An in situ gene transfection system based on hydrogenated TiO2 nanotube composite coating and its construction method and application

Abstract

The present invention provides an in situ gene transfection system based on a hydrogenated TiO2 nanotube composite coating, as well as its construction method and application. The in situ gene transfection system uses hydrogenated TiO2 nanotubes as a substrate and utilizes a layer-by-layer self-assembly technique to prepare a dopamine / graphene oxide / type IV collagen composite coating. This system can enhance the surface bioactivity of biomedical metal materials while also serving as an in situ gene transfection system for substrate-mediated local viral delivery, improving gene transfection efficiency and treatment safety, and enabling precise regulation of the treatment site. The in situ gene transfection system provides a new strategy for enhancing peri-implant epithelial sealing and preventing peri-implantitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical metal materials and nanomaterial biology, and relates to an in-situ drug loading and in-situ gene transfection system based on a super-hydrophilic hydrogenated TiO2 nanotube multifunctional composite coating, and a construction method and application thereof. Background Art

[0002] In recent years, an increasing number of scholars and dentists have recognized that peri-implant soft tissue integration is crucial for maintaining long-term implant stability and reducing the incidence of peri-implantitis. Peri-implant soft tissue integration includes epithelial attachment and underlying connective tissue attachment. The peri-implant epithelium, similar to the junctional epithelium, forms a cuff-like epithelial closure around the implant, serving as the first functional biological barrier against external stimuli. It effectively resists pathogen invasion and resists chemical and physical stimulation, playing a key role in preventing the occurrence of peri-implantitis.

[0003] However, compared with natural teeth, the width of the inner baseplate in the upper 2 / 3 area of the implant-gingival epithelium interface is less than half of the normal inner baseplate width and is discontinuous. At the same time, no hemidesmosome connection is observed, which may be an important reason for the very weak implant-gingival epithelium attachment.

[0004] At present, the discussion on how to enhance the epithelial attachment around implants mainly focuses on two aspects: one is to improve the surface bioactivity of implant materials (morphology, hydrophilicity, loading of bioactive molecules, etc.) to promote epithelial cell attachment; the other is to use gene therapy to directly increase the expression of key functional molecules of epithelial cells at the implant interface, in order to improve the structural defects of the inner basal plate and hemidesmosomes.

[0005] Guided by clinical applications, combining gene therapy with personalized design of material surfaces can achieve precise regulation of the microenvironment at the implant site. Encapsulating gene vectors in biomaterials (such as scaffolds, hydrogels, and microspheres, etc.) is a commonly used gene delivery method, but it will expose the gene throughout the entire treatment process; the second is to directly fix the gene vector on the surface of the material, that is, substrate-mediated in situ gene transfection. However, existing technical methods often cannot achieve efficient in situ gene transfection on the implant surface. Therefore, the present invention aims to develop a multifunctional drug loading system that can not only promote the biological activity of the implant surface, but also achieve efficient in situ gene transfection, so as to improve the safety of gene therapy and achieve precise regulation of the treatment site. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for constructing an in situ gene transfection system based on a hydrogenated TiO2 nanotube multifunctional composite coating, which can improve the surface biological activity of biomedical metal materials while achieving high-efficiency in situ gene transfection.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In its first aspect, the present invention provides an in situ gene transfection system based on a hydrogenated TiO2 nanotube composite coating. The in situ gene transfection system utilizes hydrogenated TiO2 nanotubes as a substrate material, and a self-assembled composite coating formed on the surface of the substrate material. The composite coating is a dopamine / graphene oxide / type IV collagen composite coating (DA / GO / COL-IV composite coating, abbreviated as DGC composite coating).

[0009] In the present invention, hydrogenated TiO2 nanotubes are used as a substrate, and a dopamine / graphene oxide / type IV collagen composite coating is prepared through layer-by-layer self-assembly technology to improve the surface biological activity of biomedical metal materials and promote epithelial attachment around implants. At the same time, as an in situ gene transfection system, substrate-mediated local viral delivery is carried out to improve gene transfection efficiency and treatment safety, and to achieve precise regulation of the treatment site. This design will provide a new strategy for improving the epithelial sealing effect around implants and preventing peri-implantitis. Among them, the surface of hydrogenated TiO2 nanotubes has been proven to contain a large number of hydroxyl groups, which can effectively bind to biological molecules. Dopamine and graphene oxide also have excellent loading capacity for bioactive substances. Type IV collagen, as an important component of the extracellular matrix and basement membrane, forms a relatively stable network structure with nestin, laminin and other extracellular matrix components, providing support and anchoring points for cells. At the same time, it can serve as an important ligand for cell membrane surface receptors to enhance the migration and adhesion of epithelial cells.

[0010] In the present invention, hydrogenated TiO2 nanotubes are the most ideal coating loading substrate. Compared with smooth pure titanium sheets and TiO2 nanotubes in air atmosphere, hydrogenated TiO2 nanotubes have a larger loading capacity of bioactive substances on the specimen surface due to their super hydrophilicity, and the coating on the specimen surface is more uniform and stable, with a better sustained-release effect.

[0011] As a preferred technical solution of the present invention, the number of layers of the composite coating is 5 to 30 layers, for example, it can be 5 layers, 8 layers, 10 layers, 12 layers, 15 layers, 18 layers, 20 layers, 22 layers, 25 layers, 28 layers or 30 layers, etc., preferably 10 to 15 layers.

[0012] When the substrate material is loaded with 5 layers of DGC coating, a film can be seen covering the surface of the TiO2 nanotubes, but it is not completely covered, and the nanotube structure under the coating can still be observed; when loaded with 10 layers of DGC coating, the nanotube structure on the surface of the specimen is completely covered, and obvious graphene oxide (GO) wrinkles can be seen; when loaded with 20 layers of DGC coating, the surface uniformity of the specimen is not as good as DGC10, and some pores are visible. Moreover, for hydrogenated TiO2 nanotubes, there is no significant difference in surface roughness and hydrophilicity when loaded with 10 layers of DGC and 20 layers of DGC. In addition, when the substrate material is the same, the total amount of type IV collagen loaded increases with the number of DGC layers, but when the DGC is 20 layers, the total amount of type IV collagen loaded is not statistically significant compared with 10 layers of DGC. Therefore, considering the material properties, preparation process and cost, the number of layers of the composite coating is preferably 10 layers.

[0013] As a preferred technical solution of the present invention, the composite coating is prepared by self-assembly.

[0014] As a preferred technical solution of the present invention, the self-assembly step includes: soaking the base material in a dopamine hydrochloride solution, a graphene oxide solution and a type IV collagen solution in sequence to prepare a base material coated with a dopamine / graphene oxide / type IV collagen composite coating.

[0015] In a second aspect, the present invention further provides a method for preparing the in situ gene transfection system as described in the first aspect, the preparation method comprising the following steps:

[0016] (1) placing the pretreated titanium sheet in an electrolyte for anodization treatment, washing and drying, and then performing high-temperature treatment; then, placing the high-temperature treated titanium sheet in a quartz tube under a hydrogen atmosphere for hydrogenation treatment to obtain hydrogenated TiO2 nanotubes;

[0017] (2) preparing dopamine hydrochloride solution, graphene oxide solution, and type IV collagen solution;

[0018] (3) Soaking the hydrogenated TiO2 nanotubes described in step (1) in a dopamine hydrochloride solution, a graphene oxide solution, and a type IV collagen solution in sequence to obtain an in situ gene transfection system coated with a dopamine / graphene oxide / type IV collagen composite coating.

[0019] As a preferred technical solution of the present invention, in the preparation method, step (3) is repeated 5 to 30 times to obtain an in situ gene transfection system coated with 5 to 30 layers of dopamine / graphene oxide / type IV collagen composite coating.

[0020] As a preferred technical solution of the present invention, the preparation method further includes the step of immersing the in situ gene transfection system coated with the dopamine / graphene oxide / type IV collagen composite coating in an anti-adenovirus antibody solution and / or a recombinant adenovirus solution.

[0021] As a preferred technical solution of the present invention, the solvent of the dopamine hydrochloride solution in step (2) is Tris / HCl buffer and deionized water, the concentration is 2-3 mg / ml (for example, 2 mg / ml, 2.2 mg / ml, 2.5 mg / ml, 2.8 mg / ml or 3 mg / ml, etc.), and the pH value is 8.5-10 (for example, 8.5, 9, 9.5 or 10).

[0022] Preferably, the solvent of the graphene oxide solution in step (2) is deionized water, the concentration is 0.005-2 mg / ml (for example, 0.005 mg / ml, 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml or 2 mg / ml, etc.), and the solution is ultrasonically treated for 120-360 min (for example, 120 min, 150 min, 180 min, 240 min, 270 min, 300 min or 360 min, etc.) before use.

[0023] Preferably, the solvent of the type IV collagen solution in step (2) is glacial acetic acid buffer and deionized water, the concentration is 20-200 μg / ml (for example, 20 μg / ml, 50 μg / ml, 80 μg / ml, 100 μg / ml, 120 μg / ml, 150 μg / ml or 200 μg / ml, etc.), and the pH value is 4-6 (for example, 4, 4.5, 5, 5.5 or 6).

[0024] As a preferred technical solution of the present invention, the soaking conditions in step (3) are:

[0025] The immersion is carried out under magnetic stirring, with a rotation speed of 50 to 500 rpm (for example, 50 rpm, 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, etc.), a reaction time of 5 to 30 min (for example, 5 min, 10 min, 20 min, 25 min or 30 min, etc.), a reaction temperature of 20 to 25° C. (for example, 20° C., 22° C., 23° C., 24° C., 25° C., etc.), a cleaning time of 30 to 90 s (for example, 30 s, 40 s, 50 s, 60 s, 70 s, 80 s or 90 s, etc.), and a cleaning number of times of 1 time, 2 times or 3 times.

[0026] As a preferred technical solution of the present invention, the preparation method comprises the following steps:

[0027] (1) placing the pretreated titanium sheet in an electrolyte for anodizing, washing, drying, and then performing high-temperature treatment;

[0028] The high temperature treatment process is as follows: placing the titanium sheet in a tube furnace, with an initial temperature of 20-25°C, a heating rate of 2-4°C / min, a heating temperature of 400-500°C, a holding temperature of 400-500°C, a holding time of 30-60min, and then cooling with the furnace;

[0029] Then, the titanium sheet after high temperature treatment is placed in a hydrogen atmosphere for hydrogenation treatment to obtain hydrogenated TiO2 nanotubes;

[0030] The hydrogenation process is as follows: the treated titanium sheet is placed in a quartz tube under a hydrogen atmosphere with a hydrogen pressure of 0.8 to 0.95×10 5 Pa, and then placed in a tube furnace for high-temperature hydrogenation, with an initial temperature of 20-25°C, a heating rate of 2-4°C / min, a heating temperature of 400-500°C, a holding temperature of 400-500°C, a holding time of 60-240min, and then cooled with the furnace;

[0031] (2) preparing dopamine hydrochloride solution, graphene oxide solution, and type IV collagen solution;

[0032] The solvent of the dopamine hydrochloride solution is Tris / HCl buffer and deionized water, with a concentration of 2-3 mg / ml and a pH value of 8.5-10; the solvent of the graphene oxide solution is deionized water, with a concentration of 0.005-2 mg / ml, and ultrasonic treatment is performed for 120-360 minutes before use; the solvent of the type IV collagen solution is glacial acetic acid buffer and deionized water, with a concentration of 20-200 μg / ml and a pH value of 4-6;

[0033] (3) soaking the hydrogenated TiO2 nanotubes described in step (1) in a dopamine hydrochloride solution, a graphene oxide solution, and a type IV collagen solution in sequence to obtain an in situ gene transfection system coated with a dopamine / graphene oxide / type IV collagen composite coating;

[0034] The soaking conditions are as follows: soaking under magnetic stirring, a rotation speed of 50 to 500 rpm, a reaction time of 5 to 30 min, a reaction temperature of 20 to 25°C, a cleaning time of 30 to 90 s, and a cleaning frequency of 1 to 3 times;

[0035] As a preferred solution, step (3) in the preparation method can be repeated continuously to form a multi-layer composite coating. The number of times step (3) is repeated can be 5 to 30 times, preferably 10 to 15 layers.

[0036] (4) immersing the in situ gene transfection system coated with the dopamine / graphene oxide / type IV collagen composite coating in an anti-adenovirus antibody solution and / or a recombinant adenovirus solution for reaction;

[0037] The anti-adenovirus antibody solution solvent is PBS, the concentration is 1 / 10000 to 1 / 500, the volume is 250 to 1000 μl, the reaction time is 12 to 20 hours, the reaction temperature is 2 to 8°C, the washing time is 10 to 300 seconds, and the number of washing times is 1 to 3 times;

[0038] The recombinant adenovirus solution is prepared using PBS at a concentration of 0.1 to 4 × 10 8 PFU / ml, volume is 250-1000 μl, reaction time is 1-8 hours, reaction temperature is 20-37°C, washing time is 10-300 seconds, and washing times are 1-3 times.

[0039] Specifically, the preparation method of the present invention can adopt the following steps:

[0040] (1) placing a pretreated commercially smooth pure titanium sheet in an electrolyte for anodizing;

[0041] (2) The sample obtained in step (1) was ultrasonically washed in anhydrous ethanol and deionized water for 2 to 5 minutes, dried, and set aside;

[0042] (3) placing the sample obtained in step (2) in a tube furnace for high temperature treatment and setting aside;

[0043] (4) sealing the sample obtained in step (3) in a quartz tube under a hydrogen atmosphere and performing a high-temperature hydrogenation treatment for standby use;

[0044] (5) preparing dopamine hydrochloride solution, graphene oxide solution, and type IV collagen solution for later use;

[0045] (6) Soaking the sample obtained in step (4) in the dopamine hydrochloride solution prepared in step (4), and then washing with deionized water for later use;

[0046] (7) Soaking the sample obtained in step (6) in the graphene oxide solution prepared in step (4), and then washing with deionized water for later use;

[0047] (8) Soaking the sample obtained in step (7) in the type IV collagen solution prepared in step (4), then washing with deionized water for later use;

[0048] (9) Repeat steps (6)-(8);

[0049] (10) The sample obtained in step (9) was immersed in an anti-adenovirus antibody solution for antibody functionalization, and then washed with PBS for later use.

[0050] (11) Soak the sample obtained in step (10) in a recombinant adenovirus solution, then wash it with PBS and set aside.

[0051] In a third aspect, the present invention provides a use of the in situ gene transfection system as described in the first aspect in the preparation of biomedical materials.

[0052] The preparation of the biomedical material has good application prospects in the sealing of implant soft tissue and the prevention and treatment of peri-implantitis.

[0053] The numerical range described in the present invention includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] (1) The preparation method of the present invention is simple to operate, has low requirements on equipment conditions, good reproducibility, strong controllability, and is suitable for industrial production;

[0056] (2) The present invention modifies the surface of biomedical metal materials and constructs a multifunctional composite coating. The coating can adjust the number of composite layers according to needs, thereby improving the surface biological activity, drug loading and sustained release effects of the biomedical metal materials; at the same time, as an in situ gene transfection system, it can perform substrate-mediated local virus delivery, improve gene transfection efficiency and treatment safety, and achieve precise regulation of the treatment site.

[0057] (3) The hydrogenated TiO2 nanotube multifunctional composite coating obtained in the present invention has excellent biological activity and has important research value, clinical significance and good clinical application prospects in the sealing of implant soft tissue and the prevention and treatment of peri-implantitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 2 are scanning electron microscope (SEM) images of the surfaces of the test pieces of each group of the present invention.

[0059] Figure 2 These are atomic force microscope (AFM) 2D images of each group of specimens of the present invention.

[0060] Figure 3 Figure 1 is a statistical diagram of the surface roughness (Ra) and water contact angle of each group of test pieces of the present invention; Figure I is a statistical diagram of the surface roughness, and Figure II is a statistical diagram of the water contact angle (in the figure, a indicates that the difference is statistically significant compared with DGC0; b indicates that the difference is statistically significant compared with DGC5; c indicates that the difference is statistically significant compared with DGC 10The differences were statistically significant; p < 0.05 was considered statistically significant).

[0061] Figure 4 The X-ray photoelectron spectroscopy (XPS) images of each group of specimens of the present invention are shown in Figure I; Figure II, Figure III, Figure IV and Figure V respectively represent the XPS spectra of H, H-DGC5, H-DGC 10 and H-DGC 20 XPS high-resolution C1s peak fitting diagram of the specimen surface.

[0062] Figure 5 These are comparison diagrams of relevant data of each group of specimens of the present invention; Figure I is a statistical diagram of type IV collagen loading data (a in the figure indicates that the difference is statistically significant compared with DGC5; p<0.05 is considered to be statistically significant); Figure II is a picrosirius red staining image; Figure III is a nanoscratch image.

[0063] Figure 6 This is the specimen A-DGC of the present invention 10 、H-DGC 10 Cross-section and coating thickness diagram; Figure I is a cross-section scanning electron microscope (SEM) image; Figure II is a statistical diagram of coating thickness data (a in the figure indicates the difference with A-DGC 10 The differences were statistically significant; p < 0.05 was considered statistically significant).

[0064] Figure 7 This is the specimen A-DGAC of the present invention 10 、H-DGAC 10 Figure I shows the sustained release diagram and SEM image of BSA-loaded suspension; Figure II shows the SEM images of the suspension after 7 and 14 days of sustained release.

[0065] Figure 8 Figure 1 is a statistical diagram of the transfection rate data after in situ virus transfection of the specimen of the present invention and a fluorescence microscope image, wherein Figure 1 is a statistical diagram of the transfection rate data (a in the figure indicates that the difference is statistically significant compared with H; b indicates that the difference is statistically significant compared with H-DGC 10 The difference was statistically significant; p < 0.05 was considered statistically significant). Figure II shows H-DGC 10 Representative fluorescence microscopy images of cells transfected with different virus titers.

[0066] Figure 9 The data of the proliferation of human oral epithelial cells (MTT method) on the surface of each group of specimens of the present invention are statistically analyzed (a in the figure indicates that the difference is statistically significant compared with T; b indicates that the difference is statistically significant compared with H; c indicates that the difference is statistically significant compared with H-DGC 10The differences were statistically significant; p < 0.05 was considered statistically significant).

[0067] Figure 10 Figure 1 is an image of the functional expression test of human oral epithelial cells on the surface of the test piece of the present invention, wherein Figure I is an immunofluorescence microscope image of human oral epithelial cells, Figure II is a statistical graph of RT-PCR data, Figure III is a Western blot image, and Figure IV is a statistical graph of Western blot data (in Figures II and IV, a indicates a statistically significant difference compared with T; b indicates a statistically significant difference compared with H; c indicates a statistically significant difference compared with H-DGC 10 The difference was statistically significant; d indicates that the difference was statistically significant compared with H-DGC. 10 -AdmCherry comparison showed statistically significant differences; p < 0.05 was considered statistically significant).

[0068] Figure 11 Schematic diagram of the overall process of the present invention; wherein, DA: dopamine hydrochloride; GO: graphene oxide; COL-Ⅳ: type IV collagen; DGC n : n-layer DA / GO / COL-Ⅳ composite nanocoating; AdLAMA3: recombinant adenovirus overexpressing laminin332α gene variant. DETAILED DESCRIPTION

[0069] The technical solution of the present invention is further illustrated below with reference to the accompanying drawings and through specific implementation methods. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0070] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0071] Example 1 Preparation of hydrogenated TiO2 nanotubes

[0072] This embodiment provides a method for preparing hydrogenated TiO2 nanotubes. The specific preparation steps are as follows:

[0073] (1) Place a commercially smooth pure titanium sheet (T) in acetone, anhydrous ethanol, and deionized water, ultrasonically wash for 5 minutes, dry, and set aside;

[0074] (2) The test piece obtained in step (1) was used as the anode and the platinum sheet was used as the cathode, and the samples were placed in an ethylene glycol electrolyte (ammonium fluoride mass fraction of 0.5 wt%, deionized water mass fraction of 10 wt%, hydrofluoric acid volume fraction of 0.3%), and anodized under magnetic stirring at a voltage of 50 V, a reaction time of 15 min, and a reaction temperature of 25°C;

[0075] (3) The specimen obtained in step (2) was ultrasonically cleaned with anhydrous ethanol and deionized water for 2 minutes, and then dried;

[0076] (4) The specimen obtained in step (3) was placed in a tube furnace for high-temperature treatment, with an initial temperature of 25°C, a heating rate of 4°C / min, a heating temperature of 500°C, a holding temperature of 500°C, and a holding time of 60 min, and then cooled with the furnace. The obtained specimen was TiO2 nanotubes in air atmosphere (marked as A);

[0077] (5) The specimen obtained in step (4) was sealed in a quartz tube under a hydrogen atmosphere with a hydrogen pressure of 0.95×10 5 Pa, and then placed in a tube furnace for high-temperature hydrogenation, with an initial temperature of 25°C, a heating rate of 4°C / min, a heating temperature of 500°C, a holding temperature of 500°C, a holding time of 240min, and then cooled with the furnace. The resulting specimen is hydrogenated TiO2 nanotubes (marked as H).

[0078] Among them, smooth pure titanium sheet is marked as T; TiO2 nanotubes in air atmosphere are marked as A; and hydrogenated TiO2 nanotubes are marked as H.

[0079] Example 2 Construction of 5-layer DGC multifunctional composite coating

[0080] This embodiment provides a method for constructing a multifunctional composite coating. Using a titanium-based material as a substrate, dopamine hydrochloride (labeled as D), graphene oxide (labeled as G), and type IV collagen (labeled as C) are sequentially loaded using layer-by-layer self-assembly technology to construct T-DGC5, A-DGC5, and H-DGC5 multifunctional composite coatings, each with five layers.

[0081] The specific preparation steps are as follows:

[0082] (1) Prepare dopamine hydrochloride solution, graphene oxide solution and type IV collagen solution:

[0083] The solvent for the dopamine hydrochloride solution was Tris / HCl buffer (10 mM) and deionized water, with a concentration of 2 mg / ml and a pH of 8.5. The solvent for the graphene oxide solution was deionized water, with a concentration of 500 μg / ml, and ultrasonic dispersion was performed for 120 minutes before use. The solvent for the type IV collagen solution was glacial acetic acid buffer and deionized water, with a concentration of 50 μg / ml and a pH of 4.5.

[0084] (2) Specimens T, A, and H obtained in Example 1 were immersed in the dopamine hydrochloride solution prepared in step (1) under magnetic stirring, with a solution volume of 20 ml, a reaction time of 5 min, and a reaction temperature of 25° C., and then washed with deionized water three times, each time for 1 min;

[0085] (3) The specimen in step (2) was immersed in the graphene oxide solution prepared in step (1) under magnetic stirring, with a solution volume of 20 ml, a reaction time of 5 min, and a reaction temperature of 25°C, and then washed with deionized water three times, each time for 1 min;

[0086] (4) The specimen in step (3) was immersed in the type IV collagen solution prepared in step (1) under magnetic stirring, with a solution volume of 20 ml, a reaction time of 5 min, and a reaction temperature of 25°C, and then washed with deionized water three times, each time for 1 min;

[0087] (5) Repeat steps (2) to (4) 5 times. The obtained specimens are vacuum dried and set aside. They are marked as T-DGC5, A-DGC5 and H-DGC5 respectively.

[0088] Example 3 Construction of 10-layer DGC multifunctional composite coating

[0089] This embodiment provides a method for constructing a multifunctional composite coating. A titanium-based material is used as a substrate, and dopamine hydrochloride (marked as D), graphene oxide (marked as G) and type IV collagen (marked as C) are sequentially loaded using layer-by-layer self-assembly technology to construct a T-DGC. 10 、A-DGC 10 and H-DGC 10 Multifunctional composite coating with 10 layers.

[0090] The specific preparation steps are as follows:

[0091] (1) Same as step (1) in Example 2;

[0092] (2) Repeat steps (2) to (4) in Example 2 10 times, and the resulting specimens are vacuum dried and set aside, and are marked as T-DGC. 10 、A-DGC 10 and H-DGC 10 .

[0093] Example 4 Construction of 20-layer DGC multifunctional composite coating

[0094] This embodiment provides a method for constructing a multifunctional composite coating. A titanium-based material is used as a substrate, and dopamine hydrochloride (marked as D), graphene oxide (marked as G) and type IV collagen (marked as C) are sequentially loaded using layer-by-layer self-assembly technology to construct a T-DGC. 20 、A-DGC 20 and H-DGC 20 Multifunctional composite coating with 20 layers.

[0095] The specific preparation steps are as follows:

[0096] (1) Same as step (1) in Example 2;

[0097] (2) Repeat steps (2) to (4) in Example 2 20 times. The obtained specimens are vacuum dried and set aside. They are marked as T-DGC. 20 、A-DGC 20 and H-DGC 20 .

[0098] Performance Test 1 Basic Performance Test

[0099] (1) Scanning electron microscope (SEM) observation

[0100] The intermediate products and final products obtained in Examples 1-4 were subjected to SEM scanning. The results are as follows: Figure 1 shown.

[0101] As can be seen in the figure, the surface of pure titanium sheet T is relatively smooth, while TiO2 nanotubes with uniform morphology and a diameter of approximately 100 nm are formed on the surfaces of groups A and H. After coating, scattered particles are visible on the surface of the pure titanium, but no film is formed. The number of particles increases with the number of coating layers.

[0102] When the surfaces of groups A and H were loaded with 5 layers of DGC coating, a film was visible covering the surface of the TiO2 nanotubes, but it was not completely covered, and the nanotube structure under the coating could still be observed; when loaded with 10 layers of DGC coating, the nanotube structure on the surface of the specimen was completely covered, and obvious graphene oxide (GO) wrinkles were visible (indicated by the red arrows); when loaded with 20 layers of DGC coating, the surface uniformity of the specimen was not as good as that of DGC. 10 When loaded with the same number of DGC coating layers, group H loaded more than group A and the coating was more uniform.

[0103] (2) Atomic force microscopy (AFM) observation and roughness and hydrophilic angle testing

[0104] The 2D images obtained by AFM are as follows Figure 2As shown in Figure 3, the results further confirmed the SEM observation results of the surface morphology of each group of specimens.

[0105] The surface roughness (Ra) of each group of specimens measured by AFM is as follows: Figure 3 As shown in Figure I, the water contact angles on the surfaces of the specimens in each group are as follows: Figure 3 As shown in Figure II. As can be seen from the figure, coating load can increase the surface roughness of the T group specimens and reduce the surface roughness of the A and H group specimens. 10 Surface roughness and DGC 20 There was no statistically significant difference between the two groups. At the same time, after the pure titanium sheet was loaded with DGC, the original hydrophobic surface (water contact angle>60°) was transformed into a hydrophilic surface (water contact angle<60°), and DGC5 and DGC 10 With DGC 20 There was no significant difference between the two groups. The water contact angle of Group A specimens increased after loading with DGC, but gradually decreased with increasing number of layers, maintaining a hydrophilic surface. However, the water contact angle of hydrogenated TiO2 nanotubes loaded with DGC increased, transforming from a superhydrophilic surface (4.2°) to a hydrophilic surface.

[0106] (3) Changes in chemical elements of the XPS test specimen before and after loading the DGC coating

[0107] Taking hydrogenated TiO2 nanotubes as an example, XPS was used to detect the changes in chemical elements before and after the DGC coating was applied to the test specimen. The results are as follows: Figure 4 shown.

[0108] exist Figure 4 Figure I shows the XPS elemental spectrum of each group of specimens. In addition to the C1s and O1s peaks, the Ti2p peak can also be detected in hydrogenated TiO2 nanotubes. After coating loading, the Ti2p peak disappears and the N1s peak appears, indicating that the TiO2 surface is covered by the coating and the DGC coating is successfully loaded. Further fitting of the XPS high-resolution C1s peak of each group of specimens yields Figure 4 From Figure II to Figure V, it can be seen that after coating loading, the CO peak content increased significantly and a C=O peak appeared, which was mainly due to the successful loading of dopamine, graphene oxide and type IV collagen.

[0109] (4) Detection of the total amount of type IV collagen loaded in different layers of DGC coating

[0110] Figure 5 Figure I in the middle shows the total amount of type IV collagen loaded on different layers of DGC coating in each group of specimens.

[0111] The results showed that when the number of DGC layers was the same, the amount of type IV collagen loaded by hydrogenated TiO2 nanotubes (H) was significantly higher than that of the other two groups. When the number of DGC layers was less than 10, the total amount of type IV collagen loaded by the three groups T, A, and H increased with the increase in the number of DGC layers. However, when the number of DGC layers was 20, the total amount of type IV collagen loaded by the T and H groups did not increase. Although the total amount of type IV collagen loaded by group A increased compared to the 10-layer group, the difference was not statistically significant.

[0112] Figure 5 Figure II in the middle is T-DGC 10 、A-DGC 10 、H-DGC 10 The surface of the specimen was stained with picrosirius red. It can be seen from the figure that the surface of the hydrogenated TiO2 nanotube group specimens is loaded with more type IV collagen.

[0113] (5) Detection of the micro-tribological characteristics of each group of specimens when loaded with 10 layers of DGC coating

[0114] Figure 5 Figure III is the nano scratch image of each group of specimens. It can be seen from the figure that T-DGC 10 、A-DGC 10 、H-DGC 10 The lateral sudden force of the surface coating of the three groups of specimens is 16mN, 30mN and 45mN respectively. 10 The surface coating strength is better.

[0115] (6) Detect the coating thickness of each group of specimens when loaded with 10 layers of DGC coating

[0116] Scanning electron microscopy was used to observe cross sections. Figure 6 As shown in Figure I, H-DGC 10 The coating thickness is thicker than A-DGC 10 Thick, Figure II shows that its thickness is about 155nm, due to T-DGC 10 The coating is too thin and no clear cross-sectional image of the coating can be observed.

[0117] The above results show that hydrogenated TiO2 nanotubes (H) are a more ideal coating loading substrate compared with pure titanium (T) and TiO2 nanotubes in air atmosphere (A), and when the DGC coating has 10 layers, the type IV collagen loading is the largest and the coating is more stable.

[0118] Example 5 Using bovine serum albumin as a drug model

[0119] This example is used to construct A-DGC 10 and H-DGC 10The in situ drug delivery system uses a titanium-based material as the substrate and bovine serum albumin (BSA, marked as A) as the drug model. It uses layer-by-layer self-assembly technology to sequentially form dopamine hydrochloride (marked as D), graphene oxide (marked as G), BSA and type IV collagen (marked as C), with a total of 10 layers.

[0120] The specific preparation steps are as follows:

[0121] (1) Prepare dopamine hydrochloride solution, graphene oxide solution, bovine serum albumin solution and type IV collagen solution: wherein the solvent of dopamine hydrochloride solution is Tris / HCl buffer (10 mM) and deionized water, the concentration is 2 mg / ml, and the pH is 8.5; the solvent of graphene oxide solution is deionized water, the concentration is 200 μg / ml, and ultrasonic dispersion is performed for 120 minutes before use; the solvent of bovine serum albumin solution is deionized water, the concentration is 1 mg / ml; the solvent of type IV collagen solution is glacial acetic acid buffer and deionized water, the concentration is 50 μg / ml, and the pH is 4.5;

[0122] (2) Specimens A and H obtained in Example 1 were immersed in the dopamine hydrochloride solution prepared in step (1) under magnetic stirring, with a solution volume of 20 ml, a reaction time of 5 min, and a reaction temperature of 25° C., and then washed with deionized water three times, each time for 1 min;

[0123] (3) The specimen in step (2) was immersed in the graphene oxide solution prepared in step (1) under magnetic stirring, with a solution volume of 20 ml, a reaction time of 5 min, and a reaction temperature of 25°C, and then washed with deionized water three times, each time for 1 min;

[0124] (4) The test piece in step (3) was immersed in the bovine serum albumin solution prepared in step (1) under magnetic stirring, the solution volume was 20 ml, the reaction time was 5 min, the reaction temperature was 25°C, and then washed with deionized water three times, each time for 1 min;

[0125] (5) The specimen in step (4) was immersed in the type IV collagen solution prepared in step (1) under magnetic stirring, with a solution volume of 20 ml, a reaction time of 5 min, and a reaction temperature of 25°C, and then washed with deionized water three times, each time for 1 min;

[0126] (6) Repeat steps (2)-(5) 10 times, and the resulting specimens are vacuum dried and set aside, and are marked as A-DGAC 10 and H-DGAC 10 .

[0127] Performance Test 2 verifies the ability of the multifunctional composite coating to load and release bioactive substances

[0128] In Example 5, based on the experimental results of Examples 1-4, A-DGC 10 、H-DGC 10 As the loading template, bovine serum albumin was used as the bioactive substance model to obtain A-DGAC 10 and H-DGAC 10 ,This performance test is used to verify the ability of DGC coating to load and ,sustained release of bioactive substances.

[0129] like Figure 7 As shown, Figure I is A-DGAC 10 、H-DGAC 10 Protein sustained release results on the specimen surface, A-DGAC 10 、H-DGAC 10 There were two phases of rapid release at 8h and 168h. At 336h, A-DGAC 10 The amount of sustained-release protein has reached 95%, but H-DGAC 10 Only 64% was sustained release. 10 、H-DGAC 10 The overall effect is sustained, but H-DGAC 10 The sustained release effect is better and the sustained release duration is longer. Figure II shows the scanning electron microscope images of the two groups of specimens at 7 and 14 days of sustained release. As the sustained release progresses, A-DGAC 10 、H-DGAC 10 The surface film coating slowly dissolves, and the base nanotubes are exposed. 10 The surface area of the coating is larger than that of A-DGAC 10 larger, and on the 14th day, there was still more film-like coating covering the nanotubes.

[0130] The above results show that compared with conventional TiO2 nanotubes, the DGC coating based on hydrogenated TiO2 nanotubes has better load-release effect.

[0131] Example 6: Using the recombinant adenovirus Ad-mCherry as an in situ gene transfection model

[0132] In this example, an in situ gene transfection system based on a multifunctional composite coating of hydrogenated TiO2 nanotubes is constructed. A titanium-based material is used as the substrate, and the recombinant adenovirus Ad-mCherry is used as the in situ gene transfection model. Layer-by-layer self-assembly technology is used to sequentially load dopamine hydrochloride (labeled as D), graphene oxide (labeled as G), and type IV collagen (labeled as C). The number of layers is 10. The composite coating is then functionalized with antibodies and loaded with the recombinant adenovirus, thereby achieving high-efficiency in situ gene transfection.

[0133] The specific preparation steps are as follows:

[0134] (1) Prepare anti-adenovirus antibody (Ab) solution using PBS as diluent at a concentration of 1 / 1000;

[0135] (2) The H-DGC specimen obtained in Example 3 was 10 Soak in the Ab solution prepared in step (1) with a solution volume of 300 μl, a reaction time of 16 h, and a reaction temperature of 4°C;

[0136] (3) The specimen obtained in step (2) was washed with PBS three times, each time for 1 min, and the obtained specimen was marked as H-DGC 10 -Ab;

[0137] (4) Prepare recombinant adenovirus solution (Ad-mCherry) with PBS as diluent and virus titer of 0.1×10 8 , 0.5×10 8 , 1.0×10 8 , 2.0×10 8 PFU / ml;

[0138] (5) The H-DGC specimen obtained in step (1-3) 10 -Ab and the specimen H-DGC obtained in Example 3 10 Soak in the Ad-mCherry gradient titer solution in step (4) with a solution volume of 300 μl, a reaction time of 4 h, and a reaction temperature of 37°C;

[0139] (6) The specimen obtained in step (5) was washed with PBS three times, each time for 1 min.

[0140] Performance Test 3 Verifies H-DGC 10 In situ gene transfection efficiency

[0141] In Example 6, H-DGC with the best loading and sustained release effect was used. 10As a loading template for in situ gene transfection, the gene transfection adenovirus Ad-mCherry was used as an in situ gene transfection model. This performance test was used to verify the H-DGC 10 in situ gene transfection efficiency.

[0142] Combine Figure 8 As can be seen from Figure II, the transfection efficiency of Ad-mCherry on the surface of hydrogenated TiO2 nanotubes (H) is low. 8 The transfection efficiency was only 32% when the PFU / ml was used. However, after hydrogenated TiO2 nanotubes were loaded with DGC coating (H-DGC 10 ), its viral transfection efficiency was significantly improved (p<0.05). 10 After antibody functionalization, its viral transfection efficiency was further improved. 8 PFU / ml, the transfection efficiency reached 89%, and the virus titer was 2.0×10 8 There is no significant statistical difference in transfection efficiency at PFU / ml. Figure II shows H-DGC 10 The virus titer was 0.1×10 8 (B1), 0.5×10 8 (B2), 1.0×10 8 (B3), 2.0×10 8 Representative fluorescence microscopy images at 50 PFU / ml (B4).

[0143] From the above results, we can see that H-DGC 10 As a loading template for in situ gene transfection, it can significantly improve transfection efficiency, reduce the amount of adenovirus used, and thus improve biosafety. Antibody functionalization is effective when the virus titer is low (0.5-1.0×10 8 PFU / ml) can further improve the efficiency of in situ gene transfection.

[0144] Example 7: Using recombinant adenovirus Ad-LAMA3 as an in situ gene transfection model

[0145] This embodiment is used to construct an in situ gene transfection overexpression system of laminin332α variant (LAMA3) based on a hydrogenated TiO2 nanotube multifunctional composite coating. It uses a titanium-based material as a substrate and a recombinant adenovirus Ad-LAMA3 as an in situ gene transfection model. It uses layer-by-layer self-assembly technology to sequentially load dopamine hydrochloride (marked as D), graphene oxide (marked as G) and type IV collagen (marked as C). The number of layers is 10. The composite coating is then functionalized with antibodies and loaded with recombinant adenovirus, thereby achieving high-efficiency in situ gene transfection.

[0146] The specific preparation steps are as follows:

[0147] (1) Prepare anti-adenovirus antibody (Ab) solution using PBS as diluent at a concentration of 1 / 1000;

[0148] (2) The H-DGC specimen obtained in Example 3 was 10 Soak in the Ab solution prepared in step (1) with a solution volume of 300 μl, a reaction time of 16 h, and a reaction temperature of 4°C;

[0149] (3) Wash the specimen obtained in step (2) with PBS three times, each time for 1 min;

[0150] (4) Prepare recombinant adenovirus (Ad-mCherry and Ad-LAMA3) solution, diluent is PBS, and the virus titer is 1.0×10 8 PFU / ml;

[0151] (5) The test pieces obtained in steps (1-3) were immersed in the solution in step (4) respectively, with a solution volume of 300 μl, a reaction time of 4 h, and a reaction temperature of 37°C;

[0152] (6) The specimens obtained in step (5) were washed with PBS three times, each time for 1 min, and labeled as H-DGC. 10 -AdmCherry and H-DGC 10 -AdLAMA3.

[0153] Performance Test 4 Human Oral Epithelial Cell Proliferation Assay

[0154] The performance test was carried out on the materials of the obtained specimens (T, H, H-DGC 10 、H-DGC 10 -AdmCherry and H-DGC 10 -AdLAMA3) surface was used to detect the proliferation of human oral epithelial cells (HOEC).

[0155] The specific experimental steps are as follows:

[0156] (1) HOEC (5×10 4 pcs / well) were inoculated on the surface of each group of specimens and cultured for 1, 3 and 5 days;

[0157] (2) Aspirate and discard the old culture medium, add 340 μl of complete culture medium containing 10% MTT solution to each well, and incubate at 37°C for 4 h;

[0158] (3) Aspirate and discard the old solution, wash three times with PBS, add 340 μl of DMSO to each well, fully dissolve the purple precipitate, transfer the solution to a 96-well plate (100 μl / well), and measure the absorbance at 570 nm.

[0159] The experimental results are as follows Figure 9 As shown, on day 1, the H group and H-DGC 10 The number of surface cells in group A was higher than that in the other three groups, but on the 3rd and 5th days, H-DGC 10 、H-DGC 10 There was no statistical difference in cell proliferation between the surface of the materials in groups T and H, indicating that both the DGC coating functionalization and the Ad-LAMA3 in situ gene transfection system had good cell compatibility.

[0160] Performance test 5 Human oral epithelial cell function expression detection

[0161] In the materials of the obtained specimens (T, H, H-DGC 10 、H-DGC 10 -AdmCherry and H-DGC 10 -AdLAMA3) surface was used to detect the functional expression of human oral epithelial cells (HOEC).

[0162] The specific experimental steps are as follows:

[0163] (1) HOEC (5×10 4 Cells were inoculated on the surface of each group of specimens and cultured for 2 days. The cells were immunofluorescently stained with laminin 332α (LAMA3) and integrin β4 (ITGB4) as primary antibodies. After staining, fluorescence microscopy was performed.

[0164] (2) Each group of specimens was placed in a 6-well plate and inoculated with HOEC (1×10 6 After culturing for 2 days, cellular RNA was extracted with TRIzol and reverse transcribed for RT-PCR detection.

[0165] (3) Each group of specimens was placed in a 6-well plate and inoculated with HOEC (1×10 6After culturing for 2 days, the cells were collected with a cell scraper and subjected to Western blot analysis.

[0166] The results are as follows Figure 10 As shown, Figure I is the immunofluorescence image of HOEC expressing LAMA3 and ITGB4 on the surface of each group of specimens. Compared with T and H, DGC coating can significantly promote the expression of LAMA3 and ITGB4 in HOEC, while H-DGC 10 After further in situ gene transfection, LAMA3 and ITGB4 fluorescence staining became more obvious, and a mature dot-like hemidesmosome structure was presented. Figure II shows the expression results of HOEC and adhesion-related and hemidesmosome-related genes on the surface of each group of specimens. Compared with T and H, although the DGC coating did not significantly promote LAMA3 gene expression, it significantly promoted the expression of integrins α3, β1, α6, β4 and plectin compared with T (p < 0.05). 10 After further in situ gene transfection with Ad-LAMA3, the expression of LAMA3 and ITGB4 genes increased significantly (p<0.05), and the expression of other adhesion and hemidesmosome-related genes also increased significantly compared with the T group. Figure III is a Western blot image of LAMA3 protein expression on the surface of HOECs in each group of specimens, and Figure IV is a Western blot data graph; at the protein level, DGC coating can significantly promote the expression of LAMA3 in HOECs, and H-DGC 10 -The LAMA3 protein expressed by cells on the surface of the AdLAMA3 specimen was the highest (p<0.05).

[0167] The above results show that H-DGC 10 The coating can promote HOEC adhesion and the expression of hemidesmosome-related genes and proteins to a certain extent. After in situ gene transfection with Ad-LAMA3, it exhibits the most superior HOEC adhesion activity and hemidesmosome formation activity.

[0168] Figure 11 This is a schematic diagram of the overall process of the construction method of the in situ gene transfection system based on the super-hydrophilic hydrogenated TiO2 nanotube multifunctional composite coating described in the present invention. DA: dopamine hydrochloride; GO: graphene oxide; COL-Ⅳ: type IV collagen; DGC n : n-layer DA / GO / COL-Ⅳ composite nanocoating; AdLAMA3: recombinant adenovirus overexpressing laminin332α gene variant.

[0169] In summary, the dopamine / graphene oxide / type IV collagen composite coating prepared by layer-by-layer self-assembly technology using hydrogenated TiO2 nanotubes as the substrate can improve the surface bioactivity of biomedical metal materials, promote epithelial attachment around implants, and achieve high-efficiency in situ gene transfection on the material surface, providing a new strategy to further improve the epithelial sealing effect around implants and prevent peri-implantitis.

[0170] The above are embodiments and experimental examples of the present invention. For ordinary technicians in this field, based on the teachings of the present invention, all equivalent changes, modifications, substitutions and variations made within the scope of the patent application of the present invention without departing from the principles and spirit of the present invention should be covered by the scope of the present invention.

Claims

1. A method for preparing an in situ gene transfection system based on a hydrogenated TiO2 nanotube composite coating, characterized in that: The in situ gene transfection system uses hydrogenated TiO2 nanotubes as a base material, and a self-assembled composite coating on the surface of the base material, wherein the composite coating is a dopamine / graphene oxide / type IV collagen composite coating; The preparation method comprises the following steps: (1) placing the pretreated titanium sheet in an electrolyte for anodization treatment, washing and drying, and then performing high-temperature treatment; then, placing the high-temperature treated titanium sheet in a hydrogen atmosphere for hydrogenation treatment to obtain hydrogenated TiO2 nanotubes; The high temperature treatment process is as follows: placing the titanium sheet in a tube furnace, with an initial temperature of 20-25°C, a heating rate of 2-4°C / min, a heating temperature of 400-500°C, a holding temperature of 400-500°C, a holding time of 30-60min, and then cooling with the furnace; (2) preparing dopamine hydrochloride solution, graphene oxide solution, and type IV collagen solution; (3) Soaking the hydrogenated TiO2 nanotubes described in step (1) in a dopamine hydrochloride solution, a graphene oxide solution, and a type IV collagen solution in sequence, repeating 10 times to obtain an in situ gene transfection system coated with 10 layers of dopamine / graphene oxide / type IV collagen composite coating; (4) Immersing the obtained in situ gene transfection system in an anti-adenovirus antibody solution and / or a recombinant adenovirus solution to react, thereby obtaining an antibody-functionalized in situ gene transfection system.

2. The preparation method according to claim 1, characterized in that The solvent of the dopamine hydrochloride solution in step (2) is Tris / HCl buffer and deionized water, with a concentration of 2-3 mg / ml and a pH of 8.5-10; The solvent of the graphene oxide solution in step (2) is deionized water with a concentration of 0.005 to 2 mg / ml, and the solution is ultrasonically treated for 120 to 360 minutes before use; The solvent of the type IV collagen solution in step (2) is glacial acetic acid buffer and deionized water, with a concentration of 20 to 200 μg / ml and a pH value of 4 to 6.

3. The preparation method according to claim 1, characterized in that The soaking conditions in step (3) are: The immersion is carried out under magnetic stirring, the rotation speed is 50 to 500 revolutions per minute, the reaction time is 5 to 30 minutes, the reaction temperature is 20 to 25° C., the cleaning time is 30 to 90 seconds, and the number of cleaning times is 1 to 3 times.

4. The preparation method according to claim 1, characterized in that The preparation method comprises the following steps: (1) placing the pretreated titanium sheet in an electrolyte for anodizing, washing, drying, and then performing high-temperature treatment; The high temperature treatment process is as follows: placing the titanium sheet in a tube furnace, with an initial temperature of 20-25°C, a heating rate of 2-4°C / min, a heating temperature of 400-500°C, a holding temperature of 400-500°C, a holding time of 30-60min, and then cooling with the furnace; Then, the titanium sheet after high temperature treatment is placed in a hydrogen atmosphere for hydrogenation treatment to obtain hydrogenated TiO2 nanotubes; The hydrogenation process is as follows: the treated titanium sheet is placed in a quartz tube under a hydrogen atmosphere with a hydrogen pressure of 0.8 to 0.95×10 5 Pa, and then placed in a tube furnace for high-temperature hydrogenation, with an initial temperature of 20-25°C, a heating rate of 2-4°C / min, a heating temperature of 400-500°C, a holding temperature of 400-500°C, a holding time of 60-240min, and then cooled with the furnace to obtain hydrogenated TiO2 nanotubes; (2) preparing dopamine hydrochloride solution, graphene oxide solution, and type IV collagen solution; The solvent of the dopamine hydrochloride solution is Tris / HCl buffer and deionized water, with a concentration of 2-3 mg / ml and a pH value of 8.5-10; the solvent of the graphene oxide solution is deionized water, with a concentration of 0.005-2 mg / ml, and ultrasonic treatment is performed for 120-360 minutes before use; the solvent of the type IV collagen solution is glacial acetic acid buffer and deionized water, with a concentration of 20-200 μg / ml and a pH value of 4-6; (3) soaking the hydrogenated TiO2 nanotubes described in step (1) in a dopamine hydrochloride solution, a graphene oxide solution, and a type IV collagen solution in sequence to obtain an in situ gene transfection system coated with a dopamine / graphene oxide / type IV collagen composite coating; The soaking conditions are as follows: soaking under magnetic stirring, a rotation speed of 50 to 500 rpm, a reaction time of 5 to 30 min, a reaction temperature of 20 to 25°C, a cleaning time of 30 to 90 s, and a cleaning frequency of 1 to 3 times; (4) immersing the in situ gene transfection system coated with the dopamine / graphene oxide / type IV collagen composite coating in an anti-adenovirus antibody solution and / or a recombinant adenovirus solution for reaction; The anti-adenovirus antibody solution solvent is PBS, the concentration is 1 / 10000 to 1 / 500, the reaction time is 12 to 20 hours, the reaction temperature is 2 to 8°C, the washing time is 10 to 300 seconds, and the washing times are 1 to 3 times; The recombinant adenovirus solution is prepared using PBS as the solvent, with a concentration of 0.1 to 4×10 8 PFU / ml, a reaction time of 1 to 8 hours, a reaction temperature of 20 to 37° C., a washing time of 10 to 300 seconds, and a washing frequency of 1 to 3 times.

5. Use of the in situ gene transfection system prepared by the preparation method according to any one of claims 1 to 4 in the preparation of biomedical materials.

Images