A method based on sulfonated polyetheretherketone (PEEK) adhesion adeno-associated virus and its application

Abstract

This invention relates to the field of adeno-associated virus (AAV) vector technology, specifically a method for adhering AAV based on sulfonated polyetheretherketone (PEEK) and its application. The material preparation method includes the following steps: S1, adding PEEK to concentrated sulfuric acid to obtain sulfonated PEEK; S2, performing a primary polydopamine coating; S3, spin-coating a nano-silica solution onto the surface of the PEEK with primary polydopamine coating obtained in step S2; S4, performing hot-pressing treatment; S5, performing a secondary polydopamine coating; S6, adding the PEEK with secondary polydopamine coating obtained in step S4 to a hydrofluoric acid solution for reaction and cleaning; S7, adding the filtered product washed with deionized water in step S6 to an AAV solution for incubation. The AAV viral vector prepared by this invention has a large amount of adhering AAV, enabling sustained release.

Description

Technical Field

[0001] This invention relates to the field of adeno-associated virus (AAV) loading technology, specifically a method for adhering AAV based on sulfonated polyether ether ketone (PEEK) and its application. Background Technology

[0002] Adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus with advantages such as a wide host range, low immunogenicity, high safety, and long-term stable expression of exogenous genes, making it one of the most commonly used viral vectors in gene therapy and vaccine development. However, AAV's low transduction efficiency in vivo limits its clinical application. Improving AAV transduction efficiency is currently a research hotspot in the field of gene therapy. Studies have shown that altering the delivery method of AAV, such as immobilizing AAV on the surface of materials, can achieve sustained release of AAV and increase the probability of contact with cells, thereby improving transduction efficiency and protecting AAV from clearance by the body's immune system. Therefore, developing a material that can effectively adsorb AAV has significant application value.

[0003] Currently, methods for immobilizing AAV on material surfaces mainly include physical adsorption, chemical coupling, and the biotin-avidin system. Physical adsorption is simple, but the amount of AAV immobilized is low and it is prone to detachment. Chemical coupling can improve the amount and stability of immobilized AAV, but requires complex chemical modification of the material surface. While the biotin-avidin system has high specificity and affinity, it is costly and complex to operate. Therefore, developing a simple, efficient, and stable method for AAV immobilization is of significant practical importance. Summary of the Invention

[0004] The purpose of this invention is to provide a method and its application based on sulfonated polyether ether ketone adhesion adeno-associated virus, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method based on sulfonated polyetheretherketone (PEEK) adhesion to adeno-associated virus (AAV) includes the following steps:

[0007] S1. Add polyether ether ketone to concentrated sulfuric acid and sonicate at 40-60℃ for 4-6 minutes. Then pour the reaction product into ice water, filter, wash and dry to obtain sulfonated polyether ether ketone.

[0008] S2. Immerse the sulfonated polyether ether ketone obtained in step S1 in a hydrochloric acid dopamine solution and react continuously for 3-6 hours to obtain polyether ether ketone coated with polydopamine once.

[0009] S3. Spin-coat nano silica solution onto the polyether ether ketone surface that has been coated with polydopamine once obtained in step S2.

[0010] S4. The polyether ether ketone coated with polydopamine after step S3 is subjected to hot pressing.

[0011] S5. The polyether ether ketone coated with polydopamine after step S4 is immersed in a dopamine hydrochloride solution and reacted continuously for 8-12 hours to obtain polyether ether ketone coated with polydopamine after a second time.

[0012] S6. Add the polydopamine-coated polyether ether ketone obtained in step S5 to hydrofluoric acid solution and react continuously for 3-6 hours. Then filter and wash the filtered product with sufficient deionized water.

[0013] S7. Add the filtered product washed with deionized water in step S6 to the adeno-associated virus solution and incubate at 35-40℃ for 2-5 hours. Then filter and wash the filtered product three times with PBS buffer solution to obtain a vector with adeno-associated virus attached.

[0014] Furthermore, the concentration of concentrated sulfuric acid in step S1 is 98 wt%.

[0015] Furthermore, in step S3, the mass fraction of nano-silica in the nano-silica solution is 1-5%.

[0016] Furthermore, in step S4, the hot pressing temperature is 150-200℃, the pressure is 1-3MPa, and the hot pressing time is 0.5-2h.

[0017] Furthermore, the concentration of the dopamine hydrochloride solution in step S2 is 0.5-2 mg / mL, and the concentration of the dopamine hydrochloride solution in step S5 is 15-20 mg / mL; the pH of the dopamine hydrochloride solution in both steps S2 and S5 is 8.5, and the pH of the dopamine hydrochloride solution is adjusted by Tris-HCl buffer.

[0018] Furthermore, the concentration of the hydrofluoric acid solution in step S6 is 1-2 wt%.

[0019] Furthermore, in step S7, the concentration of adeno-associated virus in the adeno-associated virus solution is 1 × 10⁻⁶. 13 vg / mL.

[0020] The above-mentioned vectors with adeno-associated virus (AAV) adsorbed by the method of sulfonated polyether ether ketone adsorption of AAV are used in the preparation of gene-targeted drugs, virus-like particle vaccines, and bioactive scaffolds.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. Improve AAV adsorption efficiency and transfection efficiency: This invention sulfonates polyether ether ketone material and then modifies its surface with polydopamine. By utilizing the adhesion properties and abundant functional groups of polydopamine, it is possible to achieve efficient adsorption of AAV. Compared with traditional physical adsorption methods, the material prepared by the method of this invention has a significantly improved adsorption capacity for AAV and good adsorption stability.

[0023] 2. In this invention, before polymerizing the polydopamine layer on the polyetheretherketone surface, nano-silica is spin-coated onto the polyetheretherketone surface, then polydopamine is coated, and finally the nano-silica is etched by hydrofluoric acid. Through the template effect of the nano-silica, a large number of pores are formed. The pores are conducive to the adsorption of AAV, which further improves the adsorption amount and adsorption stability of AAV.

[0024] 3. In this invention, after adsorbing nano-silica with polyether ether ketone, hot pressing is used to better connect the nano-silica with the carrier, prevent the nano-silica from falling off, ensure its stability as a template, and ensure that a sufficient number of pores can be formed.

[0025] 4. In the preparation of the adsorption carrier, the present invention has a total of two polydopamine layers. The two polydopamine layers cooperate with each other to form a more three-dimensional adsorption structure, which increases the adsorption area and improves the adhesion ability of the entire carrier material to AAV.

[0026] 5. The AAV-adsorbed material prepared by this invention has broad application prospects in gene therapy, vaccine development, tissue engineering, and regenerative medicine. For example, in gene therapy, it can be used to prepare gene-targeted drugs, delivering therapeutic genes to target cells to achieve gene therapy; in vaccine development, it can be used to prepare virus-like particle vaccines, improving the immunogenicity and safety of vaccines; in tissue engineering and regenerative medicine, it can be used to prepare bioactive scaffolds, promoting cell adhesion, proliferation, and differentiation, achieving tissue repair and regeneration; at the same time, it has good biocompatibility and will not produce significant toxicity or immune responses to cells and organisms, which is beneficial for its application in the biomedical field. Attached Figure Description

[0027] Figure 1 This is a process flow diagram of the present invention;

[0028] Figure 2 XPS image of the sample prepared in Example 1 of this invention;

[0029] Figure 3 This is a SEM image of the sample prepared in Example 1 of the present invention;

[0030] Figure 4 This is a schematic diagram showing the AAV adsorption capacity of the samples prepared in Example 1 and Comparative Examples 1-4 of this invention;

[0031] Figure 5 This is a schematic diagram showing the AAV release levels of the samples prepared in Example 1 and Comparative Examples 1-4 of this invention;

[0032] Figure 6 These are images showing the osteogenic effects of the samples prepared in Example 1 and Comparative Examples 1-4 of this invention.

[0033] Figure 7 This is a schematic diagram showing the cell viability of samples prepared in Example 1 and Comparative Examples 1-4 of this invention;

[0034] Figure 8 This is a schematic diagram showing the cell proliferation rate of the samples prepared in Example 1 and Comparative Examples 1-4 of this invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only 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.

[0036] Please see Figures 1 to 8 The present invention provides: Example 1

[0037] A method based on sulfonated polyetheretherketone (PEEK) adhesion to adeno-associated virus (AAV) includes the following steps:

[0038] S1. Add 10g of polyether ether ketone to 200ml of concentrated sulfuric acid with a concentration of 98wt%, and sonicate at 50℃ for 5min at a sonic frequency of 30kHz. Then pour the reaction product into ice water, filter, wash and dry to obtain sulfonated polyether ether ketone.

[0039] S2. The sulfonated polyether ether ketone obtained in step S1 is immersed in 120 ml of a 1 mg / mL dopamine hydrochloride solution and reacted continuously for 4 h. The pH of the dopamine hydrochloride solution is 8.5. The pH of the dopamine hydrochloride solution is adjusted by Tris-HCl buffer to obtain polyether ether ketone coated with polydopamine once.

[0040] S3. Spin-coat a 2wt% nano-silica solution onto the polyether ether ketone surface coated with polydopamine obtained in step S2.

[0041] S4. The polyether ether ketone coated with polydopamine after step S3 is subjected to hot pressing at a temperature of 180°C, a pressure of 2 MPa, and a pressing time of 1.5 h.

[0042] S5. The polyether ether ketone coated with polydopamine after step S4 is immersed in 120 ml of dopamine hydrochloride solution with a concentration of 18 mg / mL and reacted continuously for 10 h. The pH of the dopamine hydrochloride solution is 8.5. The pH of the dopamine hydrochloride solution is adjusted by Tris-HCl buffer to obtain polyether ether ketone coated with polydopamine for secondary treatment.

[0043] S6. The polyether ether ketone with polydopamine secondary coating obtained in step S5 is added to 150 ml of hydrofluoric acid solution with a concentration of 1.5 wt% and reacted continuously for 4 h. After that, the product is filtered and washed with sufficient deionized water.

[0044] S7. Add the filtered product washed with deionized water in step S6 to 60 ml of adeno-associated virus solution and incubate at 38°C for 3.5 h. The concentration of adeno-associated virus in the adeno-associated virus solution is 1 × 10⁻⁶. 13 The concentration was measured in vg / mL, then filtered. The filtered product was washed three times with PBS buffer to obtain a vector with adeno-associated virus attached. Example 2

[0045] A method based on sulfonated polyetheretherketone (PEEK) adhesion to adeno-associated virus (AAV) includes the following steps:

[0046] S1. Add 10g of polyether ether ketone to 200ml of concentrated sulfuric acid with a concentration of 98wt%, and sonicate at 40℃ for 4min with an ultrasonic frequency of 30kHz. Then pour the reaction product into ice water, filter, wash and dry to obtain sulfonated polyether ether ketone.

[0047] S2. The sulfonated polyether ether ketone obtained in step S1 is immersed in 120 ml of 0.5 mg / mL dopamine hydrochloride solution and reacted continuously for 3 h. The pH of the dopamine hydrochloride solution is 8.5. The pH of the dopamine hydrochloride solution is adjusted by Tris-HCl buffer to obtain polyether ether ketone coated with polydopamine once.

[0048] S3. Spin-coat a 1wt% nano-silica solution onto the polyether ether ketone surface that has been coated with polydopamine in step S2.

[0049] S4. The polyether ether ketone coated with polydopamine after step S3 is subjected to hot pressing at a temperature of 150°C, a pressure of 1 MPa, and a pressing time of 0.5 h.

[0050] S5. The polyether ether ketone coated with polydopamine after step S4 is immersed in 120 ml of a 15 mg / mL dopamine hydrochloride solution and reacted continuously for 8 h. The pH of the dopamine hydrochloride solution is 8.5. The pH of the dopamine hydrochloride solution is adjusted by Tris-HCl buffer to obtain polyether ether ketone coated with polydopamine for secondary treatment.

[0051] S6. The polyether ether ketone with polydopamine secondary coating obtained in step S5 is added to 150 ml of 1 wt% hydrofluoric acid solution and reacted continuously for 3 h. After filtration, the filtered product is washed with sufficient deionized water.

[0052] S7. Add the filtered product washed with deionized water in step S6 to 60 ml of adeno-associated virus solution and incubate at 35°C for 2 hours. The concentration of adeno-associated virus in the solution is 1 × 10⁻⁶. 13 The concentration was measured in vg / mL, then filtered. The filtered product was washed three times with PBS buffer to obtain a vector with adeno-associated virus attached. Example 3

[0053] A method based on sulfonated polyetheretherketone (PEEK) adhesion to adeno-associated virus (AAV) includes the following steps:

[0054] S1. Add 10g of polyether ether ketone to 200ml of concentrated sulfuric acid with a concentration of 98wt%, and sonicate at 60℃ for 6min with an ultrasonic frequency of 30kHz. Then pour the reaction product into ice water, filter, wash and dry to obtain sulfonated polyether ether ketone.

[0055] S2. The sulfonated polyether ether ketone obtained in step S1 is immersed in 120 ml of a 2 mg / mL dopamine hydrochloride solution and reacted continuously for 6 h. The pH of the dopamine hydrochloride solution is 8.5. The pH of the dopamine hydrochloride solution is adjusted by Tris-HCl buffer to obtain polyether ether ketone coated with polydopamine once.

[0056] S3. Spin-coat a 5wt% nano-silica solution onto the polyether ether ketone surface that has been coated with polydopamine in step S2.

[0057] S4. The polyether ether ketone coated with polydopamine after step S3 is subjected to hot pressing at a temperature of 200°C, a pressure of 3MPa, and a time of 2 hours.

[0058] S5. The polyether ether ketone coated with polydopamine once after step S4 is immersed in 120 ml of a 20 mg / mL dopamine hydrochloride solution with a pH of 8.5. The pH of the dopamine hydrochloride solution is adjusted by Tris-HCl buffer. The reaction is carried out continuously for 12 h to obtain polyether ether ketone coated with polydopamine twice.

[0059] S6. The polyether ether ketone with polydopamine secondary coating obtained in step S5 is added to 150 ml of hydrofluoric acid solution with a concentration of 2 wt% and reacted continuously for 6 h. After filtration, the filtered product is washed with sufficient deionized water.

[0060] S7. Add the filtered product washed with deionized water in step S6 to 60 ml of adeno-associated virus solution and incubate at 40°C for 5 h. The concentration of adeno-associated virus in the adeno-associated virus solution is 1 × 10⁻⁶. 13 The concentration was measured in vg / mL, then filtered. The filtered product was washed three times with PBS buffer to obtain a vector with adeno-associated virus attached.

[0061] The average particle size of the nano-silica used in this invention is 50-100 nm; the polyether ether ketone used in step S1 of this invention has a disc-like structure; the PBS buffer solution is derived from Solarbio (0.01 M pH 7.2-7.4).

[0062] Comparative Example 1

[0063] The difference between Comparative Example 1 and Example 1 is that the spin-coating process of nano-silica solution in step S3 was omitted, while the remaining steps are exactly the same as in Example 1.

[0064] Comparative Example 2

[0065] The difference between Comparative Example 2 and Example 1 is that the hot pressing process in step S4 was omitted, while the remaining steps are exactly the same as in Example 1.

[0066] Comparative Example 3

[0067] The difference between Comparative Example 3 and Example 1 is that the hydrofluoric acid solution treatment in step S6 was omitted, while the remaining steps are exactly the same as in Example 1.

[0068] Comparative Example 4

[0069] The difference between Comparative Example 4 and Example 1 is that the process of coating polydopamine once in step S2 was omitted, while the remaining steps are exactly the same as in Example 1.

[0070] XPS: The surface chemical composition of the sample prepared in Example 1 was measured by XPS, such as... Figure 2 As shown, in addition to the C 1s and O 1s peaks, the carrier prepared in Example 1 also showed an S 2p peak, indicating that the material had been successfully sulfonated. Furthermore, N 1s and P 2P peaks were also detected, indicating that polydopamine and adeno-associated virus had been successfully grafted and adhered to the sulfonated PEEK surface.

[0071] SEM: Electron microscope image of the sample prepared in Example 1 is shown below. Figure 3 As shown, through Figure 3 It can be seen that the coated carrier forms a large number of pores on the surface of the polydopamine layer. (Note: There are also many irregular protrusions on the surface, which are dopamine that has not polymerized).

[0072] AAV coating rate determination

[0073] The adsorption capacity of AAV by the vectors prepared in Example 1 and Comparative Examples 1-4 was determined by qPCR. The vectors prepared in Example 1 and Comparative Examples 1-4 were mixed with AAV solution and incubated, then washed three times with PBS buffer. The washed solutions were collected, and the amount of unadhered virus in the solution was determined by qPCR. The adsorption capacity of the vector for AAV was calculated, and the maximum coating rate of AAV on the vector surface was indirectly calculated.

[0074] The results are as follows Figure 4 As shown, the AAV-adhesive material prepared in Example 1 has a significantly higher AAV adsorption capacity than that of Comparative Examples 1-4, indicating that the method of the present invention can significantly improve the adhesion efficiency of AAV. The adsorption capacity of Comparative Example 2 is lower than that of Example 1. Comparative Example 2 lacks the hot-pressing process, which leads to a decrease in the adhesion strength between the nanoparticles and the material surface, making the nanoparticles easier to detach and reducing the number of pores formed. Comparative Examples 1 and 3 lack the etching of nano-silica templates and hydrofluoric acid, respectively, resulting in fewer pores and lower adsorption capacity than Comparative Example 2. In Comparative Example 4, the adsorption capacity also decreases after the formation of a primary polydopamine coating. However, the adsorption capacity is improved by forming a more three-dimensional adsorption structure through two layers of coating.

[0075] Figure 5 This document describes the release curves of AAV from the vectors containing adeno-associated virus (AAV) prepared in Examples 1 and 1-4 of this invention. Specifically, the AAV-containing vectors prepared in Examples 1 and 1-4 were placed in PBS buffer solution (3g of vector and 20ml of PBS buffer solution). The amount of AAV virus in the PBS buffer solution was measured over 1-9 days to determine the amount of AAV virus released by the vector. Figure 5 It can be seen that the release curves of the five are relatively consistent, and they all have a slow-release effect on AAV virus. However, the total release of Example 1 is higher than that of Comparative Examples 1-4.

[0076] Figure 6 These are images illustrating the osteogenic effects of the carriers prepared in Examples 1 and 1-4 of this invention. Alkaline phosphatase (ALP) is an early marker of osteoblast differentiation, appearing at the beginning of bone matrix synthesis and reaching its peak during mineralization. BCIP / NBT is a commonly used substrate for alkaline phosphatase. Under the catalysis of alkaline phosphatase, BCIP is hydrolyzed to produce a highly reactive product, which reacts with NBT to form insoluble dark blue to blue-purple NBT-formazan. 1×10⁻⁶ cells were inoculated onto the surface of the carriers prepared in Examples 1 and 1-4. 5MC3T3-E1 cells were cultured for 7 and 14 days and then stained. In this experiment, Beyotime's BCIP / NBT alkaline phosphatase staining kit was used. The AAV cells carried the BMP-2 gene. More and deeper blue-purple nodules were visible in Example 1, indicating that the bone-promoting effect was the best.

[0077] The AAV (adeno-associated virus) used in this invention was designed and synthesized by WZ Biosciences Inc., namely Shandong Weizhen Biotechnology Co., Ltd. The AAV genome was modified to introduce the bone morphogenetic protein 2 (BMP-2) gene to promote bone integration.

[0078] Good biocompatibility is fundamental to medical materials. A CCK-8 assay kit is used to assess the biocompatibility of materials. Components in CCK-8 can be reduced by dehydrogenases in mitochondria to a highly water-soluble yellow formazan product; therefore, a CCK-8 assay kit is used to test the biocompatibility of materials. Figure 7 As shown, the vectors prepared in Example 1 and Comparative Examples 1-4 all exhibited high cell viability (over 90%) after co-culturing for 1, 4, and 7 days. Furthermore, as shown in Figure 8, the cell proliferation experiment data indicated that the vectors did not inhibit cell proliferation after co-culturing with cells for 1, 4, and 7 days, and showed a time-dependent growth trend. These results demonstrate that the vectors prepared in Example 1 and Comparative Examples 1-4 have good biocompatibility.

[0079] The formulas for calculating cell viability and cell proliferation rate are shown below:

[0080] Cell viability (%) = (OD) 处理组 -OD 空白组 ) / (OD 正常对照组 -OD 空白组 ) × 100%;

[0081] Cell proliferation rate (%) = (OD) 终末处理组 - OD 空白组 ) / (OD 初始对照组（0h） - OD 空白组 ) × 100% - 100%;

[0082] OD 处理组 OD values ​​of cell pores co-cultured with one of the vectors from Example 1 and Comparative Examples 1-4;

[0083] OD 正常对照组 OD value of normal live cell wells without any carrier (including cells + culture medium + CCK-8, which is the reference for "100% survival");

[0084] OD 空白组OD values ​​of blank wells containing only culture medium + CCK-8 (eliminating background absorbance from the culture medium and reagents themselves);

[0085] OD 终末处理组 : The OD values ​​of cell pores after culturing one group of vectors from Example 1 and Comparative Examples 1-4 to the experimental endpoint (1d, 4d, 7d);

[0086] OD 初始对照组（0h） : OD value of cell pores at the start of the experiment (0h) (representing the "initial number of viable cells").

[0087] The vector with adeno-associated virus adsorbed by this invention can be applied in the following ways:

[0088] 1. Gene therapy field: Efficiently achieving targeted delivery of therapeutic genes

[0089] The core requirement of gene therapy is to precisely deliver therapeutic genes to target cells in the body and reduce off-target effects and vector-related risks. This material, through stable adsorption of AAV, can solve the problems of "uneven vector dispersion, easy degradation by in vivo enzymes, and weak targeting" in the preparation of gene-targeted drugs. AAV adsorbed on the surface of the material can reach the target tissue more stably. Under the "anchoring" effect of the material, the loss at non-target sites is reduced, thereby efficiently introducing therapeutic genes into target cells and ultimately achieving the treatment of genetic diseases, cancer, and other diseases.

[0090] 2. Vaccine Development: Improving the safety and immunogenicity of virus-like particle vaccines.

[0091] Virus-like particle (VLP) vaccines are a current hot topic in vaccine development. Their core advantage is that they do not contain viral nucleic acid and are non-infectious, but it is necessary to ensure the integrity of the particle structure to stimulate an effective immune response. In the preparation of VLP vaccines, this material can serve as a "carrier scaffold" for AAV: by adsorbing AAV and assisting it in assembling into a structurally stable VLP, it can prevent the aggregation and degradation of VLPs during preparation or storage, ensuring the structural integrity of the vaccine. On the other hand, the synergistic effect between the material and AAV can enhance the "recognition signal" of VLPs to the immune system, improve the body's ability to produce specific antibodies, and since the material itself is non-toxic, it can further reduce the risk of vaccine side effects, thus balancing safety and immune efficacy.

[0092] 3. Tissue engineering and regenerative medicine: Promoting cell function and aiding tissue repair.

[0093] The core objective of tissue engineering is to construct "bioactive scaffolds" that guide cells to adhere, proliferate, and differentiate into specific tissue cells (such as osteoblasts, chondrocytes, and skin cells) on the scaffold, ultimately achieving the regeneration and repair of damaged tissues. The AAV adsorbed in this material can serve as a "delivery carrier of cell regulatory factors" and be prepared into a bioactive scaffold. When the material is implanted in the body as a scaffold, AAV can be slowly released and delivered to surrounding cells genes that promote differentiation (such as osteogenic genes and chondrogenic genes). At the same time, the physical structure and chemical properties of the material itself can provide "growth anchors" for cells. Under these dual effects, cell adhesion efficiency is significantly improved, proliferation rate is accelerated, and cell differentiation is directionally induced, ultimately accelerating the tissue repair process.

[0094] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method of adeno-associated virus based on sulfonated polyether ether ketone adhesion, characterized by, The method comprises the following steps: S1, adding polyether ether ketone into concentrated sulfuric acid, ultrasonic reaction at 40-60℃ for 4-6min, then pouring the reaction product into ice water, filtering, washing, drying to obtain sulfonated polyether ether ketone; S2, soaking the sulfonated polyether ether ketone obtained in step S1 into dopamine hydrochloride solution, continuous reaction for 3-6h to obtain polydopamine once-coated polyether ether ketone; S3, spin coating nano-silica solution on the surface of the polydopamine once-coated polyether ether ketone obtained in step S2; S4, heat pressing the polydopamine once-coated polyether ether ketone treated in step S3; S5, soaking the polydopamine once-coated polyether ether ketone treated in step S4 into dopamine hydrochloride solution, continuous reaction for 8-12h to obtain polydopamine twice-coated polyether ether ketone; S6, adding the polydopamine twice-coated polyether ether ketone obtained in step S5 into hydrofluoric acid solution, continuous reaction for 3-6h, then filtering, and washing the filtered product with sufficient deionized water; S7, adding the filtered product washed with deionized water in step S6 into adeno-associated virus solution, incubating at 35-40℃ for 2-5h, then filtering, and washing the filtered product with PBS buffer solution for three times to obtain the virus-attached carrier.

2. The method of adeno-associated virus based on sulfonated polyether ether ketone adhesion according to claim 1, characterized in that, The concentration of the concentrated sulfuric acid in step S1 is 98wt%.

3. The method of adeno-associated virus based on sulfonated polyether ether ketone adhesion according to claim 1, characterized in that, The mass fraction of nano-silica in the nano-silica solution in step S3 is 1-5%.

4. The method of adeno-associated virus based on sulfonated polyether ether ketone adhesion according to claim 1, characterized in that, The temperature of heat pressing in step S4 is 150-200℃, the pressure is 1-3MPa, and the time of heat pressing is 0.5-2h.

5. The method of adeno-associated virus based on sulfonated polyether ether ketone adhesion according to claim 1, characterized in that, The concentration of dopamine hydrochloride solution in step S2 is 0.5-2mg / mL, and the concentration of dopamine hydrochloride solution in step S5 is 15-20mg / mL; the pH of dopamine hydrochloride solution in step S2 and step S5 is 8.5, and the pH of dopamine hydrochloride solution is adjusted by Tris-HCl buffer.

6. The method of adeno-associated virus based on sulfonated polyether ether ketone adhesion according to claim 1, characterized in that, The concentration of hydrofluoric acid solution in step S6 is 1-2wt%.

7. The method of adeno-associated virus based on sulfonated polyether ether ketone adhesion according to claim 1, characterized in that, The concentration of the adeno-associated virus in the adeno-associated virus solution in the step S7 is 1 x 10 13 vg / mL.

8. The use of the virus-attached carrier prepared by the method of adeno-associated virus adhesion based on sulfonated polyether ether ketone in the preparation of a bioactive scaffold.

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