of the present invention, the concentration of the Tris solution in step 2.1 is 10-20 mM. The Tris buffer solution can stably maintain the pH value of the system from the influence of dopamine oxidation, ensuring the controllability of the dopamine self-polymerization reaction. When the concentration is too low, the buffering capacity is insufficient, and when the concentration is too high, Tris ions will accelerate the PDA oxidation self-polymerization rate.

[0018] In some preferred embodiments of the present invention, the pH value of the Tris solution in step 2.1 is 8 to 8.5. When pH < 8, the oxidation rate of PDA slows down significantly; when pH > 8.5, the oxidation self-polymerization rate of PDA is too fast, forming large aggregates with uneven particle size.

[0019] In some preferred embodiments of the present invention, in step 2.1, dopamine hydrochloride is added to the Tris solution at a rate of 1.5~2.5 g / L. Within this dropping rate range, dopamine hydrochloride can obtain a suitable self-polymerization rate. If the dropping rate is too fast, the local concentration is too high, causing rapid cross-linking. If the concentration is too low, the self-polymerization rate will be too slow. The stirring time is 12~24 h to ensure that the reaction is complete.

[0020] In some preferred embodiments of the present invention, the vacuum drying time in step 2.2 is 4 to 8 hours.

[0021] In some preferred embodiments of the present invention, the contact reaction time in step 2.3 is 22-26 hours. Within this contact reaction time range, a suitable grafting amount of PDA-NPs can be obtained. When the reaction time is further increased, the grafting amount of PDA-NPs tends to saturate and will not increase further.

[0022] In some technical solutions of the present invention, the heparin activation step in step 3 mainly includes the following steps: First, dissolve sodium heparin in MES buffer; then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS) and disperse by ultrasonication; then incubate the solution in a 37°C constant temperature shaker to activate heparin.

[0023] In some preferred embodiments of the present invention, the 2-(N-morpholine)ethanesulfonic acid (MES) solution has a mass fraction of 0.5-2 wt% and a pH value of 5-6.

[0024] In some preferred embodiments of the present invention, the molar ratio of MES:heparin sodium:EDC•HCl:NHS is 25:(4~6):(4~6):(2~4). Within this ratio range, heparin can be sufficiently activated for grafting.

[0025] In some preferred embodiments of the present invention, the incubation time in a constant temperature oscillator at 37 °C is 20-40 min. Too short an incubation time will result in incomplete activation, while too long an incubation time will lead to a decrease in the effective activation rate due to the hydrolysis of the active intermediate.

[0026] In some technical solutions of the present invention, the reaction time of the ePTFE / PDA small-diameter artificial blood vessel with the activated HEP solution in step 3 is 22~26h.

[0027] In some technical solutions of the present invention, the freeze-drying time in step 4 is 24~48h.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. This invention uses plasma treatment technology to improve the defects of ePTFE surface lacking polar functional groups and low reactivity; moreover, plasma treatment is energy-saving and environmentally friendly, and the treatment effect is stable and reliable. The modification treatment only acts on the superficial layer of small-diameter artificial blood vessels, avoiding damage to the matrix performance of small-diameter artificial blood vessels.

[0030] 2. This invention eliminates the need for coupling agents, improving the biocompatibility of ePTFE small-diameter artificial blood vessels and reducing the risk of immune reactions.

[0031] 3. The present invention uses PDA-NPs coating to construct a multifunctional intermediate layer, which enhances interfacial bonding and biocompatibility. This not only gives it excellent biocompatibility and cell adhesion, but also constructs a continuous, dense, and strongly adhesive intermediate layer, providing sufficient reaction sites for subsequent heparin grafting, thus achieving a triple functional synergy of "activating the substrate - biocompatibility - site reserve".

[0032] 4. The three steps of this invention—plasma treatment, PDA-NPs coating, and direct heparin grafting—work in close synergy: plasma treatment provides binding sites for PDA-NPs, PDA-NPs provide a functional platform for heparin grafting, and heparin grafting imparts the core anticoagulant properties. These three steps are progressive and indispensable, ultimately achieving the comprehensive modification goal of "high binding strength, high biocompatibility, and long-lasting anticoagulation." The entire process is simple, controllable, environmentally friendly, safe, and efficient, requiring no complex equipment or stringent conditions, making it suitable for mass production. It opens up new methods for surface modification of small-diameter artificial blood vessels and has broad clinical application prospects. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 A schematic diagram illustrating the process of preparing ePTFE / PDA / HEP small-diameter blood vessels according to an embodiment of the present invention;

[0035] Figure 2 Scanning electron microscope images of platelet adsorption in the blank group, comparative example and embodiment of the present invention;

[0036] Figure 3 Coagulation test diagrams of the blank group, comparative example, and embodiments of this invention;

[0037] Figure 4 Hemolysis test diagrams of the blank group, comparative example, and embodiments of this invention;

[0038] Figure 5 Fluorescence micrographs of cytotoxicity tests in the blank control group, comparative example, and embodiments of this invention. Detailed Implementation

[0039] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. These described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0040] Example 1: A method for preparing a small-diameter artificial blood vessel of expanded polytetrafluoroethylene (ePTFE) based on heparin directly grafted onto polydopamine nanoparticles, the specific steps of which are as follows:

[0041] Step 1: After cleaning the ePTFE small-diameter artificial blood vessel, cut it into several samples with a length of 5 cm. Treat the cleaned ePTFE small-diameter artificial blood vessel with oxygen plasma, adjusting the power to 100 W and maintaining the pressure at 30 Pa. Place the cut samples in the reaction chamber of the plasma treatment equipment, and treat for 240 s. After treatment, remove the samples and place them in a desiccator for later use.

[0042] Step 2: Graft polydopamine onto the ePTFE small-diameter artificial blood vessel from Step 1. Prepare a 10 mM Tris solution, adjust the pH to approximately 8.5 with dilute hydrochloric acid, and add dopamine hydrochloride to the Tris solution at a rate of 2 g / L. Stir at room temperature for 24 h, then centrifuge to obtain the precipitate and wash it. Place the precipitate in a vacuum drying oven and vacuum dry at room temperature for 6 h to obtain polydopamine nanoparticles (PDA-NPs). Add the obtained PDA-NPs solution to PBS (pH=7.2) and ultrasonically disperse. Completely immerse the plasma-treated ePTFE small-diameter artificial blood vessel in the solution and shake in the dark at 37 ℃ for 24 h. After the reaction, remove the small-diameter artificial blood vessel and rinse it repeatedly with deionized water three times to remove unreacted DA and PDA oligomers on the surface. Then, dry it in a vacuum drying oven at 60 ℃ for 2 h. The sample modified with polydopamine is called ePTFE / PDA small-diameter artificial blood vessel.

[0043] Step 3: Dissolve heparin sodium in 1 wt% MES buffer, adjust the pH to 5, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS) and sonicate to disperse the solution. The ratio of MES:heparin sodium:EDC•HCl:NHS is 25:5:5:3. Incubate the solution in a 37 ℃ constant temperature shaker for 30 min to complete heparin (HEP) activation. Completely immerse the ePTFE+HEP small-diameter artificial blood vessel in the heparin solution and react at 37 ℃ for 24 h.

[0044] Step 4: Obtain ePTFE / PDA / HEP small-diameter artificial blood vessels. After the reaction in Step 3 is completed, the sample is removed and thoroughly washed with deionized water to remove unbound heparin, and finally freeze-dried for 24 hours. The sample modified with polydopamine and heparin is called ePTFE / PDA / HEP small-diameter artificial blood vessel.

[0045] Example 2: A method for preparing a small-diameter artificial blood vessel of expanded polytetrafluoroethylene (ePTFE) based on heparin directly grafted onto polydopamine nanoparticles, the specific steps of which are as follows:

[0046] Step 1: After cleaning the ePTFE small-diameter artificial blood vessel, cut it into several samples with a length of 5 cm. Treat the cleaned ePTFE small-diameter artificial blood vessel with oxygen plasma, adjusting the power to 100 W and maintaining the pressure at 30 Pa. Place the cut samples in the reaction chamber of the plasma treatment equipment, and treat for 360 s. After treatment, remove the samples and place them in a desiccator for later use.

[0047] Step 2: Graft polydopamine onto the ePTFE small-diameter artificial blood vessel from Step 1. Prepare a 10 mM Tris solution, adjust the pH to approximately 8.5 with dilute hydrochloric acid, and add dopamine hydrochloride to the Tris solution at a rate of 2 g / L. Stir at room temperature for 24 h, then centrifuge to obtain the precipitate and wash it. Place the precipitate in a vacuum drying oven and vacuum dry at room temperature for 6 h to obtain polydopamine nanoparticles (PDA-NPs). Add the obtained PDA-NPs solution to PBS (pH=7.2) and ultrasonically disperse. Completely immerse the plasma-treated ePTFE small-diameter artificial blood vessel in the solution and shake in the dark at 37 ℃ for 24 h. After the reaction, remove the small-diameter artificial blood vessel and rinse it repeatedly with deionized water three times to remove unreacted DA and PDA oligomers on the surface. Then, dry it in a vacuum drying oven at 60 ℃ for 2 h. The sample modified with polydopamine is called ePTFE / PDA small-diameter artificial blood vessel.

[0048] Step 3: Dissolve heparin sodium in 1 wt% MES buffer, adjust the pH to 5, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS) and sonicate to disperse the solution. The ratio of MES:heparin sodium:EDC•HCl:NHS is 25:5:5:3. Incubate the solution in a 37 ℃ constant temperature shaker for 30 min to complete heparin (HEP) activation. Completely immerse the ePTFE+HEP small-diameter artificial blood vessel in the heparin solution and react at 37 ℃ for 24 h.

[0049] Step 4: Obtain ePTFE / PDA / HEP small-diameter artificial blood vessels. After the reaction in Step 3 is completed, the sample is removed and thoroughly washed with deionized water to remove unbound heparin, and finally freeze-dried for 24 hours. The sample modified with polydopamine and heparin is called ePTFE / PDA / HEP small-diameter artificial blood vessel.

[0050] Example 3: A method for preparing a small-diameter artificial blood vessel of expanded polytetrafluoroethylene (ePTFE) based on heparin directly grafted onto polydopamine nanoparticles, the specific steps of which are as follows:

[0051] Step 1: After cleaning the ePTFE small-diameter artificial blood vessel, cut it into several samples with a length of 5 cm. Treat the cleaned ePTFE small-diameter artificial blood vessel with oxygen or air plasma, adjusting the power to 100 W and maintaining the pressure at 30 Pa. Place the cut samples in the reaction chamber of the plasma treatment equipment, and treat for 360 s. After treatment, remove the samples and place them in a desiccator for later use.

[0052] Step 2: Graft polydopamine onto the ePTFE small-diameter artificial blood vessel from Step 1. Prepare a 10 mM Tris solution, adjust the pH to approximately 8.5 with dilute hydrochloric acid, and add dopamine hydrochloride to the Tris solution at a rate of 2 g / L. Stir at room temperature for 24 h, then centrifuge to obtain the precipitate and wash it. Place the precipitate in a vacuum drying oven and vacuum dry at room temperature for 6 h to obtain polydopamine nanoparticles (PDA-NPs). Add the obtained PDA-NPs solution to PBS (pH=7.2) and ultrasonically disperse. Completely immerse the plasma-treated ePTFE small-diameter artificial blood vessel in the solution and shake in the dark at 37 ℃ for 24 h. After the reaction, remove the small-diameter artificial blood vessel and rinse it repeatedly with deionized water three times to remove unreacted DA and PDA oligomers on the surface. Then, dry it in a vacuum drying oven at 60 ℃ for 2 h. The sample modified with polydopamine is called ePTFE / PDA small-diameter artificial blood vessel.

[0053] Step 3: Dissolve heparin sodium in 1 wt% MES buffer, adjust the pH to 5, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS) and sonicate to disperse the mixture. The ratio of MES:heparin sodium:EDC•HCl:NHS is 25:5:5:3. Incubate the solution in a 37 ℃ constant temperature shaker for 20 min to complete heparin (HEP) activation. Completely immerse the ePTFE+HEP small-diameter artificial blood vessel in the heparin solution and react at 37 ℃ for 24 h.

[0054] Step 4: Obtain ePTFE / PDA / HEP small-diameter artificial blood vessels. After the reaction in Step 3 is completed, the sample is removed and thoroughly washed with deionized water to remove unbound heparin, and finally freeze-dried for 24 hours. The sample modified with polydopamine and heparin is called ePTFE / PDA / HEP small-diameter artificial blood vessel.

[0055] Comparative Example 1: A method for preparing a small-diameter artificial blood vessel of expanded polytetrafluoroethylene (ePTFE) based on direct grafting of heparin onto polydopamine nanoparticles, the specific steps of which are as follows:

[0056] Step 1: After cleaning, the ePTFE small-diameter artificial blood vessel is cut into several 5 cm long samples for further processing. This refers to the untreated ePTFE small-diameter artificial blood vessel.

[0057] Step 2: Graft polydopamine onto the ePTFE small-diameter artificial blood vessel from Step 1. Prepare a 10 mM Tris solution, adjust the pH to approximately 8.5 with dilute hydrochloric acid, and add dopamine hydrochloride to the Tris solution at a rate of 2 g / L. Stir at room temperature for 24 h, then centrifuge to obtain the precipitate and wash it. Place the precipitate in a vacuum drying oven and vacuum dry at room temperature for 6 h to obtain polydopamine nanoparticles (PDA-NPs). Add the obtained PDA-NPs solution to PBS (pH=7.2) and ultrasonically disperse. Completely immerse the plasma-treated ePTFE small-diameter artificial blood vessel in the solution and shake in the dark at 37 ℃ for 24 h. After the reaction, remove the small-diameter artificial blood vessel and rinse it repeatedly with deionized water three times to remove unreacted DA and PDA oligomers on the surface. Then, dry it in a vacuum drying oven at 60 ℃ for 2 h. The sample modified with polydopamine is called ePTFE / PDA small-diameter artificial blood vessel.

[0058] Step 3: Dissolve heparin sodium in 1 wt% MES buffer, adjust the pH to 5, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS) and sonicate to disperse the solution. The ratio of MES:heparin sodium:EDC•HCl:NHS is 25:5:5:3. Incubate the solution in a 37 ℃ constant temperature shaker for 30 min to complete heparin (HEP) activation. Completely immerse the ePTFE+HEP small-diameter artificial blood vessel in the heparin solution and react at 37 ℃ for 24 h.

[0059] Step 4: Obtain ePTFE / PDA / HEP small-diameter artificial blood vessels. After the reaction in Step 3 is completed, the sample is removed and thoroughly washed with deionized water to remove unbound heparin, and finally freeze-dried for 24 hours. The sample modified with polydopamine and heparin is called ePTFE / PDA / HEP small-diameter artificial blood vessel.

[0060] The only difference between Comparative Example 1 and Example 1 is that the blood vessels were not treated with plasma.

[0061] Comparative Example 2: Steps 1, 3, and 4 are the same as in Example 3.

[0062] Step 2: Graft polydopamine onto the ePTFE small-diameter artificial blood vessel from Step 1. Prepare a 10 mM Tris solution, adjust the pH to approximately 8.5 with dilute hydrochloric acid, and add dopamine hydrochloride to the Tris solution at a rate of 2 g / L. Stir at room temperature for 24 h, then centrifuge to obtain the precipitate and wash it. Place the precipitate in a vacuum drying oven and vacuum dry at room temperature for 6 h to obtain polydopamine nanoparticles (PDA-NPs). Add the obtained PDA-NPs solution to PBS (pH=7.2) and ultrasonically disperse it. Completely immerse the plasma-treated ePTFE small-diameter artificial blood vessel in the solution and shake it in the dark at 37 ℃ for 24 h. After the reaction, remove the small-diameter artificial blood vessel and rinse it repeatedly with deionized water three times to remove unreacted dopamine (DA) and PDA oligomers from the surface. Then, dry it in a vacuum drying oven at 60 ℃ for 2 h. Prepare an ethanol / water solution of 3-aminopropyltrimethoxysilane (APTMS), add 2-3 drops of glacial acetic acid, and quickly immerse the dopamine-coated sample in the 50% APTMS solution. After reacting at 37°C for 24 h, treat in an oven at 110°C for 30 min, and wash successively with distilled water and anhydrous ethanol on a shaker for 30 min each. Finally, vacuum dry. Subsequently, acidically hydrolyze the polyacrylate in an aqueous solution at approximately 100°C to a concentration of 10%, using an oxidation / reduction system composed of sodium sulfate aqueous solution as an initiator. The amount of initiator is 8% of the mass of acrylic acid, and the reaction is carried out overnight at 60°C to obtain the acrylic acid homopolymer.

[0063] Blank control group: The ePTFE small-diameter artificial blood vessels were cleaned and cut into several samples with a length of 5 cm, which were untreated polytetrafluoroethylene small-diameter artificial blood vessels and served as control example 1 for subsequent testing.

[0064] Test experiment:

[0065] 1. Antiplatelet assay: Samples were placed in 24-well plates and sterilized with 75% ethanol and UV light for 10 min. The samples were then hydrated in PBS solution at 37°C for 1 h. The solution was then removed, and 1 ml of platelet solution was added to each well. The plates were incubated at 37°C for 2 h. The platelet solution was removed, and the plates were washed three times with PBS solution. For qualitative platelet analysis, the plates were soaked in 2.5% glutaraldehyde aqueous solution at room temperature for 1 h, dehydrated with a series of ethanol / distilled water solutions (50%, 70%, 90%, 95%, and 100% ethanol, 5 min each), and vacuum-dried for one day before electron microscopy. For quantitative platelet analysis, 2% Triton-X100 solution was added, and the plates were incubated at 37°C for 15 min. Then, according to the L-lactate dehydrogenase (L-LDH) activity assay kit instructions, the solution was transferred to a 96-well plate, and the absorbance was measured at 450 nm.

[0066] 2. Contact Angle Test: Using an OCA15EC contact angle measuring instrument, the small-diameter artificial blood vessels of the comparative and embodiment examples were cut into vertical strips and stretched taut. Water droplets were dropped onto the flat surface, and the projected images of the droplets were photographed and the shape of the droplets was analyzed. The obtained images were analyzed using a droplet profile fitting method, and the specific droplet profile was obtained using a 5-point drawing method to determine the contact angle of the droplets on the substrate.

[0067] 3. Coagulation Index Test: After cutting the sample, lay it flat at the bottom of a 24-well plate. A transparent circular glass slide was used as a positive control. Three replicates were set up for each group. 100 μL of mouse whole blood and 20 μL of 0.2 mol / L CaCl2 solution were added dropwise to the surface of the sample and control groups, allowing the sample to react with the Ca-containing solution. 2+ After incubating whole blood at 37 °C for 30 min, 2.5 ml of deionized water was added to each well, and incubation continued at 37 °C for 5 min. Simultaneously, a 100 μL whole blood / 2.5 ml deionized water solution was prepared and incubated at 37 °C for 20 min. The sample was then mixed with a solution containing Ca... 2+ After incubation with whole blood / deionized water, the supernatant in the well plate was aspirated into a 96-well plate and allowed to stand for 15 min. The whole blood / deionized water solution was then added to the 96-well plate, and the absorbance of the solution in the 96-well plate was measured at 540 nm using a microplate reader.

[0068] 4. Hemolysis Assay: The sample was cut into 0.5 cm × 0.5 cm films. Mouse whole blood was centrifuged at 3000 rpm for 10 minutes. After removing the supernatant, the sample was washed five times with PBS buffer. The separated red blood cells were diluted 35-fold with PBS buffer. 0.2 ml of red blood cell dilution buffer was mixed with 0.8 ml of PBS as the negative control; 0.2 ml of red blood cell dilution buffer was mixed with 0.8 ml of deionized water as the positive control. Simultaneously, 0.2 ml of red blood cell dilution buffer, 0.8 ml of PBS, and the sample were placed in centrifuge tubes and incubated at 37 °C for 2 h, followed by centrifugation at 4000 rpm for 3 minutes. Finally, the wavelength of the supernatant at 540 nm was measured using a microplate reader. The result was calculated using the following formula:

[0069] Hemolysis rate (%) = (OD s -OD nc ) / (OD pc -OD nc )×100%

[0070] Where is OD s The absorbance and OD of the test sample supernatant pc and OD ncThe absorbance values ​​are those of the supernatant of the positive control group and the negative control group, respectively.

[0071] 5. Cytotoxicity assay: Cell viability of the sample was tested using CCK-8 reagent.

[0072] Test results explanation:

[0073] 1. Antiplatelet test: such as Figure 2 As shown, in the control group, without heparin grafting, a large number of platelets adhered to the blood vessel surface, and the platelets exhibited obvious aggregation and deformation, indicating that the untreated blood vessels exhibited high coagulation activity. In Comparative Example 1, due to the heparin grafting, platelet adhesion was significantly improved compared to the control group, but significant platelet aggregation and the beginnings of a fibrin network structure still existed, indicating that low-density heparin grafting was insufficient to completely induce surface thrombus formation. In Example 2, almost no platelet adhesion or aggregation was observed on the blood vessel surface; even if a small number of platelets were present, they maintained their original shape, indicating that Example 2 effectively improved the biocompatibility of the material, significantly inhibited platelet activation and aggregation, and demonstrated excellent blood contact anticoagulation effect.

[0074] 2. Contact Angle Test: The contact angle test results are shown in Table 2. After plasma treatment, the increased oxygen-containing functional groups on the surface enhanced the hydrophilicity of the blood vessel surface, resulting in a decrease in the contact angle. The contact angle of the untreated blood vessel surface reached over 120°, exhibiting hydrophobicity. After loading PDA and grafting heparin, the water contact angle decreased to below 90°, and after plasma treatment, a further decrease in the water contact angle was observed, indicating that the loading of PDA and heparin was significantly improved compared to the untreated blood vessels.

[0075] Table 1 Contact Angle Test Results

[0076] Blank group Comparative Example 1 Example 1 Example 2 Example 3 <![CDATA[Water contact angle ( o )]]> 120.80 88.53 87.73 80.28 80.43

[0077] 3. Coagulation index test: such as Figure 3 As shown, both the control group and the comparative group exhibited significant blood clot aggregation inside the blood vessels, while no obvious fibrin clots were observed in the examples. This indicates that the modified artificial blood vessel surface can effectively inhibit coagulation and thrombus formation. The coagulation index test is shown in Table 2. The BCI value of the control group was only 46.21%, demonstrating strong procoagulant activity. After combined grafting of PDA-NPs and heparin, the BCI values ​​all increased. Compared to the comparative group, the BCI value of the examples showed a further significant increase, reaching a maximum of 68.47%, indicating that the examples can effectively endow the artificial blood vessels with superior dynamic anticoagulant properties.

[0078] Table 2 Results of Coagulation Index Test

[0079] Blank group Comparative Example 1 Example 1 Example 2 Example 3 Blood clotting index (BCI) (%) 46.21 56.54 64.84 68.47 66.79

[0080] 4. Hemolytic cell experiment: such as Figure 4 As shown, the supernatant in the examples was very light in color, with no significant difference from the negative control group, and no red exudation caused by red blood cell rupture was observed, indicating that the material has good blood compatibility. Hemolysis rate data are shown in Table 4. In Examples 1-3, by constructing a PDA / heparin hydrophilic layer, the hemolysis rate was significantly reduced, reaching a minimum of 0.03%, far below the industry safety standard.

[0081] Table 3. Hemolytic cell experimental data

[0082] Blank group Comparative Example 1 Example 1 Example 2 Example 3 Hemolysis rate (%) 0.60 0.13 0.06 0.03 0.04

[0083] 5. Cytotoxicity test: The results of the cytotoxicity test are shown in Table 4. After surface treatment with the coupling agent, the cell survival rate of Comparative Example 2 decreased to 77.37%. Although the cell survival rate of Comparative Examples 1-3 decreased after plasma treatment, it was still above 80%.

[0084] Table 4. Cytotoxicity test results

[0085] Blank group Comparative Example 1 Comparative Example 2 Example 1 Example 2 Example 3 Cell viability (%) 89.91 90.94 77.37 81.80 87.31 85.96

[0086] Finally, it should be noted that although the present invention has been described in detail above with general descriptions and specific embodiments, the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a small-diameter artificial blood vessel made of expanded polytetrafluoroethylene, characterized in that, The preparation method includes at least the following steps: Step 1: After cleaning the ePTFE small-diameter artificial blood vessel, cut it into several samples with a length of 1~10 cm, and then perform plasma treatment on the cleaned ePTFE small-diameter artificial blood vessel. Step 2: Graft polydopamine nanoparticles (PDA-NPs) onto the treated ePTFE small-diameter artificial blood vessel obtained in Step 1 to prepare an ePTFE / PDA small-diameter artificial blood vessel. Step 3: Activate heparin (HEP) with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS), and then react the ePTFE / PDA small-diameter artificial blood vessel obtained in step 2 with the activated HEP solution to obtain heparin-grafted ePTFE / PDA / HEP small-diameter artificial blood vessel; Step 4: Use deionized water to thoroughly wash the ePTFE / PDA / HEP small-diameter artificial blood vessel obtained in step 3, and then freeze-dry it to obtain the expanded polytetrafluoroethylene small-diameter artificial blood vessel.

2. The method for preparing a small-diameter artificial blood vessel according to claim 1, characterized in that, The plasma processing gas mentioned in step 1 is oxygen or air.

3. The method for preparing a small-diameter artificial blood vessel according to claim 1, characterized in that, The plasma treatment power in step 1 is 80~120 W, the pressure is 20~40 Pa, and the treatment time is 4~8 min.

4. The method for preparing a small-diameter artificial blood vessel according to claim 1, characterized in that, The grafting of polydopamine nanoparticles in step 2 includes at least the following steps: Step 2.1: Prepare a tris(hydroxymethyl)aminomethane (Tris) solution, adjust the Tris solution system with dilute hydrochloric acid, add dopamine hydrochloride to the Tris solution and stir at room temperature, then centrifuge to obtain the precipitate. Step 2.2: Place the precipitate in a vacuum drying oven and dry it under vacuum at room temperature to obtain polydopamine nanoparticles (PDA-NPs). Step 2.3: Prepare a PDA-NPs solution using phosphate buffered saline (PBS), and then react the plasma-treated ePTFE small-diameter artificial blood vessel with the PDA-NPs solution to form a PDA coating on its surface, thus obtaining the ePTFE / PDA small-diameter artificial blood vessel.

5. The method for preparing a small-diameter artificial blood vessel according to claim 4, characterized in that, The concentration of the Tris solution mentioned in step 2.1 is 10~20 mM; the pH value of the Tris solution is 8~8.

5.

6. The method for preparing a small-diameter artificial blood vessel according to claim 4, characterized in that, The dopamine hydrochloride described in step 2.1 is added to the Tris solution at a rate of 1.5~2.5 g / L.

7. The method for preparing a small-diameter artificial blood vessel according to claim 4, characterized in that, The contact reaction time described in step 2.3 is 22~26h.

8. The method for preparing a small-diameter artificial blood vessel according to claim 1, characterized in that, The heparin activation step described in step 3 mainly includes the following steps: First, dissolve sodium heparin in MES buffer; then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) and N-hydroxysuccinimide (NHS) and sonicate to disperse; then incubate the solution in a 37°C constant temperature shaker to activate heparin.

9. The method for preparing a small-diameter artificial blood vessel according to claim 8, characterized in that, The molar ratio of MES:heparin sodium:EDC•HCl:NHS is 25:(4~6):(4~6):(2~4).

10. In some preferred embodiments of the present invention, the incubation time in a constant temperature oscillator at 37 °C is 20-40 min.

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