CN110734646A

CN110734646A - Preparation method of COL/PEG @ CaP biomineralization multilayer film

Info

Publication number: CN110734646A
Application number: CN201910962308.7A
Authority: CN
Inventors: 张以河; 安琪; 马泽群
Original assignee: China University of Geosciences Beijing
Current assignee: China University of Geosciences Beijing
Priority date: 2019-10-11
Filing date: 2019-10-11
Publication date: 2020-01-31

Abstract

The invention discloses a preparation method of COL/PEG @ CaP biomineralization multilayer films, which comprises the steps of taking a titanium alloy as a substrate, improving the surface adhesion of the titanium alloy through the modification of PDA, taking COL and PEG as polyelectrolytes on the PDA modified titanium alloy substrate, preparing a COL/PEG self-assembly multilayer film by utilizing a layer-by-layer self-assembly technology, and finally preparing the COL/PEG @ CaP biomineralization multilayer film through biomineralization.

Description

Preparation method of COL/PEG @ CaP biomineralization multilayer film

Technical Field

The invention belongs to the technical field of chemical composite materials, layer-by-layer self-assembly and biomineralization preparation, relates to a preparation method of a layer-by-layer self-assembly film and biomineralization ions, and particularly relates to a preparation method of COL/PEG @ CaP biomineralization multilayer films.

Background

Supramolecular chemistry has developed rapidly and is receiving increasing scientific attention. The goal of supramolecular chemistry is to synthesize materials in a non-covalent manner, with non-covalent forces occurring throughout the natural life system, such as the quaternary structure of proteins, double-stranded DNA of phospholipid bilayer membranes. In particular supramolecular nanotechnology, which shows great potential in the diagnosis and treatment of diseases. In particular, nanoparticles with tunable and diverse properties can be prepared by supramolecular self-assembly technology, which has great potential in the field of nano-pharmaceuticals. Today, nanomaterials developed by nanomedicine can be divided into three broad categories, including: inorganic, organic and organic-inorganic hybrid nanosystems. For inorganic nanoparticles, they have unique physical and chemical properties as well as a variety of morphologies and sizes. However, inorganic nanoparticles tend to be bio-toxic, which hinders the development of such nanomaterials in clinical trials. The use of organic-inorganic hybrid nanosystems combines the advantages of inorganic and organic materials. It is hopeful to synthesize materials with multiple functions and biocompatibility and apply to novel biomedical and clinical applications. The organic-inorganic hybrid nano biomaterial constructed by self-assembly not only can combine the intrinsic properties of heterogeneous structural units, but also can obtain new physical, chemical and biological functions through the supermolecular interaction between the heterogeneous structural units.

Biomineralizing materials, e.g. Polydopamine (PDA), calcium carbonate (CaCO)₃) And calcium phosphate (CaP), are suitable materials for their excellent biological applications. CaCO₃And Cap are demonstrated by their high biocompatibility, biodegradability and responsiveness to pHIt is a good material for biomedical applications. CaCO₃Compared with the traditional nano particles, the biomineralization particles have the advantages of small biotoxicity, clear metabolic pathway and sensitive stimulation response, and all the advantages provide novel tissue engineering materials for us.

Therefore, the multilayer film with high biocompatibility is prepared by using a layer-by-layer self-assembly technology, and is combined with biomineralized inorganic nano-ions, so that the size, the structure and the appearance of the obtained material can be regulated and controlled on a molecular level, the biotoxicity problem of the inorganic nano-ions is well solved, and the performances of inorganic nano-particles such as drug loading and the like can be better exerted. According to the invention, COL and PEG are used as assembly units, so that good biocompatibility is provided for materials, and meanwhile, the growth of biomineralized CaP crystals can be controlled, and the size, structure and morphology of the crystals can be adjusted, so that the crystals have the functions of releasing drugs, promoting cell differentiation and the like.

In this context, DA is dopamine, PDA is polydopamine, Tris is Tris (hydroxymethyl) aminomethane, COL is collagen, PEG is polyethylene glycol, CaCl₂Is calcium chloride, K₂HPO₄Dipotassium hydrogen phosphate and calcium phosphate.

Disclosure of Invention

In order to overcome the problems of biotoxicity and irregular crystal growth of inorganic nanoparticles, the invention provides a preparation method of COL/PEG @ CaP biomineralization multilayer films based on a titanium alloy substrate.

The technical scheme adopted by the invention for solving the technical problems is as follows:

preparation methods of COL/PEG @ CaP biomineralization multilayer films are characterized in that a titanium alloy is used as a substrate, the surface adhesion of the titanium alloy is improved through modification of PDA, COL and PEG are used as polyelectrolytes on the PDA modified titanium alloy substrate, a COL/PEG self-assembly multilayer film is prepared through a layer-by-layer self-assembly technology, and finally, the COL/PEG @ CaP biomineralization multilayer film is prepared through biomineralization.

Titanium alloys are commonly used for the manufacture of medical devices, prostheses or artificial organs for implantation in the human body and auxiliary therapeutic equipment. Through the adhesion of PDA and the assembly of COL/PEG film, the titanium alloy substrate has better biocompatibility and is more suitable for the application of cell and tissue engineering; the CaP mineral particles are formed after biomineralization to form a porous structure with adjustable morphology, so that the drug loading capacity and the drug release time of the film are improved, and meanwhile, the porous structure plays a role in promoting the directional differentiation of osteoblasts.

, in the above technical scheme, the preparation method comprises the following steps:

s1, taking medical titanium alloy as a substrate, and making the surface of the substrate hydrophilic through plasma treatment;

s2, preparing a DA solution, adjusting the pH value of the solution by adopting a Tris solution, and stirring to polymerize the DA solution to obtain a PDA solution;

s3, soaking the substrate processed by the plasma in the step S1 in a PDA solution to improve the surface adhesiveness of the substrate;

s4, respectively preparing COL solution, PEG solution and CaCl₂Solutions and K₂HPO₄A solution;

s5, placing the substrate modified by the PDA in the step S3 into a COL solution for standing, cleaning with deionized water, and drying with nitrogen;

s6, placing the substrate obtained in the step S5 into a PEG solution for standing, washing with deionized water, and drying with nitrogen;

s7, repeating the steps S5 and S6, and detecting the change of the film assembling process by utilizing infrared rays to obtain a COL/PEG multilayer film taking the titanium alloy as a substrate;

s8, placing the COL/PEG multilayer film obtained in the step S7 into CaCl₂Standing the solution, and drying the solution by nitrogen;

s9, placing the multilayer film obtained in the step S8 in K₂HPO₄Standing in the solution, preparing a CaP mineral through biological mineralization, and drying by blowing nitrogen;

s10, repeating the steps S8 and S9 to obtain the COL/PEG @ CaP biomineralization multilayer film.

Preferably, in step S2, the concentration of the DA solution is 0.5-5 mg/mL.

Preferably, in step S2, the pH of the DA solution is adjusted to 8-10 using a Tris solution.

In detail, the DA solution can spontaneously polymerize at normal temperature under alkaline conditions.

Preferably, in step S3, the substrate is soaked in the PDA solution for 10-30 min.

Preferably, in step S4, the COL solution has a concentration of 0.5-2mg/mL and its pH is adjusted to 3-4 with sodium hydroxide solution.

Preferably, in step S4, the concentration of the PEG solution is 0.5-2 mg/mL.

Preferably, in step S4, the CaCl₂The concentration of the solution is 400-600 mmol/L.

Preferably, in step S4, K is₂HPO₄The concentration of the solution is 200-400 mmol/L.

Preferably, in step S5, the substrate is placed in the COL solution and kept standing for 10-20 min.

Preferably, in step S6, the substrate is placed in the PEG solution and left to stand for 10-20 min.

Preferably, in step S7, the steps S5 and S6 are alternately repeated 5-20 times, and finally times of immersion in the PEG solution, resulting in a COL/PEG multilayer film with a titanium alloy as a base.

Preferably, in step S8, the multilayer film is placed in CaCl₂Standing in the solution for 3-10 min.

Preferably, in step S9, the multilayer film is placed in K₂HPO₄Standing in the solution for 3-10 min.

Preferably, in step S10, the steps S8 and S9 are alternately repeated 1-5 times.

The invention also provides application of the preparation method in preparation of biological tissue engineering materials in aspects.

The invention has the advantages that:

(1) the COL/PEG @ CaP biomineralization multilayer film prepared by the invention can effectively improve the biotoxicity of inorganic particles in vivo, so that the inorganic particles can more effectively exert the functions in the organism;

(2) the preparation method provided by the invention adopts a layer-by-layer self-assembly technology to prepare the polymer multilayer film with good biocompatibility, is methods of alternately depositing layer by layer, utilizes weak interaction between layers to combine with , has simple preparation process and short period, is suitable for mass production, and has -wide application prospect;

(3) the preparation method provided by the invention adjusts the nucleation, crystallization and growth of inorganic particles by using the organic polymer multilayer film through a biomineralization technology, and simultaneously adjusts the surface appearance of the inorganic particles through the change of the layer number, so that the inorganic particles have the functions of loading and releasing medicaments and promoting the directional differentiation of cells;

(4) the preparation method provided by the invention uses the collagen which is an organic polymer in the process of layer-by-layer self-assembly, and the source of the collagen is mainly in the body of a mammal, so that the immunological rejection of organisms is avoided, and the biocompatibility of the material is increased.

Drawings

FIG. 1 is a photograph of (COL/PEG) prepared in example 1 of the present invention₁₅A surface scanning electron microscope image of the @ CaP mineralized multilayer film;

FIG. 2 is a photograph of (COL/PEG) prepared in example 1 of the present invention₁₅The atomic force microscopy images and their height representation images of the @ CaP mineralized multilayer film;

FIG. 3 shows the results of (COL/PEG) analysis in example 1 of the present invention₁₅@ CaP mineralized multilayer film and example 2 (COL/PEG)₁₀@ CaP mineralized multilayer film and non-mineralized (COL/PEG) multilayer film₁₅And (COL/PEG)₁₀The effect of multilayer films on methylene blue release;

FIG. 4 shows the results of (COL/PEG) analysis in example 1 of the present invention₁₅@ CaP mineralized multilayer film and example 2 (COL/PEG)₁₀@ CaP mineralized multilayer film, unmineralized (COL/PEG)₁₀The detection result graphs of the alkaline phosphatase activity of the multilayer film and the blank titanium alloy substrate after 7 days and 14 days of cell culture;

FIG. 5 shows the results of (COL/PEG) analysis in example 1 of the present invention₁₅@ CaP mineralized multilayer film and example 2 (COL/PEG)₁₀@ CaP mineralized multilayer film, unmineralized (COL/PEG)₁₀A detection result graph of Runx2 gene expression of the multilayer film and the blank titanium alloy substrate after 7 days, 14 days and 21 days of cell culture;

FIG. 6 shows the results of (COL/PEG) analysis in example 1 of the present invention₁₅@ CaP mineralized multilayer film and example 2 (COL/PEG)₁₀@ CaP mineralized multilayer film, unmineralized (COL/PEG)₁₀The detection result graphs of the OCN gene expression of the multilayer film and the blank titanium alloy substrate after 7 days, 14 days and 21 days of cell culture;

FIG. 7 shows the results of (COL/PEG) analysis in example 1 of the present invention₁₅@ CaP mineralized multilayer film and example 2 (COL/PEG)₁₀@ CaP mineralized multilayer film, unmineralized (COL/PEG)₁₀And (3) a detection result graph of OPN gene expression of the multilayer film and the blank titanium alloy substrate after 7 days, 14 days and 21 days of cell culture.

Detailed Description

The following detailed description is provided in conjunction with the accompanying drawings and examples to illustrate the present invention, but not to limit the scope of the invention, which is defined by the claims.

Unless otherwise specified, the test reagents and materials used in the examples of the present invention are commercially available.

Unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.

Example 1

The embodiment of the invention provides a preparation method of COL/PEG @ CaP biomineralization multilayer films, which comprises the following steps:

(1) the medical titanium alloy is taken as a substrate, and the titanium alloy substrate is subjected to plasma treatment to hydroxylate the surface of the titanium alloy substrate, so that better hydrophilicity is achieved, and the subsequent assembly process is facilitated;

(2) preparing 1mg/mL DA solution, adjusting the pH value to 8.5 by using Tris solution, and polymerizing DA to form PDA solution by magnetic stirring.

(3) Soaking the titanium alloy substrate in PDA solution for 30min to improve the surface adhesion of the substrate, and making the substrate for the subsequent polyelectrolyte assembly

(4) Respectively preparing 1mg/mL COL solution, 1mg/mL PEG solution and 500mM CaCl₂Solution and 300mM K₂HPO₄The pH value of the COL solution is adjusted to 3.5 by sodium hydroxide;

(5) placing the modified titanium alloy substrate in the step (3) into a COL solution, standing for 10min, washing with deionized water, and drying with nitrogen;

(6) placing the material in the step (5) into a PEG solution, standing for 10min, washing with deionized water, and drying with nitrogen;

(7) repeating the steps (5) and (6)15 times, and detecting the change of the process of assembling the film by infrared to obtain the titanium alloy and substrate (COL/PEG)₁₅A multilayer film.

(8) Placing the multilayer film obtained in the step (7) into CaCl₂Standing the solution for 3min, and drying by nitrogen;

(9) putting the material obtained in the step (8) into K₂HPO₄Standing in the solution for 3min to allow it to be biomineralized to generate CaP mineral, and blowing with nitrogen gas.

Performance testing

(1) Preparing a methylene blue solution of 1mg/mL, and separately adding (COL/PEG) obtained in step (7)₁₅Multilayer film and (COL/PEG) obtained in step (9)₁₅The @ CaP mineralized film is placed in the solution for 24 hours to adsorb methylene blue molecules;

(2) PBS (phosphate) buffer solution with pH 7.4 was prepared, and (COL/PEG) obtained in step (7) was added separately₁₅Multilayer film and (COL/PEG) obtained in step (9)₁₅The @ CaP mineralized film is placed in the water-soluble organic polymer matrix, the release effect of the film on loaded molecules before and after mineralization is researched, and the release process is monitored by ultraviolet;

(3) Respectively mixing the blank medical titanium alloy substrate and the (COL/PEG) obtained in the step (7)₁₅Multilayer film and (COL/PEG) obtained in step (9)₁₅The @ CaP mineralized film is placed in a cell culture plate and inoculated with 5X 10⁵Mouse embryonic osteogenic precursor cells (MC3T3-E1) were cultured normally for 7 days and 14 days, and then tested for their alkaline phosphatase (ALP) protein activity and mRNA gene expression after 7 days, 14 days, and 21 days.

Example 2

The difference between this example and example 1 is that in step 7), step 5) and step 6) were alternately repeated 10 times, and the other part was completely in example 1.

Example 3:

the difference between this example and example 1 is that, in step 4), the concentration of the COL solution and that of the PEG solution are 0.5mg/mL, and the pH of the COL solution is adjusted to 3.5 with sodium hydroxide, and the rest is mm from example 1.

Example 4:

on the basis of embodiment 1, the difference in this embodiment from embodiment 1 is: repeating the steps 8 and 9) 3 times alternately to obtain a titanium alloy substrate-based (COL/PEG)₁₅@CaP₃Mineralized multilayer film, and the rest is completely from example 1.

Experimental results and discussion:

as can be seen from the combination of FIG. 1, when the (COL/PEG) @ CaP mineralized film with the titanium alloy as the substrate has 15 layers, the surface morphology of the CaP mineral is adjusted to be a continuous porous structure, which indicates that the organic multilayer film successfully adjusts the mineral formation.

As can be seen from fig. 2, when the (COL/PEG) multilayer film is successfully assembled on the substrate and the number of layers is 15, the thickness of the film is about 236nm, which shows that the piezoelectric film prepared by the present invention satisfies the expected purpose, and the results determined by are obtained by performing the same tests on the samples of example 2, example 3, and example 4, and thus, the detailed description is omitted.

As can be seen from FIG. 3, both the (COL/PEG) multilayer film and the (COL/PEG) @ CaP mineralized multilayer film can adsorb and release methylene blue; however, compared with the mineralized (COL/PEG) @ CaP mineralized multilayer film, the unmineralized (COL/PEG) multilayer film has small adsorption amount to methylene blue and short release duration, and the mineralized continuous porous structure well improves the adsorption amount of the multilayer film to small molecules and prolongs the release time of the small molecules.

As can be seen from FIG. 4, the activity of alkaline phosphatase (ALP) is markers for evaluating the early stage of osteogenic differentiation of cells, as can be seen from FIG. 4 (COL/PEG)₁₅The @ CaP mineralized multilayer films reached the highest ALP activity on day 14, all greater than the other samples.

As is clear from FIG. 5, the expression of mRNA Runx2 is an important transcription factor in bone development, and has important regulatory effects on osteoblast differentiation, chondrocyte maturation, osteoclast differentiation, and extracellular matrix secretion, as is clear from FIG. 5 (COL/PEG)₁₅@ CaP mineralized multilayer membrane, when cells were cultured for 21 days, the expression of RunX2 gene was highest, and was higher than other controls at both 7 and 14 day time points.

As can be seen from FIG. 6, OCN is osteocalcin, a protein secreted from bone bud cells forming the skeleton, and is shown in FIG. 6 (COL/PEG)₁₅The expression of OCN gene was highest at 14 days of cell culture, and was higher than other controls at both 7 and 21 day time points.

As can be seen from FIG. 7, OPN is osteopontin, and osteoblasts, osteocytes and osteoclasts all secrete OPN, which plays an important role in the mineralization and absorption process of bone matrix, as can be seen from FIG. 7, (COL/PEG)₁₅The expression of OPN gene was highest when cells were cultured for 14 days, and was higher than other controls at both 7 and 21 day time points; in conclusion, (COL/PEG)₁₅The @ CaP mineralized multilayer membrane shows the optimal capacity of inducing the osteogenic differentiation of cells

Finally, it should be noted that: it should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims

The preparation method of the COL/PEG @ CaP biomineralization multilayer film is characterized in that a titanium alloy is used as a substrate, the surface adhesion of the titanium alloy is improved through modification of PDA, COL and PEG are used as polyelectrolytes on the PDA modified titanium alloy substrate, a COL/PEG self-assembly multilayer film is prepared through a layer-by-layer self-assembly technology, and finally, the COL/PEG @ CaP biomineralization multilayer film is prepared through biomineralization.
2. The method of claim 1, comprising the steps of:

s1, taking medical titanium alloy as a substrate, and making the surface of the substrate hydrophilic through plasma treatment;

s2, preparing a DA solution, adjusting the pH value of the solution by adopting a Tris solution, and stirring to polymerize the DA solution to obtain a PDA solution;

s3, soaking the substrate processed by the plasma in the step S1 in a PDA solution to improve the surface adhesiveness of the substrate;

s4, respectively preparing COL solution, PEG solution and CaCl₂Solutions and K₂HPO₄A solution;

s5, placing the substrate modified by the PDA in the step S3 into a COL solution for standing, cleaning with deionized water, and drying with nitrogen;

s6, placing the substrate obtained in the step S5 into a PEG solution for standing, washing with deionized water, and drying with nitrogen;

s7, repeating the steps S5 and S6, and detecting the change of the film assembling process by utilizing infrared rays to obtain a COL/PEG multilayer film taking the titanium alloy as a substrate;

s8, placing the COL/PEG multilayer film obtained in the step S7 into CaCl₂Standing the solution, and drying the solution by nitrogen;

s9, placing the multilayer film obtained in the step S8 in K₂HPO₄Standing in solution, and biomineralizing to obtain CaP mineralBlowing by nitrogen;

s10, repeating the steps S8 and S9 to obtain the COL/PEG @ CaP biomineralization multilayer film.
3. The production method according to claim 2, wherein, in step S2,

the concentration of the DA solution is 0.5-5 mg/mL;

and/or adjusting the pH value of the DA solution to 8-10 by adopting a Tris solution.
4. The method for preparing a composite material according to claim 2 or 3, wherein the substrate is soaked in the PDA solution for 10-30min in step S3.
5. The production method according to claim 2 or 3, wherein, in step S4,

the concentration of the COL solution is 0.5-2mg/mL, and the pH value of the COL solution is adjusted to 3-4 by using a sodium hydroxide solution;

and/or, the concentration of the PEG solution is 0.5-2 mg/mL;

and/or, the CaCl₂The concentration of the solution is 400-600 mmol/L;

and/or, said K₂HPO₄The concentration of the solution is 200-400 mmol/L.
6. The production method according to claim 2 or 3,

in step S5, the substrate is placed into a COL solution and stands for 10-20 min;

and/or, in the step S6, the substrate is placed into the PEG solution and stands still for 10-20 min.
7. The method of claim 2 or 3, wherein in step S7, steps S5 and S6 are alternately repeated 5-20 times, and finally times of immersion in the PEG solution, to obtain the COL/PEG multilayer film with the titanium alloy as a base.
8. The production method according to claim 2 or 3,

in step S8, the multilayer film is placed in CaCl₂Standing in the solution for 3-10 min;

and/or, in step S9, the multilayer film is inserted into K₂HPO₄Standing in the solution for 3-10 min.
9. The method of claim 2 or 3, wherein in the step S10, the steps S8 and S9 are alternately repeated 1-5 times.
10. Use of the preparation method of any of claims 1-9 in the preparation of a material for biological tissue engineering.