A need short peptide modified polylactic acid implant material and a preparation method and application thereof

Abstract

The application discloses a NEED short peptide modified polylactic acid implant material and a preparation method and application thereof, and relates to the technical field of biomedical materials. The preparation method of the NEED short peptide modified polylactic acid implant material provided by the application is as follows: an interface composed of polylactic acid material is constructed on the surface of an implant material substrate, the interface is activated, and the NEED short peptide is fixed on the interface. The polylactic acid implant material prepared by the application utilizes the interface composed of the polylactic acid material and the NEED short peptide with negative electricity to enhance the adhesion stability of adhesion proteins such as fibronectin and to enhance the subsequent cell adhesion response, thereby promoting cell adhesion and implant material integration. The material can be widely applied to the fields of degradable vascular stents, bone repair materials, dental implants, tissue engineering stents and nanofiber membranes, and can also provide a new technical scheme for interface functionalization of implant materials in a metabolic abnormal environment, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a polylactic acid implant material modified with NEED short peptides. Background Technology

[0002] Polylactic acid (PLLA) biodegradable materials have been widely used in biomedical fields such as vascular scaffolds, bone repair materials, dental implants, and tissue engineering scaffolds in recent years due to their good biocompatibility, processability, and biodegradability. Among them, PLLA (poly-L-lactic acid) and PDLLA (also known as polyracemic lactic acid, polydi-lactic acid, or poly-DL-lactide) have become some of the most commonly used absorbable implant materials due to their stable mechanical properties and safe degradation products. However, PLLA and PDLLA generally suffer from strong bioinertness and insufficient interfacial activity. Their surfaces lack effective cell recognition sites and protein binding sites, resulting in low protein adsorption efficiency in the early stages of implantation, which in turn affects subsequent cell adhesion, proliferation, and tissue integration.

[0003] After implantation, the first event is the rapid adsorption of plasma proteins onto the material surface, forming a protein coronal layer. This protein layer directly determines the subsequent recognition and adhesion behavior of cells to the material surface. Among them, fibronectin (FN), as an important adhesion protein in the extracellular matrix, can regulate cell spreading, migration, endothelialization, and tissue repair by mediating the activation of signaling pathways such as focal adhesion kinase (FAK) through integrin. Therefore, the adsorption stability of FN on the material surface is considered one of the important factors determining the biocompatibility of the implant.

[0004] Existing research has found that due to the strong hydrophobicity of PLLA and PDLLA surfaces, FN adsorption stability at their interfaces is poor, making them prone to conformational changes and interfacial desorption, resulting in insufficient focal adhesion formation. Furthermore, in metabolically abnormal environments such as hyperlipidemia, elevated levels of low-density lipoprotein (LDL) further alter the local charge environment at the material interface, weakening the electrostatic interaction between FN and the material, increasing protein diffusion at the interface, reducing residence time, and ultimately leading to decreased cell adhesion and implant integration failure.

[0005] Currently, methods for modifying the surface of polylactic acid (PLLA) materials mainly include plasma treatment, surface coating, growth factor immobilization, and extracellular matrix coating. While these methods can improve surface activity to some extent, they still suffer from problems such as insufficient stability, high cost, complex processes, and a lack of targeting for interfacial charge regulation. Especially under high lipid metabolism, traditional surface modification methods struggle to maintain stable protein adsorption and cellular response. Therefore, developing a novel surface modification strategy that can regulate the interfacial charge distribution between PLLA and PDLLA, enhance stable adhesion of fibrinogen (FN), and improve subsequent cellular response is of great significance for improving the interfacial integration of implanted materials under metabolically abnormal conditions. Summary of the Invention

[0006] To address the issues of insufficient adhesion stability of PLLA / PDLLA surface proteins and decreased implant integration capacity under high-lipid environments, this invention provides a NEED-modified polylactic acid implant material, its preparation method, and its applications. This invention utilizes negatively charged NEED peptides to modify the interface of polylactic acid materials such as poly-L-lactic acid (PLLA) and poly-racemic lactic acid (PDLLA), enhancing the adhesion stability of adhesion proteins such as fibronectin (FN) and strengthening the subsequent cell adhesion response, thereby promoting cell adhesion and implant material integration. Specifically, this is achieved through the following techniques.

[0007] The present invention provides a method for preparing a polylactic acid implant material modified with NEED short peptides, the steps of which include: constructing an interface composed of polylactic acid material on the surface of an implant material substrate, activating the interface; and immobilizing the NEED short peptides on the interface.

[0008] The amino acid sequence of the NEED short peptide is a core sequence as shown in SEQ ID NO.1, or a derived short peptide, a charged modified short peptide, a linker modified short peptide, or a PEG spacer modified short peptide containing the core sequence.

[0009] The NEED short peptide used in this invention originates from the charged adhesion region of the Type III domain in FN. Through molecular dynamics simulations, interfacial electrostatic analysis, and total internal reflection fluorescence microscopy (TIRF) single-molecule trajectory tracing, the applicant discovered that specific negatively charged amino acid regions in FN play a crucial role in maintaining stable adsorption at the protein-material interface. Specifically, the "NEED" sequence, composed of asparagine (Asn, abbreviated as N), glutamic acid (Glu, abbreviated as E), glutamic acid (Glu, abbreviated as E), and aspartic acid (Asp, abbreviated as D), can form a stable local electrostatic adsorption region, enhancing the short-range interaction between FN and the material surface. Therefore, this invention preferably uses a short peptide containing the NEED core sequence as a functional modification molecule, and can further introduce glycine linkers, polyethylene glycol spacers, cysteine ​​active sites, or dopamine anchoring groups at both ends of the short peptide to improve its immobilization stability and interfacial adaptability.

[0010] This invention uses polylactic acid (PLLA) to construct the interface of implant materials. Specifically: (1) PLLA, PDLLA, and other materials are first dissolved in organic solvents such as chloroform, dichloromethane, or hexafluoroisopropanol to prepare a polymer solution with a concentration of 10-30 mg / mL. After thorough stirring to form a uniform and transparent system, (2) a polylactic acid film is then formed on the surface of glass, titanium sheets, vascular stents, or other medical substrates using methods such as spin coating, spraying, dip coating, electrospinning, or solvent evaporation. (3) After film formation, the film is dried at room temperature or in a vacuum environment to remove residual organic solvents.

[0011] Traditional polylactic acid (PLA) materials suffer from poor stability due to their strong hydrophobic surface and lack of active functional groups and effective charge regulation sites, leading to rapid diffusion and desorption of extracellular matrix proteins such as fibroblasts (FN) at the material interface. To address this issue, this invention further activates the interface of the PLA material to improve short peptide immobilization efficiency. For example, oxygen plasma, air plasma, or alkaline treatment can be used to introduce active groups such as hydroxyl and carboxyl groups onto the material surface.

[0012] After surface activation, this invention immobilizes NEED short peptides at the PLLA / PDLLA interface using physical adsorption or chemical coupling. For example, the EDC / NHS chemical coupling method can be used. First, the carboxyl groups on the material surface are activated using the EDC / NHS system, and then the NEED short peptide is introduced into the reaction system, allowing the amino groups of the short peptide to form stable amide bonds with the material surface. This method can significantly improve the long-term stability of the short peptide and reduce the desorption phenomenon of the short peptide in physiological environments. In addition, dopamine self-polymerization, chemical oxidation treatment, and other methods can also be used to achieve short peptide immobilization.

[0013] Ultimately, this invention discovered that by utilizing negatively charged NEED short peptides (amino acid sequence Asn-Glu-Glu-Asp, abbreviated as "NEED") derived from the FN adhesion region, the local electrostatic environment and charge distribution at the interface of polylactic acid materials can be regulated, thereby forming an electrostatic microenvironment that promotes stable adsorption of FN.

[0014] In a high-fat environment, LDL alters the surface potential of the material interface, weakening the electrostatic adsorption between fibronectin (FN) and the material. This leads to enhanced FN diffusion, reduced residence time, and weakened focal adhesion signal. The NEED short peptide in this invention can effectively regulate the distribution of negative charge at the interface and reduce LDL-induced interfacial charge perturbation, thereby enhancing the stable adsorption performance of fibronectin (FN) at the interface and promoting subsequent cell adhesion, focal adhesion formation, and tissue integration.

[0015] The NEED-modified interface not only enhances FN adsorption but also improves FN conformational stability and increases the exposure of integrin binding sites, thereby promoting cellular recognition of the material interface. After stable FN adsorption, it further promotes integrin β1 expression and focal adhesion complex formation, activates the FAK / p-FAK signaling pathway, and enhances cytoskeleton remodeling. Cellular experiments of this invention show that the NEED-modified PLLA / PDLLA interface significantly increases the spreading area, cell adhesion quantity, and expression levels of adhesion-related molecules such as ITGβ1, MAP2K1, RSC, and VEGFA in human umbilical vein endothelial cells (HUVECs). Even in a high-density lipoprotein (LDL) environment, the NEED-modified interface maintains a high level of cell adhesion signaling, indicating that this invention has good adaptability to metabolically abnormal environments.

[0016] The single-molecule tracking test results of this invention show that the waiting time of FN after NEED modification is significantly increased, the jump step size is significantly reduced, and the mean square displacement (MSD) exponent decreases, indicating that the interfacial diffusion ability of FN is reduced while the adsorption stability is enhanced.

[0017] Furthermore, the interface composed of polylactic acid material is constructed by spin coating, spraying, dip coating, electrospinning or solvent evaporation.

[0018] Furthermore, when using spin coating, the spin coating method includes: spreading at 200-1000 rpm and 2000-6000 rpm successively to form a uniform film, wherein the thickness of the interface is 50 nm-10 μm.

[0019] Furthermore, the activation treatment is one or more of the following: plasma treatment, ultraviolet ozone treatment, alkali treatment, dopamine self-polymerization treatment, and chemical oxidation treatment.

[0020] Furthermore, the plasma treatment includes oxygen plasma treatment and air plasma treatment.

[0021] Optionally, the oxygen plasma treatment conditions are: power 10-200 W, treatment time 1-10 min.

[0022] Furthermore, the alkaline treatment method is as follows: treatment with 0.01-5 mol / L sodium hydroxide solution for 10 s-2 h.

[0023] Furthermore, the NEED short peptide can be immobilized on the interface by physical adsorption, self-assembly, EDC / NHS chemical coupling, or dopamine bridging.

[0024] Furthermore, the EDC / NHS chemical coupling method is as follows: the activated interface is immersed in an EDC / NHS mixture for 15-60 min, with an EDC concentration of 0.01-0.5 mol / L and an NHS concentration of 0.01-0.5 mol / L; then the interface is immersed in a solution containing the NEED short peptide for coupling for 2-24 h at a coupling temperature of 4-25℃.

[0025] Furthermore, the NEED short peptide is also connected to one or more of the following at the N-terminus and / or C-terminus of the core sequence: a glycine linker arm, a cysteine ​​active site, a dopamine anchoring group, a biotin labeling group, or a polyethylene glycol spacer chain.

[0026] Furthermore, the polylactic acid material includes one or more of poly-L-lactic acid (PLLA), poly-racemic lactic acid (PDLLA), polylactic acid-glycolic acid copolymer (PLGA), and polycaprolactone (PCL).

[0027] The present invention also provides a polylactic acid implant material modified with NEED short peptides prepared by the above preparation method.

[0028] The polylactic acid implant material modified with NEED short peptides prepared in this invention has the functions of enhancing fibronectin (FN) stable adsorption, reducing protein interfacial diffusion, enhancing focal adhesion formation, promoting cell adhesion and improving endothelialization ability by fixing a NEED short peptide modification layer on the surface.

[0029] The present invention also provides an application of polylactic acid implant material modified with the above-mentioned NEED short peptide, for the preparation of biodegradable vascular scaffolds, dental implants, bone repair membranes, tissue engineering scaffolds, nanofiber membranes, microneedles, extracellular matrix (ECM) composite materials or medical catheters.

[0030] In practical applications, NEED-modified polylactic acid material interfaces can promote integrin β1 expression and focal adhesion complex formation, and activate the FAK / p-FAK signaling pathway. They can also increase the spreading area, adhesion quantity, and adhesion-related gene expression levels of human umbilical vein endothelial cells (HUVECs), endothelial progenitor cells, osteoblasts, fibroblasts, or mesenchymal stem cells, ultimately improving the integration ability of implanted materials in a high-lipid environment.

[0031] Furthermore, the NEED-modified interface is suitable for modifying the interface of implantable materials in metabolically abnormal environments such as hyperlipidemia, diabetes, atherosclerosis, or chronic inflammation.

[0032] Compared with the prior art, the advantages of the present invention are:

[0033] 1. This invention introduces a NEED short peptide modification layer at the PLLA / PDLLA interface, forming a stable local charge microenvironment on the material surface. This enhances the short-range electrostatic interaction between FN and the material interface, achieving precise control over the surface charge environment and protein adsorption behavior. It can significantly enhance the adhesion stability of adhesion proteins such as fibronectin implanted on the PLLA / PDLLA surface, as well as their responsiveness to subsequent cells. In particular, it still exhibits good interfacial stability and biological activity under abnormal high-lipid metabolism, thus possessing significant technical advantages and application value.

[0034] 2. By introducing a NEED short peptide modification layer at the PLLA / PDLLA interface, this invention can also reduce the conformational fluctuations of FN during adsorption, increase the exposure of its bioactive sites, and provide a more stable interface basis for subsequent cell recognition.

[0035] 3. The polylactic acid implant material provided by this invention can effectively enhance the cell adhesion and endothelialization capabilities of its surface. Since FN is an important extracellular matrix protein mediating cell recognition and adhesion, its stable adsorption can further promote integrin β1 expression and focal adhesion complex formation. The NEED-modified interface in this invention can significantly enhance the activation of adhesion-related signaling pathways such as FAK and p-FAK, promote cytoskeleton remodeling and cell spreading, thereby improving the binding ability between cells and the material interface. Cell experiments show that the PLLA / PDLLA surface modified with NEED short peptides can significantly increase the spreading area of ​​HUVEC cells, increase the number of early cell adhesions, and upregulate the expression levels of adhesion and angiogenesis-related genes such as ITGβ1, MAP2K1, RSC, and VEGFA.

[0036] 4. This invention exhibits excellent adaptability to metabolically abnormal environments such as hyperlipidemia. The NEED short peptide in this invention can effectively buffer LDL-induced interfacial charge disturbances and maintain stable FN residence on the material surface, thereby mitigating the adverse effects of a high-lipid environment on implant integration. Compared to traditional surface treatment methods, this invention maintains high levels of FAK / p-FAK expression and cell spreading ability even under high-lipid conditions, indicating superior adaptability to metabolically abnormal environments. This has significant clinical implications for implantation therapy in patients with hyperlipidemia, diabetes, and atherosclerosis.

[0037] 5. The NEED short peptides used in this invention have small molecular weights, well-defined structures, and are easy to synthesize artificially. They offer advantages such as low cost, high stability, and good batch-to-batch consistency. Compared to traditional strategies such as growth factor immobilization and extracellular matrix coating, this invention eliminates the need for complex bioactive molecules, effectively reducing preparation costs and immune risks. Furthermore, the NEED short peptides can be stably immobilized on material surfaces through various methods, including physical adsorption, EDC / NHS coupling, and dopamine bridging. The process is simple and easily scaled up for industrial production.

[0038] 6. This invention possesses excellent material applicability and clinical scalability. Besides PLLA and PDLLA, this invention can also be applied to the surfaces of PLGA, PCL, titanium, magnesium alloys, and other medical polymer or metal implant materials. It can be widely used in biodegradable vascular scaffolds, bone repair materials, dental implants, tissue engineering scaffolds, and nanofiber membranes. Therefore, this invention not only improves the bioactivity of implant material interfaces but also provides a new technical solution for the functionalization of implant material interfaces under metabolically abnormal environments, demonstrating broad application prospects. Attached Figure Description

[0039] Figure 1 The flowchart of the present invention illustrates the construction process of the PLLA / PDLLA interface modified with NEED short peptide, as well as the detection steps of FN interface adhesion behavior, cell adhesion and focal adhesion formation and adhesion-related gene expression, and the main results of enhancing FN stable adsorption and promoting cell adhesion.

[0040] Figure 2 The diagram shows the original PLA film (top left), PLLA powder (top middle), and PDLLA powder (top right) of the polylactic acid (PLA) modified layer, as well as the glass substrates that are unmodified (bottom left), PLLA modified (bottom middle), and PDLLA modified (bottom right).

[0041] Figure 3 The images show a scanning electron microscope (SEM) schematic of a glass substrate (top), a PLLA-modified glass substrate (middle), and a PDLLA-modified glass substrate (bottom).

[0042] Figure 4 For: TIRF single-molecule tracking of FN jump step size and waiting time on PLLA substrates pretreated with PBS and NEED short peptides under control plasma or LDL conditions. n=3.

[0043] Figure 5 The purpose was to perform RT-qPCR analysis on the expression of ITGβ1, MAP2K1, RSC, and VEGFA in HUVECs cultured on PLLA substrate pretreated with PBS and NEED short peptides under control plasma or LDL conditions. n=3.

[0044] Figure 6 In this study, HUVECs were cultured on PLLA substrates pretreated with PBS and NEED short peptides under control plasma or LDL conditions, and FAK and p-FAK were quantitatively analyzed by Western blot. n=3.

[0045] Figure 7 For: TIRF single-molecule tracking of the jump step size and waiting time of FN on a PDLLA substrate pretreated with PBS and NEED short peptides under control plasma or LDL conditions. n=3.

[0046] Figure 8 The purpose was to perform RT-qPCR analysis on the expression of ITGβ1, MAP2K1, RSC, and VEGFA in HUVECs cultured on PDLLA substrates pretreated with PBS and NEED short peptides under control plasma or LDL conditions. n=3.

[0047] Figure 9 In this study, HUVECs were cultured on PDLLA substrates pretreated with PBS and NEED short peptides under control plasma or LDL conditions, and FAK and p-FAK were quantitatively analyzed by Western blot. n=3.

[0048] Figure 10 The purpose was to perform RT-qPCR analysis on the expression of ITGβ1, MAP2K1, RSC, and VEGFA in HUVECs cultured on titanium substrates pretreated with PBS and NEED peptides under control plasma or LDL conditions. n=3.

[0049] Figure 11 In this study, HUVECs were cultured on titanium substrates pretreated with PBS and NEED short peptides under control plasma or LDL conditions, and FAK and p-FAK were quantitatively analyzed by Western blot. n=3. Detailed Implementation

[0050] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] In some embodiments provided by this invention, the method for preparing polylactic acid implant material modified with NEED short peptides specifically involves constructing an interface composed of polylactic acid material on the surface of the implant material substrate, activating the interface, and fixing the NEED short peptides onto the interface.

[0052] In the above preparation method, the amino acid sequence of the NEED short peptide is a core sequence as shown in SEQ ID NO.1, or a derived short peptide, a charged modified short peptide, a linker modified short peptide, or a PEG spacer modified short peptide containing the core sequence.

[0053] Optionally, the interface composed of polylactic acid material is constructed by spin coating, spraying, dip coating, electrospinning or solvent evaporation.

[0054] When using spin coating, the spin coating method includes spreading the film at 200-1000 rpm and 2000-6000 rpm respectively to form a uniform film, wherein the thickness of the interface is 50 nm-10 μm.

[0055] Optionally, the activation treatment can be one or more of plasma treatment, ultraviolet ozone treatment, alkali treatment, dopamine self-polymerization treatment, and chemical oxidation treatment.

[0056] Plasma treatment includes oxygen plasma treatment and air plasma treatment.

[0057] When oxygen plasma treatment is selected, the conditions for oxygen plasma treatment are: power 10-200 W, treatment time 1-10 min.

[0058] When alkaline treatment is used, the treatment method is as follows: treat with 0.01-5 mol / L sodium hydroxide solution for 10 s-2 h.

[0059] Optionally, the NEED short peptide can be immobilized on the interface by physical adsorption, self-assembly, EDC / NHS chemical coupling, or dopamine bridging.

[0060] When using EDC / NHS chemical coupling, the specific method is as follows: the activated interface is immersed in an EDC / NHS mixture for 15-60 min, with an EDC concentration of 0.01-0.5 mol / L and an NHS concentration of 0.01-0.5 mol / L; then the interface is immersed in a solution containing the NEED short peptide for coupling for 2-24 h at a coupling temperature of 4-25℃.

[0061] The NEED short peptide selected in this invention may also be linked to one or more of the following at the N-terminus and / or C-terminus of the core sequence: a glycine linker arm, a cysteine ​​active site, a dopamine anchoring group, a biotin labeling group, or a polyethylene glycol spacer chain.

[0062] Furthermore, the polylactic acid material includes one or more of poly-L-lactic acid (PLLA), poly-racemic lactic acid (PDLLA), polylactic acid-glycolic acid copolymer (PLGA), and polycaprolactone (PCL).

[0063] Experimental Example 1: Preparation of polylactic acid implant material modified with NEED short peptides and its effect on FN adhesion and cellular response

[0064] 1. Preparation of NEED-modified PLLA interfaces (e.g.) Figure 1 (As shown)

[0065] (1) The pre-cleaned glass substrate (or titanium substrate, i.e. implant material substrate) is ultrasonically cleaned with acetone, ethanol and deionized water for 15 min in sequence, and then dried with nitrogen gas for later use.

[0066] Weigh 100 mg of PLLA and add it to 10 mL of chloroform. Stir magnetically for 1 h at room temperature to form a homogeneous and transparent polymer solution.

[0067] The NEED short peptide was prepared by solid-phase synthesis, and its amino acid sequence is Asn-Glu-Glu-Asp. The NEED short peptide was dissolved in PBS buffer for later use, with a final concentration of 200 μg / mL.

[0068] (2) The PLLA solution was dropped onto the surface of a glass substrate, and a PLLA film was constructed by spin coating. The first stage of spin coating was carried out at 500 rpm for 10 s to spread the film, and the second stage of spin coating was carried out at 3000 rpm for 30 s to form a uniform interface layer. After film formation, the sample was placed in a vacuum drying oven overnight to remove residual organic solvents.

[0069] like Figure 2 Hydrophilicity and hydrophobicity measurement results and Figure 3 The results of scanning electron microscopy show that the hydrophobicity of the glass surface increased after PLLA modification, and layered organic matter was visible on the surface.

[0070] (3) The PLLA interface was activated by oxygen plasma. The treatment power was 100 W and the treatment time was 5 min, so as to introduce active functional groups such as hydroxyl and carboxyl groups on the material surface.

[0071] (4) Immerse the activated PLLA interface in a mixed EDC / NHS solution with an EDC concentration of 0.2 mol / L and an NHS concentration of 0.1 mol / L, and react at room temperature for 30 min. Then gently rinse the surface with PBS buffer to remove unreacted reagents.

[0072] (5) Immerse the activated PLLA sample in a NEED peptide solution and react at 4°C for 12 h to allow the NEED peptide to be stably fixed on the PLLA surface via amide bonds. After the reaction, thoroughly rinse the sample surface with PBS to remove unbound peptides and store under sterile conditions for later use.

[0073] 2. FN adhesion and cell response test (e.g.) Figure 1 (As shown)

[0074] Total internal reflection fluorescence microscopy (TIRF) was used to further verify the single-molecule adhesion behavior of fibrinogen (FN) at the interface. First, FN was labeled with Cy5 fluorescent dye, and free fluorescent molecules were removed by ultrafiltration and centrifugation. Then, Cy5-labeled FN was added to the surfaces of unmodified PLLA and NEED-modified PLLA groups, and real-time imaging was performed using TIRF microscopy; the excitation wavelength was 640 nm, and the acquisition interval was 50 ms. Single-particle tracking (SPT) was used to analyze the jump step size, waiting time, and MSD changes of FN single molecules.

[0075] like Figure 4 The results showed that in the unmodified PLLA interface, FN exhibited a larger jump step size and a shorter waiting time. This indicates that FN has strong interfacial diffusion ability but poor residence stability. In the NEED-modified group, however, the jump step size of FN was significantly reduced, and the waiting time was significantly prolonged. This indicates that the NEED peptide can effectively inhibit FN interfacial diffusion and enhance protein residence stability. After adding LDL to simulate a high-fat environment, FN diffusion in the unmodified PLLA group was further enhanced, while the NEED-modified interface could still maintain a high FN residence ability, indicating that it has good adaptability to high-fat environments.

[0076] Human umbilical vein endothelial cells (HUVECs) were used to evaluate cellular responses at the NEED-modified interface. HUVECs were cultured at 1 × 10⁻⁶ cells / year. 5Cells / wells were seeded at a density of 24 h on the surface of each group of materials. Total RNA was then extracted using the TRIzol method, reverse transcribed, and detected by real-time quantitative polynucleotide chain reaction (RT-qPCR). Genes detected included integrin β1 (ITGβ1), mitogen-activated protein kinase 1 (MAP2K1), regulatory solute carrier protein (RSC), and vascular endothelial growth factor A (VEGFA), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as an internal control gene.

[0077] like Figure 5 The results showed that, compared with the unmodified PLLA group, the expression of ITGβ1, MAP2K1, RSC, and VEGFA was significantly upregulated in the NEED-modified PLLA group. Specifically, the enhanced expression of ITGβ1 and MAP2K1 indicates that the NEED short peptide can promote focal adhesion formation; the increased VEGFA expression suggests that it is beneficial to subsequent angiogenesis and endothelialization. Furthermore, under high LDL conditions, the NEED-modified group maintained a high level of adhesion-related gene expression, indicating that the NEED short peptide-modified polylactic acid implant material provided in this invention can effectively alleviate the inhibitory effect of a high-lipid environment on cell adhesion function.

[0078] HUVEC at 1×10 5 Cells / wells were seeded at a density of 1 / 2 cells / well on the surface of each group of materials and cultured for 24 h. Total protein was then extracted, and the expression levels of focal adhesion kinase (FAK) and phosphorylated focal adhesion kinase (p-FAK) were detected by Western blot, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as an internal control gene. Figure 6 The results showed that, in both control and LDL environments, the p-FAK / FAK expression ratio was significantly upregulated in the NEED-modified PLLA group compared to the unmodified PLLA group. This indicates that the NEED-modified interface can promote FAK phosphorylation activation and enhance cell adhesion.

[0079] Experimental Example 2: Construction of NEED-modified PDLLA interface and verification of its biological performance

[0080] Polylactic acid implant material modified with NEED short peptides was constructed using the same method as in Example 1. Subsequently, FN single-molecule TIRF imaging and HUVEC cell RT-qPCR and Western blot analysis were performed.

[0081] like Figure 7 TIRF single-molecule imaging results showed that the FN jumping step size decreased and the waiting time increased in the NEED-modified PDLLA interface, indicating enhanced FN interface retention. Even under high LDL conditions, the NEED-modified group maintained high FN adsorption stability.

[0082] like Figure 8 The RT-qPCR results showed that, compared with the unmodified PDLLA group, the expression of adhesion-related genes such as ITGβ1, MAP2K1, RSC and VEGFA was significantly upregulated in the NEED-modified PDLLA group, and the spreading ability of HUVEC cells was enhanced.

[0083] like Figure 9 The Western blot results showed that, in both the control and LDL environments, the expression ratio of FAK in the NEED-modified PDLLA group was significantly upregulated compared to the unmodified PDLLA group. This indicates that the present invention is also applicable to PDLLA interface modification and can effectively improve the cell compatibility and adaptability of the material to a high-lipid environment.

[0084] Experimental Example 3: Construction of NEED-Modified Titanium Interface and Validation of its Biological Properties

[0085] Titanium (Ti) material modified with NEED peptides was constructed using a physical adsorption method. The NEED peptides were dissolved in PBS buffer to a final concentration of 200 μg / mL. The titanium substrate was placed in the peptide solution and reacted at room temperature for 1 h, followed by RT-qPCR and Western blot analysis of HUVEC cells.

[0086] like Figure 10 The RT-qPCR results showed that, compared with the unmodified titanium group, the expression of adhesion-related genes such as ITGβ1, MAP2K1, RSC and VEGFA was significantly upregulated in the NEED-modified titanium group, and the spreading ability of HUVEC cells was enhanced.

[0087] like Figure 11 Western blot results showed that, in both control and LDL environments, the p-FAK / FAK expression ratio was significantly upregulated in the NEED-modified titanium group compared to the unmodified titanium group. This indicates that the present invention is also applicable to titanium interface modification and can effectively improve the cell compatibility and adaptability of materials to high-lipid environments.

[0088] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing a polylactic acid implant material modified with NEED short peptides, characterized in that, The steps include: constructing an interface composed of polylactic acid material on the surface of the implant material substrate, activating the interface; and immobilizing NEED short peptides on the interface. The amino acid sequence of the NEED short peptide is a core sequence as shown in SEQ ID NO.1, or a derived short peptide, a charged modified short peptide, a linker modified short peptide, or a PEG spacer modified short peptide containing the core sequence.

2. The method for preparing polylactic acid implant material modified with NEED short peptides according to claim 1, characterized in that, Interfaces composed of polylactic acid materials are constructed by spin coating, spraying, dip coating, electrospinning, or solvent evaporation. Furthermore, when using spin coating, the spin coating method includes: spreading at 200-1000 rpm and 2000-6000 rpm successively to form a uniform film, wherein the thickness of the interface is 50 nm-10 μm.

3. The method for preparing polylactic acid implant material modified with NEED short peptides according to claim 1, characterized in that, The activation treatment method is one or more of the following: plasma treatment, ultraviolet ozone treatment, alkali treatment, dopamine self-polymerization treatment, and chemical oxidation treatment.

4. The method for preparing polylactic acid implant material modified with NEED short peptides according to claim 3, characterized in that, The plasma treatment includes oxygen plasma treatment and air plasma treatment; Furthermore, the conditions for the oxygen plasma treatment are: power 10-200 W, treatment time 1-10 min.

5. The method for preparing polylactic acid implant material modified with NEED short peptides according to claim 3, characterized in that, The alkaline treatment method is as follows: treat with 0.01-5 mol / L sodium hydroxide solution for 10 s-2 h.

6. The method for preparing polylactic acid implant material modified with NEED short peptides according to claim 1, characterized in that, The NEED short peptide is immobilized on the interface by physical adsorption, self-assembly, EDC / NHS chemical coupling, or dopamine bridging. Furthermore, the EDC / NHS chemical coupling method is as follows: the activated interface is immersed in an EDC / NHS mixture for 15-60 min, with an EDC concentration of 0.01-0.5 mol / L and an NHS concentration of 0.01-0.5 mol / L; then the interface is immersed in a solution containing the NEED short peptide for coupling for 2-24 h at a coupling temperature of 4-25℃.

7. The method for preparing polylactic acid implant material modified with NEED short peptides according to claim 1, characterized in that, The NEED peptide is further connected to one or more of the following at the N-terminus and / or C-terminus of the core sequence: a glycine linker, a cysteine ​​active site, a dopamine anchoring group, a biotin labeling group, or a polyethylene glycol spacer chain.

8. The method for preparing polylactic acid implant material modified with NEED short peptides according to claim 1, characterized in that, The polylactic acid material includes one or more of poly-L-lactic acid, polyracemic lactic acid, polylactic acid-glycolic acid copolymer, and polycaprolactone.

9. A polylactic acid implant material modified with NEED short peptides prepared by the preparation method according to any one of claims 1-8.

10. The application of the polylactic acid implant material modified with NEED short peptide as described in claim 9, characterized in that, It is used to prepare biodegradable vascular scaffolds, dental implants, bone repair membranes, tissue engineering scaffolds, nanofiber membranes, microneedles, extracellular matrix composites or medical catheters; Furthermore, it can be used to prepare implant materials for use in metabolically abnormal environments; Furthermore, the abnormal metabolic environment includes, but is not limited to, hyperlipidemia, diabetes, atherosclerosis, or chronic inflammation.

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