Directional thrombolytic pro-endothelization composite small-diameter artificial blood vessel and preparation method thereof

Abstract

The application provides a directional thrombolytic pro-endothelialization composite small-caliber artificial blood vessel and a preparation method thereof. The method activates and chemically modifies the surface of an expanded polytetrafluoroethylene (ePTFE) artificial blood vessel through plasma treatment, introduces active carboxyl groups and covalently grafts a pro-endothelial adhesion polypeptide REDV; then, by using the strong adhesion of a polydopamine (PDA) coating, nano-hydrogel particles loaded with thrombolytic drugs t-PANPs are fixed on the inner surface of the blood vessel. The application actively recruits endothelial cells by the bottom layer of the REDV polypeptide, and at the same time, realizes on-demand and controlled release of t-PA for thrombolysis by the upper layer of the nano-hydrogel, thereby solving the problems of easy thrombosis and intimal hyperplasia of the small-caliber artificial blood vessel. The preparation process condition is mild, the performance is stable, the synergistic effect of antithrombosis and pro-endothelialization is realized, and the application has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of artificial blood vessel technology, specifically to a method for preparing a composite expanded polytetrafluoroethylene (ePTFE) small-diameter artificial blood vessel that combines targeted thrombolysis and endothelialization promotion. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death worldwide, and vascular transplantation is a crucial means of saving the lives of critically ill patients in clinical treatment. As the core implantable device for replacing diseased blood vessels, the performance of artificial blood vessels directly affects the success rate of surgery and the long-term prognosis of patients. An ideal artificial blood vessel should possess excellent biocompatibility, mechanical properties that match those of the patient's own blood vessel, long-term antithrombotic ability, and the ability to promote endothelial cell repair. Expanded polytetrafluoroethylene (ePTFE), due to its excellent chemical stability, mechanical strength, and mature processing technology, has become one of the most widely used artificial blood vessel materials in clinical practice.

[0003] However, the highly hydrophobic and bioinert surface of ePTFE materials makes them prone to non-specific protein adsorption and platelet adhesion after implantation, which can lead to acute thrombosis and long-term intimal hyperplasia. This defect severely limits its application in small-diameter (<6 mm) vascular grafts. To improve its surface properties, researchers typically employ surface modification techniques such as chemical grafting, plasma treatment, and biomolecular immobilization to enhance its hydrophilicity and bioactivity.

[0004] Current common surface modification strategies mostly focus on achieving a single function, such as introducing active groups through plasma treatment or improving hydrophilicity by coating with polydopamine (PDA). However, these methods often struggle to simultaneously meet the synergistic requirements of antithrombosis and endothelial repair. Furthermore, the immobilization of bioactive molecules (such as heparin and peptides) in existing technologies often relies on complex coupling chemistry, potentially introducing cytotoxic risks, and the stability and functional durability of the modified layer remain challenging. Therefore, how to construct a stable, biosafe, multifunctional coating on the ePTFE surface that combines antithrombotic and active endothelial repair functions has become a key problem urgently needing breakthroughs in this field.

[0005] In recent years, composite modification strategies combining surface activation, biomimetic coatings, and drug sustained release have attracted widespread attention. For example, plasma pretreatment enhances surface activity, followed by PDA coating-mediated biomolecule immobilization, and further loading with thrombolytic drugs or growth factors, potentially achieving a synergistic improvement in surface properties. However, this strategy still faces challenges in practical applications, such as low process integration, uncontrollable drug release, and difficulty in maintaining the activity of functional molecules over the long term. Therefore, developing a rational, mild, and non-toxic ePTFE surface functionalization method that integrates antithrombotic and endothelialization effects is of significant value in promoting the clinical application of small-diameter artificial blood vessels. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and propose a method for preparing a composite small-diameter artificial blood vessel that combines targeted thrombolysis and endothelialization. This method provides a simplified procedure, mild conditions, better biocompatibility, and superior overall performance, which is beneficial for clinical application.

[0007] To achieve the above technical objectives, this invention provides a method for preparing a targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel, specifically including the following steps:

[0008] Step 1: After cleaning the expanded polytetrafluoroethylene artificial blood vessel (ePTFE), cut it into several samples with a length of 1~10cm. After plasma treatment of the ePTFE artificial blood vessel substrate, the surface of the blood vessel is sequentially silanized, amination, and carboxylated to obtain an artificial blood vessel with active carboxyl groups introduced.

[0009] Step 2: In the MES buffer system, the arginine-glutamic acid-aspartic acid-valine (Arg-Glu-Asp-Val, abbreviated as REDV) polypeptide is covalently grafted onto the inner surface of blood vessels by the coupling effect of N-hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), thus preparing functionalized blood vessels (ePTFE-REDV) with endothelial adhesion-promoting polypeptides fixed.

[0010] Step 3: Mix the peptide containing a specific enzyme digestion sequence, acrylamide monomer, and thrombolytic drug t-PA. Under the action of an initiator and a catalyst, prepare a nanohydrogel. After the reaction, the product is dialyzed and freeze-dried to obtain t-PA loaded nanogel particles (t-PA NPs).

[0011] Step 4: Immerse the functionalized blood vessel (ePTFE-REDV) obtained in Step 2 into a polydopamine solution to form a polydopamine coating, and then immerse it in a solution of the nanogel (t-PA NPs) prepared in Step 3, so that the nanogel is loaded onto the inner surface of the blood vessel through adsorption. After cleaning and drying, the directional thrombolysis and endothelialization composite small-diameter artificial blood vessel is obtained.

[0012] In some technical solutions of the present invention, the plasma treatment gas in step 1 is oxygen or air, the plasma treatment power is 80~120 W, the pressure is 20~40 Pa, and the treatment time is 4~8 min.

[0013] In some technical solutions of the present invention, in step 1, at least one of trichlorosilane (HSiCl3), methyltrichlorosilane (CH3SiCl3), and silicon tetrachloride (SiCl4) is selected to silanize the surface of blood vessels.

[0014] In some technical solutions of the present invention, at least one of (3-aminopropyl)trimethoxysilane (APTMS), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and (3-aminopropyl)triethoxysilane (APTES) is selected for amination in step 1.

[0015] In some technical solutions of the present invention, in step 1, at least one of succinic anhydride, maleic anhydride, adipic anhydride, and glutaric anhydride is selected for carboxylation.

[0016] In some preferred embodiments of the present invention, in step 1, silicon tetrachloride (SiCl4) solution, (3-aminopropyl)triethoxysilane (APTES) solution, and glutaric anhydride buffer solution are used sequentially to silanize, amination, and carboxylate the surface of the blood vessel.

[0017] In some preferred embodiments of the present invention, the solvent of the silicon tetrachloride (SiCl4) solution is toluene; the concentration of the silicon tetrachloride (SiCl4) solution is 5~20 g / L; and the reaction time of the silicon tetrachloride (SiCl4) solution is 5~10 min.

[0018] In some preferred embodiments of the present invention, the solvent of the (3-aminopropyl)triethoxysilane (APTES) solution is ethanol; the volume concentration of the APTES solution is 1% to 5%; and the reaction time of the APTES solution is 10 to 20 min.

[0019] In some preferred embodiments of the present invention, the buffer solution of glutaric anhydride is a phosphate buffer solution of glutaric anhydride. Preferably, the phosphate buffer solution of glutaric anhydride contains 1% to 5% glutaric anhydride by volume; the pH value of the phosphate buffer solution of glutaric anhydride is 7 to 8; and the reaction time of the phosphate buffer solution of glutaric anhydride is 1 to 2 hours.

[0020] In some technical solutions of this invention, in step 2, the functionalized artificial blood vessel is immersed in sulfo-NHS and EDC for reaction, with a reaction time of 10-60 min. In step 2, through the combined action of sulfo-NHS and EDC, the surface carboxyl groups introduced in step 1 can be further activated, enabling them to react with the amino groups on the REDV polypeptide, thereby covalently grafting the REDV polypeptide onto the inner surface of the blood vessel and constructing a specific endothelialization-promoting interface. If the reaction time of sulfo-NHS and EDC is too short (<10 min), the activation may be insufficient, and only a small number of carboxyl groups on the surface may be converted into active sulfo-NHS esters. If the reaction time is too long (60 min, or even several hours), the activated ester may hydrolyze, resulting in a reduction in effective activation sites, and prolonged reaction with coupling agents may lead to rearrangement of surface groups or non-specific adsorption.

[0021] In some preferred embodiments of the present invention, the concentration of sulfo-NHS in step 2 is 1-10 mM.

[0022] In some preferred embodiments of the present invention, the EDC concentration in step 2 is 0.5-20 mM.

[0023] In some technical solutions of this invention, the REDV polypeptide described in step 2 is dissolved in a phosphate buffer solution with a concentration of 0.5-5 mM / mL; further, the reaction time of the blood vessel with the REDV polypeptide in the phosphate buffer solution is 12-48 h. The REDV polypeptide, as an endothelialization ligand, is covalently grafted onto the inner surface of the blood vessel, actively recognizing and promoting endothelial cell adhesion and proliferation. If the reaction time is too short (<12 h), the surface carboxyl-activated esters do not react sufficiently with the REDV polypeptide, resulting in low grafting density and uneven distribution, leading to insufficient endothelial cell adhesion sites and limited endothelialization effect. If the reaction time is too long (48 h), the grafting density tends to saturate, and further extension only brings marginal benefits. Furthermore, long-term immersion may cause hydrolysis or conformational changes in the grafted polypeptide, reducing biological activity and increasing the risk of contamination. Preferably, 12-48 h ensures high-density grafting and optimal cell response while maintaining production efficiency.

[0024] In some technical solutions of this invention, the enzymatic digestion sequence of the polypeptide in step 3 is selected from Gly-(D)Phe-Pro-Arg-Gly-Phe or (D-Phe)-Pro-Arg-Pro-(Gly)4. The polypeptide acts as a nanogel crosslinking agent, endowing it with thrombin responsiveness and enabling on-demand release of t-PA.

[0025] In some technical solutions of the present invention, the preparation of the nano-hydrogel in step 3 specifically includes the following steps:

[0026] Step 3.1: The polypeptide is dissolved in sodium bicarbonate buffer solution at a concentration of 0.5~5 mg / mL;

[0027] Step 3.2: Dissolve the acrylamide monomer 1 in N,N-dimethylformamide (DMF) and add it dropwise to the polypeptide solution obtained in step 3.1. The reaction time is 1-4 h.

[0028] Step 3.3: Dissolve acrylamide monomer 2 in sodium bicarbonate buffer solution at a total concentration of 1-5 mg / mL;

[0029] Step 3.4: Dissolve t-PA in phosphate buffered saline (PBS) and mix it with the mixed solution prepared in step 3.2 and the solution prepared in step 3.3. Stir at 2-8 °C for 5-20 min.

[0030] Step 3.5: Add peptide crosslinking agent and N,N,N',N'-tetramethylethylenediamine sequentially and stir. Then add ammonium persulfate aqueous solution to initiate free radical polymerization. React at 2-8 °C for 2-8 h under a nitrogen atmosphere.

[0031] Step 3.6: After the reaction is complete, dialyze for 24-48 h, and freeze-dry to obtain the t-PA-loaded nanogel particles (t-PA NPs).

[0032] The cross-linking network and structure of the nanogel directly affect the t-PA encapsulation effect. When the polymerization time is too short (<2 h), the monomer conversion rate is low, the cross-linking is insufficient, the gel structure is loose, and the mechanical strength is poor, resulting in unstable t-PA encapsulation and easy burst release. When the polymerization time is too long (8 h), the cross-linking density is too high, which may limit drug diffusion and release and reduce response sensitivity. The optimal time is 2~8 h to form a moderately cross-linked stable network, ensuring that the nanogel is firmly loaded on the vascular surface and releases t-PA in response, thereby effectively exerting the active thrombolytic function and synergistically promoting endothelialization with REDV without interference.

[0033] In some preferred embodiments of the present invention, the acrylamide monomer 1 described in step 3.2 contains at least an amine reactive group and a polymerizable double bond. Further, N-succinimidyl acrylate is preferred, through which a polymerizable acryloyl group is introduced onto the polypeptide molecule via a coupling reaction between this monomer and the amino group at the polypeptide terminus, thus preparing a macromolecular crosslinking agent that can participate in subsequent free radical polymerization. A reaction time of 1-4 hours ensures sufficient modification and guarantees the efficiency of subsequent crosslinking reactions.

[0034] In some preferred embodiments of the present invention, the acrylamide monomer 2 mentioned in step 3.3 is selected from at least one of acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride, N-isopropylacrylamide, N,N-dimethylacrylamide, or hydroxyethyl methacrylate. Preferably, the acrylamide monomer 2 is selected from acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride. Further, acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride are added in an equimolar ratio. Acrylamide, as the main chain monomer of the polymerization reaction, provides a hydrophilic framework and mechanical support for the nanohydrogel; N-(3-aminopropyl)methacrylamide hydrochloride, as a functionalized comonomer, has its terminal amino groups that can introduce positive charges or serve as subsequent modification sites to regulate the interaction between the gel and the t-PA drug, thereby optimizing the drug loading and release behavior.

[0035] In some technical solutions of this invention, the amount of peptide added in step 3 accounts for 0.5~5 mol% of the total amount of acrylamide monomer 1 and acrylamide monomer 2. The amount of crosslinking agent added has a significant impact on the crosslinking network structure of the nanohydrogel. Too low an amount (<0.5 mol%) results in insufficient crosslinking density, a loose gel structure, and easy burst release of t-PA; too high an amount (>5 mol%) results in excessive crosslinking, a dense network, hindering drug diffusion and release, and reducing responsiveness. Preferably, 0.5~5 mol% can form a moderately crosslinked stable network, balancing drug loading and responsive release performance.

[0036] In some technical solutions of this invention, the polydopamine solution in step 4 is prepared by adding dopamine hydrochloride to a Tris solution with a concentration of 10-20 mM and a pH of 8.0-9.0, and stirring at room temperature for 12-24 h. Further, the concentration of the polydopamine solution is 1-2 g / L. The amount of polydopamine added directly affects the loading of t-PA nanogels on the blood vessel surface. Higher polydopamine concentrations result in increased coating thickness and more active sites such as catechol groups on the surface, thereby enhancing the adsorption and anchoring ability of t-PA NPs and improving the loading efficiency of the nanogel. However, excessively high concentrations (>2 g / L) may lead to excessively thick coatings or agglomeration, which in turn affects the uniformity of loading and gel release behavior. Therefore, it is necessary to balance coating activity and loading stability within a preferred concentration range to ensure that t-PA NPs are firmly bound and their release is controllable.

[0037] In some technical solutions of the present invention, the functionalized blood vessel (ePTFE-REDV) in step 4 is soaked and shaken in polydopamine solution for 12-36 h, then washed with deionized water and vacuum dried at 60°C.

[0038] In some technical solutions of the present invention, the functionalized blood vessel (ePTFE-REDV) described in step 4 is immersed in a PBS solution containing t-PANPs for 6 to 24 hours.

[0039] The present invention also provides a targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel, which is prepared by the above-described preparation method.

[0040] In some technical solutions of the present invention, the inner surface of the composite small-diameter artificial blood vessel includes a REDV polypeptide-modified layer and a t-PA NPs-loaded layer; the t-PA NPs-loaded layer is loaded onto the REDV polypeptide-modified layer through a polydopamine coating; the t-PANPs are encapsulated in a nanohydrogel containing a polypeptide with a specific enzyme digestion sequence as a crosslinking agent. Further, the enzyme digestion sequence of the polypeptide is Gly-(D)Phe-Pro-Arg-Gly-Phe or (D-Phe)-Pro-Arg-Pro-(Gly)4.

[0041] In some preferred embodiments of the present invention, the t-PA loading accounts for 0.5~5 wt% of the dry weight of the nanohydrogel.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The composite small-diameter artificial blood vessel prepared in this invention encapsulates a loaded thrombolytic drug (t-PA) within a nanohydrogel containing a peptide with a specific enzymatic cleavage sequence as a cross-linking agent, endowing it with thrombin responsiveness. The nanohydrogel can degrade under the action of thrombin, thereby enabling t-PA to respond to blood flow thrombus signals in a timely manner, achieving targeted and controlled drug release, avoiding the bleeding risk caused by systemic non-specific drug release, and improving the safety and effectiveness of treatment. Simultaneously, the inner layer covalently immobilized REDV peptide acts as an endothelial cell-specific ligand, promoting endothelial cell adhesion and spread through integrin-mediated signaling pathways. The two layers are stably bound by polydopamine, without interference and with synergistic effects.

[0044] 2. This invention utilizes plasma treatment technology to overcome the deficiencies of ePTFE surface, such as lack of polar functional groups and low reactivity. Plasma treatment is energy-efficient, environmentally friendly, and provides stable and reliable results. The modification treatment only acts on the superficial layer of small-diameter artificial blood vessels, avoiding damage to the substrate properties. Furthermore, the strong adhesion and secondary reactivity of the polydopamine (PDA) coating are used to fix the drug-loaded nanogel to the blood vessel surface, simultaneously bonding it firmly to the endothelialization-promoting functional layer (i.e., the ePTFE-REDV surface) formed by covalent grafting of REDV peptides. This combination creates a stable, multifunctional integrated interface on the blood vessel surface. Plasma activation ensures firm grafting of the underlying REDV layer to actively recruit endothelial cells, while the strong adhesion of PDA anchors the upper drug-loaded gel for on-demand thrombolysis, ultimately achieving synergistic and long-term stable antithrombotic and endothelialization functions.

[0045] 3. The preparation method of this invention has clear steps and mild conditions. The plasma treatment, surface grafting, solution polymerization and other technologies used are all mature and controllable, which is conducive to clinical translation and application. It opens up new methods for surface modification of small-diameter artificial blood vessels and has good application prospects. Attached Figure Description

[0046] 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.

[0047] Figure 1 Scanning electron microscope image of the surface of the ePTFE-REDV-tPA small-diameter blood vessel prepared in this invention;

[0048] Figure 2 Contact angle diagram of the ePTFE-REDV-tPA small-diameter blood vessel prepared by this invention;

[0049] Figure 3 The image shows a coagulation experiment of the ePTFE-REDV-tPA small-diameter blood vessel prepared in this invention.

[0050] Figure 4 This image shows the cell proliferation of the ePTFE-REDV-tPA small-diameter blood vessels prepared according to the present invention. Detailed Implementation

[0051] 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.

[0052] Example 1: A method for preparing a targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel, the specific steps of which are as follows:

[0053] Step 1: The polytetrafluoroethylene artificial blood vessel is subjected to air atmosphere plasma treatment at a power of 100 W, a pressure of 30 Pa, and a treatment time of 360 s. After treatment, the surface is silanized, aminationd, and carboxylated sequentially using reagents such as silicon tetrachloride (SiCl4), (3-aminopropyl)triethoxysilane (APTES), and glutaric anhydride to introduce active carboxyl groups.

[0054] Step 2: After activating the carboxylated artificial blood vessel in MES buffer, it was immersed in 5 mM sulfo-NHS and 2 mMEDC solution for 15 min, and then immersed in REDV phosphate buffer for 24 h at pH 8 to obtain ePTFE-REDV.

[0055] Step 3: Dissolve the peptide (Gly-(D)Phe-Pro-Arg-Gly-Phe) at a concentration of 1 mg / mL in 50 mM sodium bicarbonate buffer. Dissolve N-succinimide acrylate in DMF and add it dropwise to the peptide solution. React at room temperature for 2 h. After the reaction, dialyze and freeze-dry to obtain the peptide crosslinking agent. Dissolve acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride in sodium bicarbonate buffer at an equimolar ratio. Dissolve t-PA in PBS at a certain concentration and mix with the above monomer solution. Add the peptide crosslinking agent and N,N,N',N'-tetramethylethylenediamine sequentially at 4°C and stir. Then add ammonium persulfate aqueous solution to immediately initiate free radical polymerization. React at 4°C under N2 environment for 5 h. After the reaction, dialyze and freeze-dry to obtain drug-loaded nanogels (t-PA NPs).

[0056] Step 4: Prepare a 10 mM Tris solution with a pH of 8.5. Add dopamine hydrochloride at a concentration of 2 g / L to the Tris solution and stir at room temperature for 24 h. Immerse the ePTFE-REDV sample in the above solution, shake for 24 h, wash with deionized water, and vacuum dry at 60 °C. Immerse the ePTFE-REDV-PDA sample in a PBS solution containing t-PA NPs for 12 h, then thoroughly wash the surface with PBS and dry in an N2 gas stream to obtain the targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel.

[0057] Example 2: A method for preparing a targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel, the specific steps of which are as follows:

[0058] Step 1: The polytetrafluoroethylene artificial blood vessel is subjected to air atmosphere plasma treatment at a power of 100 W, a pressure of 30 Pa, and a treatment time of 360 s. After treatment, the surface is silanized, aminationd, and carboxylated sequentially using reagents such as silicon tetrachloride (SiCl4), (3-aminopropyl)triethoxysilane (APTES), and glutaric anhydride to introduce active carboxyl groups.

[0059] Step 2: After activating the carboxylated artificial blood vessel in MES buffer, it was immersed in 5 mM sulfo-NHS and 2 mMEDC solution for 15 min, and then immersed in REDV phosphate buffer for 8 h at pH 8 to obtain ePTFE-REDV.

[0060] Step 3: Dissolve the peptide (Gly-(D)Phe-Pro-Arg-Gly-Phe) at a concentration of 1 mg / mL in 50 mM sodium bicarbonate buffer. Dissolve N-succinimide acrylate in DMF and add it dropwise to the peptide solution. React at room temperature for 2 h. After the reaction, dialyze and freeze-dry to obtain the peptide crosslinking agent. Dissolve acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride in sodium bicarbonate buffer at an equimolar ratio. Dissolve t-PA in PBS at a certain concentration and mix with the above monomer solution. Add the peptide crosslinking agent and N,N,N',N'-tetramethylethylenediamine sequentially at 4°C and stir. Then add ammonium persulfate aqueous solution to immediately initiate free radical polymerization. React at 4°C under N2 environment for 8 h. After the reaction, dialyze and freeze-dry to obtain drug-loaded nanogels (t-PA NPs).

[0061] Step 4: Prepare a 10 mM Tris solution with a pH of 8.5. Add dopamine hydrochloride at a concentration of 2 g / L to the Tris solution and stir at room temperature for 24 h. Immerse the ePTFE-REDV sample in the above solution, shake for 24 h, wash with deionized water, and vacuum dry at 60 °C. Immerse the ePTFE-REDV-PDA sample in a PBS solution containing t-PA NPs for 12 h, then thoroughly wash the surface with PBS and dry in an N2 gas stream to obtain the targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel.

[0062] Example 3: A method for preparing a targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel, the specific steps of which are as follows:

[0063] Step 1: The polytetrafluoroethylene artificial blood vessel is subjected to air atmosphere plasma treatment at a power of 100 W, a pressure of 30 Pa, and a treatment time of 360 s. After treatment, the surface is silanized, aminationd, and carboxylated sequentially using reagents such as silicon tetrachloride (SiCl4), (3-aminopropyl)triethoxysilane (APTES), and glutaric anhydride to introduce active carboxyl groups.

[0064] Step 2: After activating the carboxylated artificial blood vessel in MES buffer, it was immersed in 5 mM sulfo-NHS and 2 mMEDC solution for 1 h, and then immersed in REDV phosphate buffer for 24 h at pH 8 to obtain ePTFE-REDV.

[0065] Step 3: Dissolve the peptide (Gly-(D)Phe-Pro-Arg-Gly-Phe) at a concentration of 1 mg / mL in 50 mM sodium bicarbonate buffer. Dissolve N-succinimide acrylate in DMF and add it dropwise to the peptide solution. React at room temperature for 2 h. After the reaction, dialyze and freeze-dry to obtain the peptide crosslinking agent. Dissolve acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride in sodium bicarbonate buffer at an equimolar ratio. Dissolve t-PA in PBS at a certain concentration and mix with the above monomer solution. Add the peptide crosslinking agent and N,N,N',N'-tetramethylethylenediamine sequentially at 4 °C and stir. Then add ammonium persulfate aqueous solution to immediately initiate free radical polymerization. React at 4 °C under N2 environment for 12 h. After the reaction, dialyze and freeze-dry to obtain drug-loaded nanogels (t-PA NPs).

[0066] Step 4: Prepare a 10 mM Tris solution with a pH of 8.5. Add dopamine hydrochloride at a concentration of 2 g / L to the Tris solution and stir at room temperature for 24 h. Immerse the ePTFE-REDV sample in the above solution, shake for 24 h, wash with deionized water, and vacuum dry at 60 °C. Immerse the ePTFE-REDV-PDA sample in a PBS solution containing t-PA NPs for 12 h, then thoroughly wash the surface with PBS and dry in an N2 gas stream to obtain the targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel.

[0067] Comparative Example 1: After cleaning, ePTFE small-diameter artificial blood vessels were cut into several samples with a length of 5 cm. After being treated with oxygen atmosphere plasma for 120 s, a Tris solution with a pH of 8.5 was prepared. Dopamine hydrochloride was added to the Tris solution at a concentration of 2 g / L. The mixture was stirred at room temperature for 24 h. The ePTFE samples were then immersed in the above solution and shaken for 24 h. After washing with deionized water, the samples were dried under vacuum at 60 °C to obtain ePTFE-PDA as a comparative example for subsequent testing.

[0068] Comparative Example 2: A method for preparing a targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel, the specific steps of which are as follows:

[0069] Step 1: The polytetrafluoroethylene artificial blood vessel is subjected to air atmosphere plasma treatment at a power of 100 W, a pressure of 30 Pa, and a treatment time of 360 s. After treatment, the surface is silanized, aminationd, and carboxylated sequentially using reagents such as silicon tetrachloride (SiCl4), (3-aminopropyl)triethoxysilane (APTES), and glutaric anhydride to introduce active carboxyl groups.

[0070] Step 2: After activating the carboxylated artificial blood vessel in MES buffer, it was immersed in a solution of 5 mM sulfo-NHS and 2 mMEDC for 15 min, and then immersed in REDV phosphate buffer for 24 h at pH 8 to obtain ePTFE-REDV.

[0071] 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 blank control group for subsequent testing.

[0072] Test experiment:

[0073] 1. Scanning Electron Microscopy Morphology Observation: The microstructure of the sample surface was observed using a Zeiss Gemini SEM 300 field emission scanning electron microscope (SEM). Samples were cut to appropriate sizes, fixed to the sample stage with conductive adhesive, and then sputtered with gold to enhance conductivity. During observation, an accelerating voltage of 5 kV and a working distance of 13 mm were set, and surface morphology images were acquired at different magnifications.

[0074] 2. Contact Angle Test: Using the OCA15EC contact angle measuring instrument, the small-diameter artificial blood vessels of the comparative and embodiment were cut into vertical strips and stretched taut. Water droplets were dropped into the flat parts of the surface, and the projected images of the droplets were photographed and the shape of the droplets was analyzed. The obtained images were analyzed using the droplet contour fitting method, and the specific droplet contour was obtained using the 5-point drawing method to determine the contact angle of the droplets on the substrate.

[0075] 3. In vitro anticoagulation test: Place the sample flat in a 24-well plate, using a transparent circular glass slide as a positive control. Each group has three replicates. Add fresh mouse whole blood and calcium chloride solution sequentially to each well and the control surface, and incubate at 37°C for 30 min to allow blood coagulation. After incubation, add deionized water and continue incubation for 5 min to allow uncoagulated red blood cells to hemolyze. A whole blood / deionized water mixture is used as a complete hemolysis control. Transfer the supernatant from each well to a 96-well plate, let it stand at room temperature for 15 min, and then measure the absorbance at 540 nm using a microplate reader. The coagulation index (BCI) is calculated using the formula BCI(%) = OD_t / OD_w, where OD_t is the absorbance of the sample or positive control group, and OD_w is the absorbance of the complete hemolysis control group. A higher BCI value indicates better anticoagulation performance of the material surface.

[0076] 4. Cell proliferation and live / dead cell staining assays: The extract method was used to assess the proliferative activity and cell compatibility of the samples with endothelial cells (HEUVC). Sterilized samples were added to endothelial cell culture medium at the extraction ratio (sample surface area / culture medium volume = 3 cm² / mL) and extracted at 37°C for 24 h. The extract was collected for later use. HEUVC cells in the logarithmic growth phase were then added at a rate of 2 × 10⁶ cells per well. 4Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h to allow cell adhesion. After aspirating the original culture medium, extracts from each sample were added, and the cells were cultured for 1, 3, and 5 days, with three replicates per group. At each culture time point, the extracts were aspirated, and the cells were gently washed twice with PBS. Staining working solution was added to each well, and the cells were incubated at 37°C in the dark for 30 min. After staining, images of live cells (green fluorescence) were acquired using a fluorescence microscope at an excitation wavelength of 490 nm, and images of dead cells (red fluorescence) were acquired at an excitation wavelength of 535 nm. Quantitative analysis was performed on five randomly selected fields of view using image analysis software. Cell proliferation on the surface of each sample was assessed by calculating the percentage of green fluorescence-positive area to the total field of view (cell coverage, %), and the average value was taken as the final result.

[0077] Test results explanation:

[0078] 1. Observation of the surface morphology of artificial blood vessels: The surface morphology of artificial blood vessels is as follows: Figure 1 As shown, the ePTFE surface after only cleaning exhibits a typical fiber-nodular porous structure. The fiber surface is smooth and clean, without obvious adhering substances, reflecting the intrinsic morphology of unmodified ePTFE. In Comparative Example 1, a uniform thin coating is observed on the fiber surface, with slightly blurred fiber outlines. Fine particles formed by PDA aggregation are visible in some areas, proving that PDA was successfully deposited on the vascular surface. Comparative Example 2 has a similar morphology to the Comparative Example. Grafting REDV peptides onto the PDA coating did not cause significant morphological changes; the fiber network structure remained intact, and a uniform thin coating was still visible on the surface, proving that the modification process was mild and did not damage the substrate structure. In Example 1, in addition to the PDA coating, uniformly sized spherical particles (t-PA nanogels) were observed distributed on the fiber surface and between pores. The particle distribution was relatively uniform, proving that the drug-loaded nanogels were successfully loaded onto the vascular inner surface, and that the loading process did not lead to aggregation or morphological damage. In summary, the SEM images verified the successful implementation of each modification step: the PDA coating was uniformly applied, the REDV grafting did not damage the surface, and t-PANPs were successfully loaded, providing morphological basis for subsequent functional evaluation.

[0079] 2. Contact Angle Analysis: Contact angle test results showed that the contact angle of the blank group was 130.06°, exhibiting typical strong hydrophobicity. The contact angle of Comparative Example 1 decreased to 83.98°, indicating that plasma activation and polydopamine deposition significantly improved surface hydrophilicity. The contact angle of Comparative Example 2 was 80.77°, similar to the comparative example, indicating that REDV peptide grafting did not significantly change surface wettability, and the modification process was mild. The contact angle of Example 1 further decreased to 64.01°, exhibiting optimal hydrophilicity. This is attributed to the surface-loaded t-PA nanohydrogel being rich in hydrophilic groups, further enhancing surface hydration capacity. The trend of contact angle variation is related to the design intent of each modification step. Figure 1 Therefore, the increased hydrophilicity helps reduce non-specific protein adsorption, laying the foundation for subsequent anticoagulation and endothelialization functions.

[0080] Table 1 Contact Angle Test Results

[0081] 3. In vitro anticoagulation test analysis: such as Figure 3 The in vitro anticoagulation experiment shown evaluated the anticoagulation performance of each group of samples using coagulation index (BCI) and blood clot morphology photographs. A higher BCI value indicates lower blood coagulation on the material surface and better anticoagulation performance. The coagulation morphology photograph of the blank group showed large and complete blood clots on its surface, exhibiting typical procoagulant characteristics of hydrophobic materials. The comparative example showed no significant difference from the blank group, with obvious coagulation still visible in the morphology, indicating that simply introducing a hydrophilic PDA coating, while improving wettability, is insufficient to endow the material with active anticoagulation function. The coagulation morphology photograph of Comparative Example 2 still showed a certain degree of coagulation; the main function of this group was to promote endothelialization rather than anticoagulation. Example 1 had a significantly higher BCI than other groups, and the coagulation morphology photograph also showed that its surface blood clot was the smallest and least complete, proving that the t-PA nanogel-loaded material endowed the vessel with active thrombolytic function, effectively inhibiting blood coagulation and achieving excellent anticoagulation performance.

[0082] Table 2 Results of Coagulation Index Test

[0083]

[0084] 4. Cell proliferation assay analysis: The cell proliferation assay quantitatively assessed the support capacity of each group of samples for endothelial cells using live / dead cell fluorescence staining and relative proliferation rate. The results are as follows: Figure 4 As shown, using the proliferation level of the blank group on day 5 as a baseline (set as 100%), after 1 day of culture, the relative proliferation rate of the blank group was less than 17%, while the relative proliferation rates of Comparative Example 2 and Example 1 both exceeded 35%, indicating that REDV peptide grafting effectively promoted the initial adhesion of endothelial cells. With increasing culture time, the relative proliferation rate of Comparative Example 2 and Example 1 further increased compared to the blank group. Fluorescence images showed that Example 1 had high cell confluence and dense distribution of live cells, indicating that REDV modification significantly enhanced the endothelial cell proliferation capacity. The introduction of t-PA nanogel further enhanced this effect, proving that the composite functional blood vessel constructed in this invention also possesses excellent endothelialization-promoting properties.

[0085] Table 3 Cell proliferation experiment data

[0086]

[0087] 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 targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel, characterized in that, The preparation method includes at least the following preparation steps: Step 1: After cleaning the expanded polytetrafluoroethylene artificial blood vessel (ePTFE), cut it into several samples with a length of 1~10cm. After plasma treatment of the ePTFE artificial blood vessel substrate, the surface of the blood vessel is sequentially silanized, amination, and carboxylated to obtain an artificial blood vessel with active carboxyl groups introduced. Step 2: In the MES buffer system, the arginine-glutamic acid-aspartic acid-valine (Arg-Glu-Asp-Val, abbreviated as REDV) polypeptide is covalently grafted onto the inner surface of blood vessels by the coupling effect of N-hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), thus preparing functionalized blood vessels (ePTFE-REDV) with endothelial adhesion-promoting polypeptides fixed. Step 3: Mix the peptide containing a specific enzyme digestion sequence, acrylamide monomer, and thrombolytic drug t-PA. Under the action of an initiator and a catalyst, prepare a nanohydrogel. After the reaction, the product is dialyzed and freeze-dried to obtain t-PA loaded nanogel particles (t-PA NPs). Step 4: Immerse the functionalized blood vessel (ePTFE-REDV) obtained in Step 2 into a polydopamine solution to form a polydopamine coating, and then immerse it in a solution of the nanogel (t-PA NPs) prepared in Step 3, so that the nanogel is loaded onto the inner surface of the blood vessel through adsorption. After cleaning and drying, the directional thrombolysis and endothelialization composite small-diameter artificial blood vessel is obtained.

2. The preparation method according to claim 1, characterized in that, In step 1, at least one of trichlorosilane (HSiCl3), methyltrichlorosilane (CH3SiCl3), and silicon tetrachloride (SiCl4) is selected to silanize the surface of blood vessels; at least one of (3-aminopropyl)trimethoxysilane (APTMS), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and (3-aminopropyl)triethoxysilane (APTES) is selected for amination; and at least one of succinic anhydride, maleic anhydride, adipic anhydride, and glutaric anhydride is selected for carboxylation.

3. The preparation method according to claim 1, characterized in that, In some technical solutions of the present invention, in step 2, the functionalized artificial blood vessel is immersed in sulfo-NHS and EDC for reaction, and the reaction time is 10~60 min.

4. The preparation method according to claim 1, characterized in that, In step 2, the REDV polypeptide is dissolved in a phosphate buffer solution with a concentration of 0.5-5 mM / mL; the reaction time between the blood vessel and the REDV polypeptide in the phosphate buffer solution is 12-48 h.

5. The preparation method according to claim 1, characterized in that, The restriction enzyme sequence of the polypeptide in step 3 is selected from Gly-(D)Phe-Pro-Arg-Gly-Phe or (D-Phe)-Pro-Arg-Pro-(Gly)4.

6. The preparation method according to claim 1, characterized in that, The preparation of the nanohydrogel in step 3 specifically includes the following steps: Step 3.1: The polypeptide is dissolved in sodium bicarbonate buffer solution at a concentration of 0.5~5 mg / mL; Step 3.2: Dissolve the acrylamide monomer 1 in N,N-dimethylformamide (DMF) and add it dropwise to the polypeptide solution obtained in step 3.

1. The reaction time is 1-4 h. Step 3.3: Dissolve acrylamide monomer 2 in sodium bicarbonate buffer solution at a total concentration of 1-5 mg / mL; Step 3.4: Dissolve t-PA in phosphate buffered saline (PBS) and mix it with the mixed solution prepared in step 3.2 and the solution prepared in step 3.

3. Stir at 2-8 °C for 5-20 min. Step 3.5: Add peptide crosslinking agent and N,N,N',N'-tetramethylethylenediamine sequentially and stir. Then add ammonium persulfate aqueous solution to initiate free radical polymerization. React at 2-8 °C for 2-8 h under a nitrogen atmosphere. Step 3.6: After the reaction is complete, dialyze for 24-48 h, and freeze-dry to obtain the t-PA-loaded nanogel particles (t-PA NPs).

7. The preparation method according to claim 6, characterized in that, The amount of peptide added in step 3 is 0.5 to 5 mol of the total amount of acrylamide monomer 1 and acrylamide monomer 2.

8. The preparation method according to claim 6, characterized in that, The acrylamide monomer 1 in step 3.2 contains at least an amine reactive group and a polymerizable double bond, and more preferably N-succinimide acrylate; the acrylamide monomer 2 in step 3.3 is selected from at least one of acrylamide and N-(3-aminopropyl)methacrylamide hydrochloride, N-isopropylacrylamide, N,N-dimethylacrylamide or hydroxyethyl methacrylate.

9. A targeted thrombolysis and endothelialization composite small-diameter artificial blood vessel, wherein the artificial blood vessel is prepared by any one of the preparation methods according to claims 1-8.

10. The preparation method according to claim 9, characterized in that, The inner surface of the composite small-diameter artificial blood vessel includes a REDV peptide-modified layer and a t-PA NPs-loaded layer; the t-PA NPs-loaded layer is loaded onto the REDV peptide-modified layer through a polydopamine coating; the t-PANPs are encapsulated in a nanohydrogel with a peptide containing a specific enzyme digestion sequence as a crosslinking agent; the enzyme digestion sequence of the peptide is Gly-(D)Phe-Pro-Arg-Gly-Phe or (D-Phe)-Pro-Arg-Pro-(Gly)4.

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