CN114989475B - Preparation method and product application of biological functionalized surface modified polyether-ether-ketone material - Google Patents

Abstract

The invention relates to a preparation method and product application of a bio-functionalized surface modified polyether-ether-ketone material. The material is a surface activated artificial bone repair implant. The preparation process comprises the following steps: preparing a customized polyether-ether-ketone artificial bone repair implant; secondly, preprocessing the surface of the polyether-ether-ketone artificial bone repair implant; and thirdly, fixing hyperbranched polylysine on the surface of the polyether-ether-ketone artificial bone repair implant, and loading drug molecules and fluorescent imaging molecules through electrostatic action to form a bioactive coating. The preparation method disclosed by the invention can obviously improve the biocompatibility of the polyether-ether-ketone material, simultaneously endow the polyether-ether-ketone material with biological activity, promote the proliferation of cells on the surface of the customized polyether-ether-ketone artificial bone repair implant, and the surface-activated polyether-ether-ketone artificial bone repair implant has more excellent bone growth promoting capability.

Description

Preparation method and product of biofunctionalized surface-modified polyetheretherketone material application

technical field

The invention relates to a preparation method and product application of a biologically functionalized surface-modified polyetheretherketone material, belonging to the technical field of biomedical polymer materials.

Background technique

Polyetheretherketone (PEEK) has good in vitro and in vivo biocompatibility, non-toxic, non-teratogenic, non-mutagenic, and carcinogenic effects, and can transmit radiation, and will not produce artifacts in magnetic resonance scanning. At present, PEEK and its composite materials have become a research hotspot for material scientists and orthopedic experts, and intervertebral fusion devices, artificial joint prostheses, etc. have been applied clinically and achieved good recent follow-up results. However, PEEK is biologically inert and hydrophobic, lacks biological activity, is difficult to chemically modify, and cannot be well osseointegrated with the host bone after implantation, which greatly limits its wide clinical application.

On the premise of not destroying the many excellent properties of PEEK materials, it is an effective way to solve the above problems by using surface modification methods to endow them with biological activity, that is, to prepare bioactive coatings by physical or chemical methods to functionalize PEEK. In previous literature reports, many coatings (including calcium phosphate, bioactive small molecules, hydroxyapatite, titanium, etc.) have practical value in enhancing the osseointegration of PEEK implants. However, there are still many problems to be solved in these methods, including easy degradation of the coating, complex and time-consuming chemical steps, and poor adhesion between the coating and the substrate. In addition, the surface modification of PEEK implants is currently mainly focused on multifunctional properties such as improving bioactivity, promoting cell growth, or avoiding infection, which are also important functions necessary to promote the physiological osseointegration of PEEK implants. At present, there are few research reports on the surface functionalization of PEEK to obtain key functions such as anti-infection and growth promotion at the same time, and the actual clinical application is even scarcer.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the biological inertia of the PEEK material, treat the surface of PEEK by chemical grafting at room temperature, and non-covalently combine fluorescent molecules and drug molecules only by soaking to form a coating, endowing it with anti-infection promotion Growth and other biological functions.

In order to achieve the above object, the technical solution of the present invention is:

The polyether ether ketone material is prepared by injection molding, 3D printing, prepreg and other processes with a specific shape, and the outer layer or the whole is polyether ether ketone material, and the surface of the polyether ether ketone material is biologically treated. Functional modification treatment.

The modification by biofunctionalization is specifically, after the polyetheretherketone material is treated with plasma, the activation layer is chemically grafted on the surface of the polyetheretherketone material through a soaking process to modify it.

The soaking process is to generate carboxyl groups on the surface by acidification, and the carboxyl groups react with hyperbranched polylysine amidation to form a positively charged coating, that is, an activation layer; because the coating contains positively charged hyperbranched polylysine Amino acids, which can be conjugated with negatively charged fluorescent molecules and/or growth-promoting drugs for non-covalent high-efficiency loading.

The fluorescent molecules are fluorescein isothiocyanate, FAM maleimide, rhodamine, sulforhodamine 101, 5-carboxymethylrhodamine, 1-aminonaphthalene-8-carboxylic acid Texas At least one of red and 5-(iodoacetamido)fluorescein.

The growth-promoting drug is at least one of tacrolimus, pulinin potassium, oxiracetam, and alendronate sodium.

The preparation method of the polyether ether ketone material is specifically:

1) Prepare the polyetheretherketone material into the desired shape of the repaired area through injection molding, 3D printing, prepreg and other processes;

2) Activate the surface: place the PEEK material in a plasma processor for surface activation.

3) Covalent bonding: The PEEK material treated by plasma is immediately placed in a 0.1-5mol/L acrylic acid solution by weight, heated, taken out and rinsed with water three times; to prepare a hyperbranched polylysine solution, add 10- Activate with 50 parts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, add 10-50 parts of N-hydroxysuccinimide for activation, and put in acrylic acid-treated PEEK material , react overnight at room temperature, rinse with water three times after taking it out, and dry it.

4) Non-covalent loading: Prepare 1-10 parts of negatively charged fluorescent molecules or drugs into a solution, put the PEEK material in, stir, take out and dry.

The functionalized surface-modified polyetheretherketone material has the properties of efficiently loading growth-promoting drugs, thereby promoting cell growth, and resisting infection.

The promoting cell growth is promoting the growth of mouse embryonic osteoblast precursor cells.

The anti-infection performance is specifically anti-Staphylococcus aureus and Escherichia coli.

The beneficial effects of the present invention are:

The present invention provides a stable and efficient bioactive modification, that is, after plasma treatment, it can be chemically grafted only by soaking at room temperature, and the acrylated PEEK does not change the mechanical properties of the material body, and at the same time has the ability to promote osteoblast formation. Various biological properties of bone differentiation and significant bactericidal effect. The invention has the advantages of simple process, high efficiency and good repeatability, and can improve the shortcomings of the current clinical application of PEEK orthopedic implants.

Description of drawings

Figure 1: The physical picture of PEEK before and after acrylic acid modification;

Figure 2: Characterization of water contact angle of PEEK before and after modification with acrylic acid;

Figure 3: Anti-infection properties of PEEK modified against Escherichia coli and Staphylococcus aureus.

Figure 4: The physical picture of PEEK modified by sulfuric acid;

Detailed ways

The examples given are for better description of the present invention, but the content of the present invention is not limited to the examples given. Non-essential improvements and adjustments made by those skilled in the art to the embodiments based on the above content of the invention still belong to the protection scope of the present invention.

Example 1

The polyether ether ketone melt was printed into the shape required for cranial repair by a 3D printer and placed in a plasma processor, the parameters were adjusted, and the glow discharge was performed for 20 minutes. The plasma-treated PEEK skull repair implants were immediately placed in the prepared 0.5mol/L acrylic acid solution and soaked for 30 minutes, taken out and rinsed with water three times; the hyperbranched polylysine solution was prepared and activated by adding 50 mmol/L EDC for 20 minutes , then add 50mmol/L NHS to activate for 1h, put in the acrylic-treated PEEK skull repair implant, react overnight at room temperature, take it out, rinse it with water three times, and dry it. The negatively charged fluorescent molecule rhodamine and the drug alendronate sodium were prepared into a 3 mg/mL solution, and the PEEK skull repair implant was put in, stirred at room temperature for 30 minutes, and then taken out and dried.

PEEK without any treatment is marked as PEEK, and PEEK after chemical grafting is marked as PEEK-AAc, and its appearance is shown in Figure 1. At room temperature, the static contact angle was measured on PEEK and modified PEEK, and the results are shown in Figure 2. After grafting treatment, the contact angle of PEEK decreased from 80° to 20°, indicating that the hydrophilicity of PEEK was significantly improved after surface treatment.

The cells used in this example are mouse osteoblast precursor cells, but other mouse adult stem cells (adipogenic mesenchymal stem cells, bone marrow mesenchymal stem cells, etc.) and other stem cells with osteogenic differentiation potential are also suitable for the present invention. The test samples used were divided into three groups: Bare PEEK (unmodified PEEK), PEEK-AAc (chemically grafted PEEK), PEEK-AAc-AS (chemically grafted PEEK loaded with drug alendronate sodium). The three groups of samples were sterilized and placed in a 48-well plate with 3 replicate holes for each group. Mouse embryonic osteoblast precursor cells (MC3T3-E1) were inoculated on the materials of each group, and the cell density was controlled (2×10 ⁴ /ml), 500 μL per well; 1 day and 3 days after inoculation, each PEEK sample was washed with phosphate-buffered saline (PBS); cell proliferation was studied using the CCK-8 method at predetermined time points. In the unmodified PEEK group, there was no cell proliferation on the surface of the material during the 1-day and 3-day test periods. In the PEEK-AAc group, the proliferation of cells was promoted on day 1 and day 3. In addition, compared with the PEEK-AAc group, the effect of promoting cell proliferation in the PEEK-AAc-AS group loaded with the growth-promoting drug alendronate was significantly improved.

In this embodiment, the antibacterial performance of the material is evaluated by the dilution coating plate method. The specific operation is as follows: put the PEEK sheet into a 24-well plate, and dilute the concentration of the bacterial solution to 1×10 ⁶ CFU/mL with LB medium. Add 25 μL of bacterial solution dropwise on the surface of each PEEK material, fill the gap between the wells of the orifice plate with PBS to slow down the evaporation of water, and place it in a 37°C incubator for 4 hours; after taking it out, add 1975 μL of bacterial solution to each well PBS, transfer material and liquid simultaneously to a new centrifuge tube. Place the centrifuge tube in an ultrasonic water bath (power 40W) and sonicate for 5 minutes, then oscillate with a vortex oscillator for 30 seconds to fully elute the adhered bacteria; after autoclaving the prepared LB solid medium solution, Keep warm at 50°C, pour about 15mL of liquid into the plate before it solidifies, and then obtain a solid medium after cooling and solidifying evenly; after gradient dilution of the bacterial solution collected above, take 100 μL and drop it on the center of the plate, and use Spread the cloth evenly, and place it in an incubator at 37°C for 16 hours; after the colonies grow, take pictures and count the number of colonies on the plate. Taking the pure PEEK group as the control, the antibacterial rate calculation method is: antibacterial rate=(control group CFU-experimental group CFU)/control group CFU×100%. The test results are shown in Figure 3. The unmodified PEEK material has no antibacterial ability, and the antibacterial rates of E. coli and Staphylococcus aureus in the PEEK-AAc group can reach 69% and 43%, respectively, indicating that the material grafted with HBPL on the surface It has very good antibacterial effect on Gram-positive bacteria and Gram-negative bacteria.

Example 2

The polyether ether ketone melt was printed into the shape required for cranial repair by a 3D printer and placed in a plasma processor, the parameters were adjusted, and the glow discharge was performed for 20 minutes. The plasma-treated PEEK skull repair implants were immediately placed in the prepared 0.5mol/L sulfuric acid solution and soaked for 30 minutes, taken out and rinsed with water three times; the hyperbranched polylysine solution was prepared and activated by adding 50 mmol/L EDC for 20 minutes , then add 50mmol/L NHS to activate for 1h, put into the acrylic-treated PEEK skull repair implant, react overnight at room temperature, take it out, rinse it with water three times, and dry it. The negatively charged fluorescent molecule rhodamine or the drug alendronate sodium was prepared into a 3 mg/mL solution, the PEEK skull repair implant was put into it, stirred at room temperature for 30 minutes, and the biofunctional surface modified polymer was obtained after drying. Ether ether ketone material, its appearance is shown in Figure 4.

Comparative example 1

Prepare PEEK skull repair implant according to the method in the prior art (such as CN114214592A, a kind of surface treatment method that strengthens the biocompatibility of 3D printing PEEK material) polyether ether ketone (PEEK) surface is treated, improves surface smoothness, To eliminate dust particles on the surface of 3D printed PEEK, the specific treatment process is as follows: sandblasting: the 3D printed PEEK material is first sandblasted; grinding and polishing: the sandblasted PEEK material is ground and polished under sandpaper and diamond abrasive paste ; Ultrasonic cleaning: put the sandblasted PEEK material in an acetone solution for ultrasonic cleaning; use physical vapor deposition (PVD) technology to deposit pure Ta on the surface of polyetheretherketone to obtain a PEEK-based coating material . The total preparation time of Comparative Example 1 is relatively 2-3 days, the processing process requires a lot of equipment, the energy consumption is large, and the preparation process is complicated and cumbersome. Moreover, heavy metals are in the implant coating, and there is a risk of release after long-term implantation.

Comparative example 2

Prepare PEEK skull repair implants according to the methods in the prior art (such as CN111116964A, polyether ether ketone material with biological functionalization surface modification and its preparation method and application) The preparation method is specifically: polyether ether ketone The material was immersed in a strong acid solution for 0.5h, then soaked in deionized water for 24h to remove excess acid, and then ultrasonically cleaned with deionized water for 3 times, each time for 20min, and dried to obtain a surface-modified polyether ether ketone material. Although the total preparation time is short, the hydrophilic property of PEEK is only improved after acidification, and the surface is unstable, so it cannot act on the human body for a long time. However, through the preparation method and product application of a biologically functionalized surface-modified polyetheretherketone material disclosed in the present invention, the surface of PEEK can be treated by chemical grafting at room temperature within a total of 24 hours, and non-covalent Combine fluorescent molecules and drug molecules to form a coating, endowing it with various biological functions including anti-infection and promoting growth.

Claims (7)

1. A preparation method of a bio-functionalized surface modified polyether-ether-ketone material is characterized in that the material is a customized polyether-ether-ketone material, which is prepared by injection molding, 3D printing and presoaking technology, has a specific shape and is provided with an outer layer or is integrally polyether-ether-ketone, and the surface of the polyether-ether-ketone material is subjected to bio-functionalized modification treatment; the biological functionalization modification treatment is that after the polyetheretherketone material is treated by plasma, an activation layer is chemically grafted on the surface of the polyetheretherketone material by a soaking process, wherein the soaking process is as follows: immersing the polyether-ether-ketone material subjected to plasma treatment into an acid solution to generate carboxyl on the surface through acidification, immersing the polyether-ether-ketone material into a hyperbranched polylysine solution activated by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS to enable the carboxyl to react with the hyperbranched polylysine in amidation to form a positively charged coating, and finally immersing the coating into a negatively charged fluorescent imaging molecule and/or a growth-promoting drug solution to be combined with a non-covalent load of the negatively charged fluorescent molecule and/or the growth-promoting drug.

2. The method of preparing a biofunctionalized surface modified polyetheretherketone material of claim 1, wherein said polyetheretherketone material is capable of being used as an artificial bone repair implant.

3. The method for preparing a biofunctionalized surface modified polyetheretherketone material as claimed in claim 1, wherein the acid used for acidification is: at least one of sulfuric acid, acrylic acid, phosphoric acid and acetic acid.

4. The method for preparing the bio-functionalized surface modified polyether ether ketone material according to claim 1, wherein the fluorescent molecule is at least one of fluorescein isothiocyanate, FAM maleimide, rhodamine, sulfonylrhodamine 101, 5-carboxymethyl rhodamine, 1-aminonaphthalene-8-carboxylic acid texas red and 5- (iodoacetamido) fluorescein.

5. The method for preparing a bio-functionalized surface modified polyetheretherketone material according to claim 1, wherein the growth-promoting drug is at least one of tacrolimus, potassium pralidoxime, oxiracetam and alendronate sodium.

6. The method for preparing a biofunctionalized surface modified polyetheretherketone material according to any one of claims 1 to 5, wherein the polyetheretherketone material is plasma treated and then combined with acrylic acid, and then covalently reacted with hyperbranched polylysine, and finally forms a stable surface coating by non-covalent interactions with fluorescent molecules and growth promoting drugs.

7. The method of preparing a biofunctionalized surface modified polyetheretherketone material as claimed in any one of claims 1 to 5, wherein said method of preparing comprises the steps of:

1) Preparing polyether-ether-ketone into a shape required by a repair area through injection molding, 3D printing and presoaking processes;

2) Activating the surface: placing a polyether-ether-ketone material in a plasma treatment instrument for surface activation;

3) Covalent bonding: immediately placing the polyether-ether-ketone material subjected to plasma treatment in an acrylic acid solution with the concentration of 0.1-5mol/L by weight, heating, taking out and flushing with water for three times; preparing hyperbranched polylysine solution, adding 10-50 parts of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride for activation, adding 10-50 parts of N-hydroxysuccinimide for activation, adding polyether-ether-ketone treated by acrylic acid for reaction at normal temperature overnight, taking out, washing with water for three times, and drying;

4) Non-covalent loading: preparing 1-10 parts of negatively charged fluorescent molecules or growth promoting drugs into a solution, putting polyether-ether-ketone into the solution, stirring, taking out and drying the solution to obtain the biological functionalized surface modified polyether-ether-ketone material.