CN114010837A - Macromolecular micelle coating for sequentially delivering immunomodulatory factors on nanorod-arrayed surface, and preparation method and application thereof - Google Patents
Macromolecular micelle coating for sequentially delivering immunomodulatory factors on nanorod-arrayed surface, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a high molecular micelle coating for sequentially delivering an immunoregulatory factor on a nanorod-arrayed surface, a preparation method and application thereof, wherein two micelles of S1P loaded with an anti-inflammatory effect and an LPS inflammatory factor loaded with a pro-inflammatory effect are sequentially deposited on a hydroxyapatite nanorod-arrayed coating subjected to micro-arc oxidation and hydrothermal treatment in an electrodeposition mode to obtain an inner-outer double-layer nano-micelle immunomodulation composite coating loaded with S1P (inner layer) and LPS (outer layer), and the composite coating has the following characteristics: the method can firstly release the proinflammatory factor LPS deposited on the outer layer, induce macrophage to proinflammatory polarization, then release the inflammation inhibiting factor S1P deposited on the inner layer, induce macrophage to promote healing polarization, and the hydroxyapatite nanorod array configured surface layer constructed by the bone-imitated matrix and the components can obviously promote the osteogenesis of osteogenesis related cells, so that the two are combined to improve the osseointegration performance of the implant after implantation.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a nanorod-arrayed polymeric micelle coating with a surface sequentially delivering immunomodulatory factors, and a preparation method and application thereof.
Background
Titanium (Ti) and its alloys have high mechanical strength, chemical stability, corrosion resistance and biocompatibility, and are commonly used to construct orthopedic implants. However, clinically Ti-based implants still have high failure rates of 15% -20%. In the early failure case, the failure reasons are related to surgical skills, the health condition of the patient and the like, and the failure of the implant to achieve osseointegration with the body bone tissue at the material bone interface is also one of important reasons, and the degree of osseointegration between the prosthesis and the body bone tissue affects the service life of the prosthesis. In fact, Ti is biologically inert, and after being implanted into the body, the biologically inert material is not only not integrated with the bone tissue, but also wrapped by fibrous connective tissue to be separated from the bone tissue, so that the implant is loosened or falls off, and finally the implantation of the biological material fails. Therefore, the surface of the implant needs to be modified, and the related biological reaction between the bone interface of the material and the body cells is regulated and controlled to be in the direction of facilitating the integration of the bone tissues.
Osteointegration begins with the formation of a blood clot on the implant surface, undergoing an immune response by macrophages, angiogenesis by endothelial cells and new osteogenesis by osteoblasts. The macrophages play an important representative role in host immune response, and when the orthopedic implant contacts body fluid in an orthopedic surgery, the macrophages in the body fluid firstly reach a local part to identify the implant, gather and proliferate other immune cells and chemotaxis other immune cells to secrete a large amount of cytokines, so the macrophages play a core role in the subsequent healing process of implanting the implant into an organism. The classification of macrophages is generally classified into M1 type or M2 type according to their function. M1 macrophages are also known as "classical activated" pro-inflammatory macrophages, which can be polarized induced by interferon-gamma (IFN-gamma) or Lipopolysaccharide (LPS); m1 macrophages secrete a number of proinflammatory-related cytokines (interleukins IL-1 β, IL-6, IL-23), produce Inducible Nitric Oxide Synthase (iNOS) and toxic Reactive Oxygen Species (ROS), and tumor necrosis factor α (TNF- α) to promote exacerbation of local inflammation. In contrast, M2 type macrophages of "bypass activation type" can be induced by cytokines such as IL-4, IL-10, S1P and IL-13, M2 type macrophages can secrete a large amount of anti-inflammatory cytokines such as IL-10 and arginase 1(ARG1) and various growth factors such as transforming growth factor beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor (PDGF), osteopontin, 1, 25-dihydroxyvitamin D3 and Bone Morphogenetic Protein (BMP), thereby inhibiting local inflammatory response, and inducing extracellular matrix deposition and new bone formation, thereby promoting osseointegration performance. The normal process of osteointegration, the biological process of pro-inflammatory response mediated by macrophages of the M1 and M2 types followed by inhibition of inflammation, is essential for osteointegration, but requires maintenance of normal immune homeostasis.
Accordingly, there is a need for targeted design and construction of an endosteal implant to mimic the normal healing process to sequentially deliver pro-inflammatory factors and anti-inflammatory factors, so as to regulate macrophage-mediated osseointegration process and significantly improve the osseointegration performance of the titanium implant.
Disclosure of Invention
The invention aims to provide a nanorod-arrayed polymer micelle coating with a surface sequentially delivering immunomodulatory factors, and a preparation method and application thereof, so as to overcome the problem that the surface of a titanium or titanium alloy implant in the prior art is insufficient in osseointegration performance with host bones due to biological inertia; the prepared biomedical material surface layer can regulate and control good balance between proinflammatory (M1 type) and healing promoting (M2 type) macrophage subgroups through sequential release of immune regulatory factors, and promotes osseointegration performance of an implant after implantation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing a macromolecular micelle coating for sequentially delivering an immunomodulatory factor on a nanorod-arrayed surface comprises the following steps:
step 1: micro-arc oxidation is carried out on a pure titanium or titanium alloy matrix in electrolyte containing phosphorus ions and calcium ions, so that a microporous titanium dioxide coating is formed on the surface of the matrix;
step 2: preparing a hydroxyapatite nanorod-structured coating on the microporous titanium dioxide coating obtained in the step 1 by adopting a hydrothermal treatment method;
and step 3: dissolving saturated fatty acid, N-hydroxysuccinimide as a catalyst and excessive dicyclohexylcarbodiimide in an anhydrous dimethyl sulfoxide solution to obtain a mixed solution, wherein the concentration of the saturated fatty acid in the mixed solution is 0.05-0.1 mol/L, the concentration of the N-hydroxysuccinimide in the mixed solution is 0.05-0.1 mol/L, and the concentration of the dicyclohexylcarbodiimide in the mixed solution is 0.075-0.2 mol/L, and then stirring at normal temperature for 12 hours to obtain a solution containing saturated fatty acid active ester;
and 4, step 4: dissolving gelatin in deionized water, heating to 50-65 ℃ for dissolving, cooling to room temperature to obtain a gelatin aqueous solution with the concentration of 10-20 g/L, adding a dimethyl sulfoxide solution, fully stirring for reaction to obtain a gelatin solution, adding 0.2-0.4 time of the volume of the dimethyl sulfoxide to the gelatin aqueous solution, dropwise adding the solution containing the saturated fatty acid active ester obtained in the step (3) into the gelatin solution, wherein the volume ratio of the gelatin solution to the solution containing the saturated fatty acid active ester is 5: 1-10: 1, adjusting the pH to 8-8.5 by using sodium hydroxide or hydrochloric acid, reacting for 12-24 h, centrifuging the obtained solution to remove precipitates, collecting supernatant, dialyzing for 3 days, and freeze-drying for later use to obtain a gelatin-g-saturated fatty acid copolymer;
and 5: loading the gelatin-g-saturated fatty acid copolymer prepared in the step 4 with an anti-inflammatory factor S1P by a coprecipitation method, dissolving in water, and uniformly mixing with the chitosan hydrochloride aqueous solution for later use;
step 6: loading the proinflammatory factor LPS on the gelatin-g-saturated fatty acid copolymer prepared in the step 4 in a self-assembly mode, dissolving the copolymer in water, and uniformly mixing the copolymer with the chitosan hydrochloride aqueous solution for later use;
and 7: and (3) taking the mixed solution prepared in the steps (5) and (6) as an electrolyte, and sequentially depositing the mixed solution on the hydroxyapatite nanorod-structured coating prepared in the step (2) in an electrodeposition mode to form a nanorod-arrayed polymer micelle coating with the surface sequentially delivering the immune regulatory factors.
Further, the step 1 specifically comprises:
taking a pure titanium or titanium alloy matrix as an anode and a stainless steel electrolytic tank as a cathode, performing micro-arc oxidation at the temperature of 295-305K, washing with deionized water, and drying to form a microporous titanium dioxide coating on the surface of the pure titanium or titanium alloy matrix; the parameters of the micro-arc oxidation are as follows: the arc frequency is 100-120 Hz, the positive voltage is 350-450V, and the duty ratio is 15-25%; the concentration of sodium hydroxide in the electrolyte is 0.01-0.02 mol/L, the concentration of calcium acetate is 0.3-0.5 mol/L, and the concentration of beta-phosphoglyceride disodium salt pentahydrate is 0.03-0.05 mol/L.
Further, the step 2 specifically includes:
step 2.1: putting the substrate of the microporous titanium dioxide coating obtained in the step 1 into a sodium hydroxide aqueous solution with the concentration of 0.02-0.04 mol/L, and sealing for primary hydrothermal treatment;
step 2.2: removing liquid in the product obtained in the step 2.1, adding uniformly stirred aqueous solutions of calcium sodium ethylenediaminetetraacetate, beta-glycerol phosphate disodium salt pentahydrate and sodium hydroxide, and sealing for secondary hydrothermal treatment, wherein the concentration of the calcium sodium ethylenediaminetetraacetate in the uniformly stirred aqueous solutions of the calcium sodium ethylenediaminetetraacetate, the beta-glycerol phosphate disodium salt pentahydrate and the sodium hydroxide is 0.1-0.2 mol/L, the concentration of the beta-glycerol phosphate disodium salt pentahydrate is 0.02-0.04 mol/L, and the concentration of the sodium hydroxide is 0.2-0.4 mol/L; and after the secondary hydrothermal treatment is finished, cleaning and drying the product for later use.
Furthermore, the temperature of the primary hydrothermal treatment is 365-395K, and the time is 2-3 h.
Furthermore, the temperature of the secondary hydrothermal treatment is 385-415K, and the time is 22-24 h.
Further, the step 5 specifically includes: dissolving the anti-inflammatory factor S1P and the gelatin-g-saturated fatty acid copolymer in glacial acetic acid, volatilizing a solvent to obtain a blend of the anti-inflammatory factor S1P and the gelatin-g-saturated fatty acid copolymer at a molecular level, then dissolving the blend in deionized water, encapsulating the anti-inflammatory factor S1P to a hydrophobic core of a micelle in a dissolving self-assembly process of the gelatin-g-saturated fatty acid copolymer to obtain a micelle solution carrying the anti-inflammatory factor S1P, wherein the concentration of the micelle solution carrying the anti-inflammatory factor S1P is 10-20 mg/mL, in the step 5, the concentration of a chitosan hydrochloride aqueous solution is 10-20 mg/mL, and mixing the micelle solution carrying the anti-inflammatory factor S1P and the chitosan hydrochloride aqueous solution according to a volume ratio of 2: 1-1: 2.
Further, the step 6 specifically includes: the method comprises the steps of simultaneously dissolving the proinflammatory factor LPS and the gelatin-g-saturated fatty acid copolymer in deionized water, self-assembling the proinflammatory factor LPS and the gelatin-g-saturated fatty acid copolymer into micelles together based on hydrophilic and hydrophobic effects and electrostatic effects to obtain a micellar solution carrying the proinflammatory factor LPS, wherein the concentration of the micellar solution carrying the proinflammatory factor LPS is 10-20 mg/mL, the concentration of a chitosan hydrochloride aqueous solution in the step 6 is 10-20 mg/mL, and the micellar solution carrying the proinflammatory factor LPS is mixed with a chitosan hydrochloride aqueous solution according to the volume ratio of 2: 1-1: 2.
Further, the step 7 specifically includes: and (3) sequentially depositing the micelles loaded with the anti-inflammatory factor S1P and the micelles loaded with the pro-inflammatory factor LPS on the surface of the hydroxyapatite nanorod-structured coating by using a constant voltage electrodeposition process and taking the mixed solution in the steps 5 and 6 as an electrolyte, wherein the deposition voltage is 2V-8V, and the deposition time is 5S-10 min.
A high-molecular micelle coating for sequentially delivering immune regulatory factors on a nanorod-arrayed surface is prepared by the preparation method.
An application of a high-molecular micelle coating for sequentially delivering an immunomodulatory factor on a nanorod-arrayed surface as an implant coating material.
Compared with the prior art, the invention has the following beneficial technical effects:
the preparation method of the macromolecular micelle coating for sequentially delivering the immune regulatory factor on the nanorod arrayed surface comprises the steps of firstly preparing a porous titanium dioxide coating containing phosphorus and calcium on the surface layer of titanium or an alloy thereof by adopting a micro-arc oxidation process, then growing a Hydroxyapatite (HA) nanorod-configured coating on the porous titanium dioxide coating containing phosphorus and calcium in situ by using a hydrothermal treatment method, and then respectively preparing a loaded anti-inflammatory factor S1P and an anti-inflammatory factor S1And (3) depositing the drug-loaded micelle on the nanorod-arrayed coating by using a constant voltage electrodeposition process to obtain the biomedical material of the macromolecular micelle coating, which is anti-inflammatory and sequentially delivers the immune regulatory factor, of the factor LPS micelle. In the preparation method, the micro-arc oxidation electrolyte and the hydrothermal solution have simple components, do not contain easily decomposed components, and have stable and controllable process and strong repeatability; using chitosan/amphiphilic gelatin copolymer mixed solution as electrolyte, passing-NH on chitosan3+Radical and-COO on gelatin-The electrostatic interaction between the groups forms a colloidal polyelectrolyte compound, and the colloidal polyelectrolyte compound is deposited on the nanorod array coating in a constant-voltage electrodeposition mode, so that the process is stable and simple, and can be used for large-scale production and preparation. The raw materials used for preparing the coating, such as saturated fatty acid, gelatin, chitosan and the like, are nontoxic and safe, have no side effect on human bodies, can be directly purchased in the market and are easily obtained, and therefore the popularization and the application of the technology are guaranteed.
Further, the deposition voltage is 2V to 10V. If the voltage is too low, the micelle cannot be deposited on the nanorod coating; if the voltage is too large, the deposition speed is too fast, and too high voltage can also cause a large amount of hydrogen to be generated, which is not favorable for the deposition of micelles. The nanorod-arrayed high-molecular micelle coating with the surface sequentially delivering the immunomodulatory factors prepared by the preparation method has a double-layer structure, and the inner layer is a nanorod array with a bone-like matrix configuration and has the effect of promoting bone formation; the outer layer is a macromolecule coating loaded with inflammatory factors, which can imitate the normal healing process to sequentially deliver proinflammatory factors and inflammation-inhibiting factors so as to regulate and control the macrophage-mediated osseointegration process, thereby improving the osseointegration performance of the titanium implant. The adopted raw materials are nontoxic and safe, and have no side effect on human bodies.
The coating prepared by the invention can firstly release the proinflammatory factor LPS deposited on the outer layer, induce macrophage polarization to proinflammatory (M1 type), then release the inflammation-inhibiting factor S1P deposited on the inner layer, and induce macrophage polarization to healing promotion (M2 type), and the Hydroxyapatite (HA) nanorod array configuration surface layer constructed by imitating bone matrix and components can obviously promote the bone formation behavior of bone formation related cells, and the two are combined to improve the bone integration performance of an implant after implantation.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is an SEM photograph of the surface morphology of the HA nanorod structured coating prepared by the micro-arc oxidation-hydrothermal treatment in example 1;
FIG. 2 is a SEM photograph of the cross-sectional morphology of the HA nanorod structured coating prepared by the micro-arc oxidation-hydrothermal treatment in example 1;
FIG. 3 is an SEM photograph of the surface morphology of a titanium implant deposited with two drug loading factor coatings prepared in example 2;
FIG. 4 is a SEM photograph of the cross-sectional morphology of a titanium implant deposited with two drug-loaded factor coatings prepared in example 3;
fig. 5 is a fourier infrared spectrum of a titanium implant deposited with two layers of drug loading factor coating prepared in example 2.
Detailed Description
The present invention is described in detail below:
a method for preparing a macromolecular micelle coating for sequentially delivering an immunomodulatory factor on a nanorod-arrayed surface comprises the following steps:
step 1: carrying out micro-arc oxidation on a pure titanium or titanium alloy matrix in an electrolyte containing phosphorus ions and calcium ions to form a microporous titanium dioxide coating on the surface of the titanium-based matrix;
the method specifically comprises the following steps: taking a pure titanium or titanium alloy matrix as an anode and a stainless steel electrolytic tank as a cathode, performing micro-arc oxidation at the temperature of 295-305K, washing with deionized water, and drying to form a microporous titanium dioxide coating on the surface of the titanium matrix; the parameters of the micro-arc oxidation are as follows: the arc frequency is 100-120 Hz, the positive voltage is 350-450V, and the duty ratio is 15-25%; the electrolyte includes: 0.01-0.02 mol/L of sodium hydroxide, 0.3-0.5 mol/L of calcium acetate and 0.03-0.05 mol/L of beta-phosphoglyceride disodium salt pentahydrate.
Step 2: preparing a hydroxyapatite nanorod-structured coating on the microporous titanium dioxide coating obtained in the step 1 by adopting a hydrothermal treatment method;
the method specifically comprises the following steps:
step 2.1: putting the microporous titanium dioxide coating obtained in the step 1 into 15-20 mL of a 0.02-0.04 mol/L sodium hydroxide aqueous solution, sealing and carrying out primary hydrothermal treatment at 365-395K for 2-3 h;
step 2.2: adding sodium calcium ethylene diamine tetracetate, a beta-phosphoglyceride disodium salt pentahydrate and sodium hydroxide into water, uniformly stirring, sealing the product obtained in the step 2.1, and performing secondary hydrothermal treatment at 385-415K for 22-24 h; wherein the concentration of the calcium sodium ethylene diamine tetraacetate is 0.1-0.2 mol/L, the concentration of the beta-phosphoglyceride disodium salt pentahydrate is 0.02-0.04 mol/L, and the concentration of the sodium hydroxide is 0.2-0.4 mol/L; cleaning and drying the product for later use;
and step 3: activation of saturated fatty acids: dissolving saturated fatty acid, N-hydroxysuccinimide and excessive dicyclohexylcarbodiimide in an anhydrous dimethyl sulfoxide solution, wherein the concentration of the saturated fatty acid in the mixed solution is 0.05-0.1 mol/L, the concentration of the N-hydroxysuccinimide is 0.05-0.1 mol/L, and the concentration of the dicyclohexylcarbodiimide is 0.075-0.2 mol/L. Stirring at normal temperature for 12h to obtain a solution containing saturated fatty acid active ester.
And 4, step 4: preparation of amphiphilic high molecular polymer: dissolving gelatin in deionized water to obtain a gelatin aqueous solution with the concentration of 10-20 g/L, heating to dissolve at 50-65 ℃, cooling to room temperature, adding dimethyl sulfoxide, fully stirring to react to obtain a gelatin solution, adding the dimethyl sulfoxide with the volume 0.2-0.4 times that of the gelatin aqueous solution, dropwise adding the solution containing the saturated fatty acid active ester obtained in the step (3) into the gelatin solution, wherein the volume ratio of the gelatin solution to the solution containing the saturated fatty acid active ester is 5: 1-10: 1, adjusting the pH to 8-8.5 by using sodium hydroxide or hydrochloric acid, and reacting for 12-24 h. The obtained solution is centrifuged to remove precipitates, supernatant is taken out and dialyzed for 3 days, and freeze drying is carried out for standby application, so as to obtain the gelatin-g-saturated fatty acid copolymer.
And 5: preparation of electrolyte loaded with anti-inflammatory factor S1P (sphingosine 1-phosphate): dissolving the anti-inflammatory factor S1P and the gelatin-g-saturated fatty acid copolymer in glacial acetic acid, volatilizing a solvent to obtain a blend of the anti-inflammatory factor S1P and the gelatin-g-saturated fatty acid copolymer at a molecular level, then dissolving the blend in deionized water, and encapsulating the anti-inflammatory factor S1P to a hydrophobic core of a micelle in a self-assembly process of the gelatin-g-saturated fatty acid copolymer, so as to obtain a micelle solution carrying the anti-inflammatory factor S1P, wherein the concentration of the micelle solution carrying the anti-inflammatory factor S1P is 10-20 mg/mL. Preparing chitosan hydrochloride aqueous solution with the concentration of 10-20 mg/mL. Mixing the micelle solution carrying the anti-inflammatory factor S1P with the chitosan hydrochloride aqueous solution according to the volume ratio of 2: 1-1: 2.
Step 6: the method comprises the steps of simultaneously dissolving a proinflammatory factor LPS (lipopolysaccharide) and a gelatin-g-saturated fatty acid copolymer in deionized water, and self-assembling the proinflammatory factor LPS and the gelatin-g-saturated fatty acid copolymer into micelles together based on hydrophilic and hydrophobic effects and electrostatic effects to obtain a micellar solution carrying the proinflammatory factor LPS, wherein the concentration of the micellar solution carrying the proinflammatory factor LPS is 10-20 mg/mL, and the chitosan hydrochloride aqueous solution with the concentration of 10-20 mg/mL is prepared. Mixing the micelle solution loaded with the proinflammatory factor LPS with the chitosan hydrochloride aqueous solution according to the volume ratio of 2: 1-1: 2.
And 7: and (3) sequentially depositing the micelles loaded with the anti-inflammatory factor S1P (inner layer) and the micelles loaded with the pro-inflammatory factor LPS (outer layer) on the surface of the hydroxyapatite nanorod-structured coating prepared in the step 2 by using a constant voltage electrodeposition process and taking the mixed solution in the steps 5 and 6 as electrolyte, wherein the deposition voltage is 2V-8V, and the deposition time is 5S-10 min.
The invention also discloses application of the macromolecular micelle coating with the nanorod arrayed surface sequentially delivering the immune regulatory factor as an implant coating material.
The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict.
The following detailed description is illustrative of the embodiments and is intended to provide further details of the invention. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention.
Example 1
A method for preparing a nano-rod arrayed biomedical material with a high-molecular micelle coating for sequentially delivering an immune regulatory factor on the surface comprises the following steps:
step 1, micro-arc oxidation of titanium and titanium alloy:
the micro-arc oxidation parameters are set as follows: the frequency of the micro-arc oxidation arc is 110Hz, the power supply is positive voltage 385V, and the duty ratio is 20%. In the micro-arc oxidation process, the titanium alloy is used as an anode, the stainless steel electrolytic tank is used as a cathode, and the components and the concentration of the electrolyte are as follows: 0.015mol/L sodium hydroxide (NaOH), calcium acetate (Ca (CH)3COO)2)0.4mol/L, 0.04mol/L of beta-phosphoglyceride disodium salt pentahydrate (beta-GP). In the preparation process, a cooling system is adopted to control the temperature of the micro-arc oxidation electrolyte at 300K. And (3) obtaining a sample coated with the microporous titanium dioxide coating after micro-arc oxidation, and putting the prepared sample into a drying oven for later use after the sample is cleaned by alcohol and deionized water.
Step 2, adding porous TiO containing phosphorus and calcium2Carrying out hydrothermal treatment on the coating to obtain the HA nanorod structured coating:
step 2.1, primary hydrothermal treatment
Firstly, preparing a solution required by primary hydrothermal, wherein the solution is a sodium hydroxide aqueous solution with the concentration of 0.03mol/L, putting a titanium sheet coated with a titanium dioxide coating after micro-arc oxidation into reaction kettles, then adding 15mL of the sodium hydroxide aqueous solution into each reaction kettle, screwing down the reaction kettles, putting the reaction kettles into a drying oven, and finally setting temperature and time parameters. The temperature was adjusted to 380K and the time was set to 2.5 h.
Step 2.2, Secondary hydrothermal treatment
Adding calcium sodium ethylene diamine tetraacetate, a beta-phosphoglyceride disodium salt pentahydrate and sodium hydroxide into water, uniformly stirring, and sealing the product obtained in the step 2.1 for secondary hydrothermal treatment, wherein the concentration of the calcium sodium ethylene diamine tetraacetate is 0.15mol/L, the concentration of the beta-phosphoglyceride disodium salt pentahydrate is 0.03mol/L, and the concentration of the sodium hydroxide is 0.3 mol/L; the secondary hydrothermal temperature was set to 400K and the time was set to 23 h. And (3) obtaining a titanium sheet coated with the HA nanorod-structured coating after two times of hydrothermal treatment, taking the sample out of the reaction kettle, washing the sample with deionized water, and putting the sample into a drying oven for later use.
Step 3, activation of saturated fatty acid
Dissolving saturated fatty acid, N-hydroxysuccinimide and dicyclohexylcarbodiimide in an anhydrous dimethyl sulfoxide solution together, wherein the concentration of the saturated fatty acid is 0.05mol/L, the concentration of the N-hydroxysuccinimide is 0.05mol/L, the concentration of the dicyclohexylcarbodiimide is 0.075mol/L, and stirring for 12h at normal temperature.
Dissolving gelatin in deionized water to obtain 10g/L gelatin solution, heating at 50 deg.C for dissolving, cooling to room temperature, adding 0.2 times volume of dimethyl sulfoxide solution, and stirring. And (3) dropwise adding the solution containing the saturated fatty acid active ester obtained in the step (3) into a gelatin solution, wherein the volume ratio of the gelatin solution to the solution containing the saturated fatty acid active ester is 5:1, adjusting the pH value to 8 by using sodium hydroxide or hydrochloric acid, and reacting for 24 hours. The obtained solution is centrifuged to remove the precipitate, the supernatant is dialyzed for 3 days, and the supernatant is frozen and dried for later use.
Step 5, preparation of anti-inflammatory factor S1P loaded electrolyte
S1P and gelatin-g-saturated fatty acid copolymer are dissolved in glacial acetic acid, and the solvent is volatilized to obtain a blend of the two at a molecular level. And dissolving the blend in deionized water to prepare a solution with the drug-loaded micelle concentration of 10 mg/mL. Chitosan hydrochloride is dissolved in water to prepare a chitosan hydrochloride solution with the concentration of 10 mg/mL. The two solutions were mixed at a 1:1 volume ratio for use.
Step 6, preparation of LPS electrolyte loaded with anti-inflammatory factors
LPS and gelatin-g-saturated fatty acid are simultaneously dissolved in deionized water to prepare a solution with the drug-loaded micelle concentration of 15 mg/mL. Chitosan hydrochloride is dissolved in water to prepare a chitosan hydrochloride solution with the concentration of 15 mg/mL. The two solutions were mixed at a 1:1 volume ratio for use.
Step 7, high-molecular micelle coating for sequential delivery of immune regulatory factors on nanorod arrayed surface
Taking a pure titanium or titanium alloy matrix as a cathode and a platinum sheet as an anode, firstly taking the mixed solution prepared in the step 5 as an electrolyte, depositing for 5s at a constant voltage of 8V, taking a sample out of the electrolyte, washing the sample clean by deionized water, continuously taking the taken sample as the cathode and the platinum sheet as the anode, continuously depositing for 30s in a constant voltage mode of 3V by using the mixed solution prepared in the step 6, taking the sample out of the electrolyte, washing the sample clean by deionized water and drying to obtain the high-molecular micelle coating with the titanium-based surface capable of sequentially delivering the immune regulatory factors.
Example 2
A method for preparing a nano-rod arrayed biomedical material with a high-molecular micelle coating for sequentially delivering an immune regulatory factor on the surface comprises the following steps:
step 1, micro-arc oxidation of titanium and titanium alloy:
the micro-arc oxidation parameters are set as follows: the frequency of the micro-arc oxidation arc is 100Hz, the power supply is positive pressure 350V, and the duty ratio is 15%. In the micro-arc oxidation process, pure titanium is used as an anode, a stainless steel electrolytic tank is used as a cathode, and the components and the concentration of the electrolyte are as follows: 0.02mol/L of sodium hydroxide (NaOH), calcium acetate (Ca (CH)3COO)2)0.3mol/L, 0.05mol/L of beta-phosphoglyceride disodium salt pentahydrate (beta-GP). In the preparation process, a cooling system is adopted to control the temperature of the micro-arc oxidation electrolyte to be 295K. And (3) obtaining a sample coated with the microporous titanium dioxide coating after micro-arc oxidation, and putting the prepared sample into a drying oven for later use after the sample is cleaned by alcohol and deionized water.
Step 2, adding porous TiO containing phosphorus and calcium2Carrying out hydrothermal treatment on the coating to obtain the HA nanorod structured coating:
step 2.1, primary hydrothermal treatment
Firstly, preparing a solution required by primary hydrothermal, wherein the solution is a sodium hydroxide aqueous solution with the concentration of 0.02mol/L, putting a titanium sheet coated with a titanium dioxide coating after micro-arc oxidation into reaction kettles, then adding 20mL of the sodium hydroxide aqueous solution into each reaction kettle, screwing down the reaction kettles, putting the reaction kettles into a drying oven, and finally setting temperature and time parameters. The temperature was adjusted to 365K and the time was set to 3 h.
Step 2.2, Secondary hydrothermal treatment
Adding calcium sodium ethylene diamine tetraacetate, a beta-phosphoglyceride disodium salt pentahydrate and sodium hydroxide into water, uniformly stirring, and sealing the product obtained in the step 2.1 for secondary hydrothermal treatment, wherein the concentration of the calcium sodium ethylene diamine tetraacetate is 0.1mol/L, the concentration of the beta-phosphoglyceride disodium salt pentahydrate is 0.02mol/L, and the concentration of the sodium hydroxide is 0.4 mol/L; the secondary hydrothermal temperature was set to 415K and the time was set to 24 h. And (3) obtaining a titanium sheet coated with the HA nanorod-structured coating after two times of hydrothermal treatment, taking the sample out of the reaction kettle, washing the sample with deionized water, and putting the sample into a drying oven for later use.
Step 3, activation of saturated fatty acid
Dissolving saturated fatty acid, N-hydroxysuccinimide and dicyclohexylcarbodiimide in anhydrous dimethyl sulfoxide solution together, wherein the concentration of the saturated fatty acid is 0.1mol/L, the concentration of the N-hydroxysuccinimide is 0.1mol/L, the concentration of the dicyclohexylcarbodiimide is 0.2mol/L, and stirring at normal temperature for 12 h.
Dissolving gelatin in deionized water to prepare 15g/L gelatin solution, heating to dissolve at 60 deg.C, cooling to room temperature, adding 0.3 times volume of dimethyl sulfoxide solution, and stirring. And (3) dropwise adding the solution containing the saturated fatty acid active ester obtained in the step (3) into a gelatin solution, wherein the volume ratio of the gelatin solution to the solution containing the saturated fatty acid active ester is 7:1, adjusting the pH value to 8.2 by using sodium hydroxide or hydrochloric acid, and reacting for 12 hours. The obtained solution is centrifuged to remove the precipitate, the supernatant is dialyzed for 2 days, and the supernatant is frozen and dried for standby.
Step 5, preparation of anti-inflammatory factor S1P loaded electrolyte
S1P and gelatin-g-saturated fatty acid copolymer are dissolved in glacial acetic acid, and the solvent is volatilized to obtain a blend of the two at a molecular level. And dissolving the blend in deionized water to prepare a solution with the drug-loaded micelle concentration of 20 mg/mL. Chitosan hydrochloride is dissolved in water to prepare chitosan hydrochloride solution with the concentration of 20 mg/mL. The two solutions were mixed at a volume ratio of 1:2 for use.
Step 6, preparation of LPS electrolyte loaded with anti-inflammatory factors
LPS and gelatin-g-saturated fatty acid are simultaneously dissolved in deionized water to prepare a solution with the drug-loaded micelle concentration of 15 mg/mL. Chitosan hydrochloride is dissolved in water to prepare chitosan hydrochloride solution with the concentration of 20 mg/mL. The two solutions were mixed at a 2:1 volume ratio for use.
Step 7, high-molecular micelle coating for sequential delivery of immune regulatory factors on nanorod arrayed surface
Taking a pure titanium or titanium alloy matrix as a cathode and a platinum sheet as an anode, firstly taking the mixed solution prepared in the step 5 as an electrolyte, depositing for 10s at a constant voltage of 4V, taking a sample out of the electrolyte, washing the sample clean by deionized water, continuously taking the taken sample as the cathode and the platinum sheet as the anode, continuously depositing for 10min in a constant voltage mode of 2V by using the mixed solution prepared in the step 6, taking the sample out of the electrolyte, washing the sample clean by deionized water and drying to obtain the high-molecular micelle coating with the titanium-based surface capable of sequentially delivering the immune regulatory factors.
Example 3
A method for preparing a nano-rod arrayed biomedical material with a high-molecular micelle coating for sequentially delivering an immune regulatory factor on the surface comprises the following steps:
step 1, micro-arc oxidation of titanium and titanium alloy:
the micro-arc oxidation parameters are set as follows: the frequency of the micro-arc oxidation arc is 120Hz, the power supply is positive voltage of 450V, and the duty ratio is 25%. In the micro-arc oxidation process, pure titanium is used as an anode, a stainless steel electrolytic tank is used as a cathode, and the components and the concentration of the electrolyte are as follows: 0.01mol/L of sodium hydroxide (NaOH), calcium acetate (Ca (CH)3COO)2)0.5mol/L, 0.03mol/L of beta-phosphoglyceride disodium salt pentahydrate (beta-GP). In the preparation process, a cooling system is adopted to control the temperature of the micro-arc oxidation electrolyte to be 305K. Micro-arc oxidation to obtain coated micro-particlesAnd cleaning the prepared sample with alcohol and deionized water, and putting the sample into a drying oven for later use.
Step 2, adding porous TiO containing phosphorus and calcium2Carrying out hydrothermal treatment on the coating to obtain the HA nanorod structured coating:
step 2.1, primary hydrothermal treatment
Firstly, preparing a solution required by primary hydrothermal, wherein the solution is a sodium hydroxide aqueous solution with the concentration of 0.04mol/L, putting a titanium sheet coated with a titanium dioxide coating after micro-arc oxidation into reaction kettles, then adding 15mL of the sodium hydroxide aqueous solution into each reaction kettle, screwing down the reaction kettles, putting the reaction kettles into a drying oven, and finally setting temperature and time parameters. The temperature was adjusted to 395K and the time was set to 2 h.
Step 2.2, Secondary hydrothermal treatment
Adding calcium sodium ethylene diamine tetraacetate, a beta-phosphoglyceride disodium salt pentahydrate and sodium hydroxide into water, uniformly stirring, and sealing the product obtained in the step 2.1 for secondary hydrothermal treatment, wherein the concentration of the calcium sodium ethylene diamine tetraacetate is 0.2mol/L, the concentration of the beta-phosphoglyceride disodium salt pentahydrate is 0.04mol/L, and the concentration of the sodium hydroxide is 0.3 mol/L; the secondary hydrothermal temperature was set at 405K and the time was set at 22 h. And (3) obtaining a titanium sheet coated with the HA nanorod-structured coating after two times of hydrothermal treatment, taking the sample out of the reaction kettle, washing the sample with deionized water, and putting the sample into a drying oven for later use.
Step 3, activation of saturated fatty acid
Dissolving saturated fatty acid, N-hydroxysuccinimide and dicyclohexylcarbodiimide in an anhydrous dimethyl sulfoxide solution together, wherein the concentration of the saturated fatty acid is 0.05mol/L, the concentration of the N-hydroxysuccinimide is 0.075mol/L, the concentration of the dicyclohexylcarbodiimide is 0.1mol/L, and stirring for 12h at normal temperature.
Dissolving gelatin in deionized water to obtain 20g/L gelatin solution, heating at 65 deg.C for dissolving, cooling to room temperature, adding 0.4 times volume of dimethyl sulfoxide solution, and stirring. And (3) dropwise adding the solution containing the saturated fatty acid active ester obtained in the step (3) into a gelatin solution, wherein the volume ratio of the gelatin solution to the solution containing the saturated fatty acid active ester is 10:1, adjusting the pH value to 8.5 by using sodium hydroxide or hydrochloric acid, and reacting for 20 hours. The obtained solution is centrifuged to remove the precipitate, the supernatant is dialyzed for 1 day, and the supernatant is frozen and dried for later use.
Step 5, preparation of anti-inflammatory factor S1P loaded electrolyte
S1P and gelatin-g-saturated fatty acid copolymer are dissolved in glacial acetic acid, and the solvent is volatilized to obtain a blend of the two at a molecular level. And dissolving the blend in deionized water to prepare a solution with the drug-loaded micelle concentration of 15 mg/mL. Chitosan hydrochloride is dissolved in water to prepare a chitosan hydrochloride solution with the concentration of 15 mg/mL. The two solutions were mixed at a 1:1 volume ratio for use.
Step 6, preparation of LPS electrolyte loaded with anti-inflammatory factors
LPS and gelatin-g-saturated fatty acid are simultaneously dissolved in deionized water to prepare a solution with the drug-loaded micelle concentration of 15 mg/mL. Chitosan hydrochloride is dissolved in water to prepare a chitosan hydrochloride solution with the concentration of 10 mg/mL. The two solutions were mixed at a volume ratio of 1:2 for use.
Step 7, high-molecular micelle coating for sequential delivery of immune regulatory factors on nanorod arrayed surface
Taking a pure titanium or titanium alloy matrix as a cathode and a platinum sheet as an anode, firstly taking the mixed solution prepared in the step 5 as an electrolyte, depositing for 60s at a constant voltage of 3V, taking a sample out of the electrolyte, washing the sample cleanly by using deionized water, continuously taking the taken sample as the cathode and the platinum sheet as the anode, continuously depositing for 60s in a constant voltage mode of 3V by using the mixed solution prepared in the step 6, taking the sample out of the electrolyte, washing the sample cleanly by using the deionized water, and drying to obtain the high-molecular micelle coating with the titanium-based surface capable of sequentially delivering the immune regulatory factors.
Example 4
A method for preparing a nano-rod arrayed biomedical material with a high-molecular micelle coating for sequentially delivering an immune regulatory factor on the surface comprises the following steps:
step 1, micro-arc oxidation of titanium and titanium alloy:
the micro-arc oxidation parameters are set as follows: the frequency of the micro-arc oxidation arc is 110Hz, the power supply is positive voltage of 380V, and the duty ratio is 20%. In the micro-arc oxidation process, pure titanium is used as an anode, a stainless steel electrolytic tank is used as a cathode, and the components and the concentration of the electrolyte are as follows: 0.02mol/L of sodium hydroxide (NaOH), calcium acetate (Ca (CH)3COO)2)0.4mol/L, 0.05mol/L of beta-phosphoglyceride disodium salt pentahydrate (beta-GP). In the preparation process, a cooling system is adopted to control the temperature of the micro-arc oxidation electrolyte at 300K. And (3) obtaining a sample coated with the microporous titanium dioxide coating after micro-arc oxidation, and putting the prepared sample into a drying oven for later use after the sample is cleaned by alcohol and deionized water.
Step 2, adding porous TiO containing phosphorus and calcium2Carrying out hydrothermal treatment on the coating to obtain the HA nanorod structured coating:
step 2.1, primary hydrothermal treatment
Firstly, preparing a solution required by primary hydrothermal, wherein the solution is a sodium hydroxide aqueous solution with the concentration of 0.02mol/L, putting a titanium sheet coated with a titanium dioxide coating after micro-arc oxidation into reaction kettles, then adding 17mL of the sodium hydroxide aqueous solution into each reaction kettle, screwing down the reaction kettles, putting the reaction kettles into a drying oven, and finally setting temperature and time parameters. The temperature was adjusted to 380K and the time was set to 2 h.
Step 2.2, Secondary hydrothermal treatment
Adding calcium sodium ethylene diamine tetraacetate, a beta-phosphoglyceride disodium salt pentahydrate and sodium hydroxide into water, uniformly stirring, and sealing the product obtained in the step 2.1 for secondary hydrothermal treatment, wherein the concentration of the calcium sodium ethylene diamine tetraacetate is 0.15mol/L, the concentration of the beta-phosphoglyceride disodium salt pentahydrate is 0.02mol/L, and the concentration of the sodium hydroxide is 0.4 mol/L; the secondary hydrothermal temperature was set to 415K and the time was set to 24 h. And (3) obtaining a titanium sheet coated with the HA nanorod-structured coating after two times of hydrothermal treatment, taking the sample out of the reaction kettle, washing the sample with deionized water, and putting the sample into a drying oven for later use.
Step 3, activation of saturated fatty acid
Dissolving saturated fatty acid, N-hydroxysuccinimide and dicyclohexylcarbodiimide in an anhydrous dimethyl sulfoxide solution together, wherein the concentration of the saturated fatty acid is 0.06mol/L, the concentration of the N-hydroxysuccinimide is 0.1mol/L, the concentration of the dicyclohexylcarbodiimide is 0.2mol/L, and stirring for 12h at normal temperature.
Dissolving gelatin in deionized water to obtain 10g/L gelatin solution, heating at 65 deg.C for dissolving, cooling to room temperature, adding 0.2 times volume of dimethyl sulfoxide solution, and stirring. And (3) dropwise adding the solution containing the saturated fatty acid active ester obtained in the step (3) into a gelatin solution, wherein the volume ratio of the gelatin solution to the solution containing the saturated fatty acid active ester is 8:1, adjusting the pH value to 8.2 by using sodium hydroxide or hydrochloric acid, and reacting for 24 hours. The obtained solution is centrifuged to remove the precipitate, the supernatant is dialyzed for 3 days, and the supernatant is frozen and dried for later use.
Step 5, preparation of anti-inflammatory factor S1P loaded electrolyte
S1P and gelatin-g-saturated fatty acid copolymer are dissolved in glacial acetic acid, and the solvent is volatilized to obtain a blend of the two at a molecular level. And dissolving the blend in deionized water to prepare a solution with the drug-loaded micelle concentration of 10 mg/mL. Chitosan hydrochloride is dissolved in water to prepare a chitosan hydrochloride solution with the concentration of 10 mg/mL. The two solutions were mixed at a volume ratio of 1:2 for use.
Step 6, preparation of LPS electrolyte loaded with anti-inflammatory factors
LPS and gelatin-g-saturated fatty acid are simultaneously dissolved in deionized water to prepare a solution with the drug-loaded micelle concentration of 10 mg/mL. Chitosan hydrochloride is dissolved in water to prepare a chitosan hydrochloride solution with the concentration of 15 mg/mL. The two solutions were mixed at a 2:1 volume ratio for use.
Step 7, high-molecular micelle coating for sequential delivery of immune regulatory factors on nanorod arrayed surface
Taking a pure titanium or titanium alloy matrix as a cathode and a platinum sheet as an anode, firstly taking the mixed solution prepared in the step 5 as an electrolyte, depositing for 10s at a constant voltage of 8V, taking a sample out of the electrolyte, washing the sample clean by deionized water, continuously taking the taken sample as the cathode and the platinum sheet as the anode, continuously depositing for 60s in a constant voltage mode of 2V by using the mixed solution prepared in the step 6, taking the sample out of the electrolyte, washing the sample clean by deionized water and drying to obtain the high-molecular micelle coating with the titanium-based surface capable of sequentially delivering the immune regulatory factors.
The titanium alloy sample of the macromolecular micelle coating capable of sequentially delivering the immune regulatory factors comprises TiO which are sequentially arranged from the surface of a substrate to the outside2A coating, an HA nanorod coating and a drug-loaded polymer coating. Referring to fig. 1, the scanning pictures of the hydroxyapatite nanorod coating obtained after micro-arc oxidation and hydrothermal treatment in examples 1 and 2 show that the nanorods are in a regular hexagonal prism shape. Referring to fig. 2, which is a scanning picture of the cross section of the hydroxyapatite nanorod coating obtained after micro-arc oxidation and hydrothermal treatment in examples 1 and 2, we can see that the surface of the nanorod is smooth and has no coverage of a polymer film. As shown in fig. 3, under the preparation conditions of example 2, after two layers of the drug-loading factor coating are deposited, it can be seen that the gaps of the nanorods are filled with micelles. As shown in fig. 4, under the preparation conditions of example 3, after two drug-loaded factor coatings were deposited, a thin polymer film was observed on the nanorod array. As shown in fig. 5, in the correlation fourier infrared characterization chart, after the drug-loading factor coating is deposited, peaks of amide bond and carbon-hydrogen bond of the polymer coating can be observed except that hydroxyapatite related characteristic peaks include phosphate and hydroxyl peak. The combination of the surface related SEM pictures can show that the titanium alloy implant with the drug-loaded coating deposited on the nanorod array is successfully prepared.
The examples are illustrative and not shown, and in summary, within the scope of the present invention, micro-arc oxidation and hydrothermal treatment are used to prepare drug-loaded micelles and the drug-loaded micelles are deposited on the surface of titanium and alloy by electrodeposition, and a bilayer structure coating can be obtained on the surface of titanium and alloy: the inner layer is a nano rod-shaped hydroxyapatite configuration coating which is formed on an oxide film containing calcium and phosphorus elements growing on the titanium surface in situ, the components of the configuration coating are closer to the components of human skeleton, and the configuration coating can improve the biocompatibility of titanium and titanium alloy implants and promote bone conduction to a certain extent; the surface layer is a macromolecule coating loaded with inflammatory factors, and can imitate a normal healing process to sequentially deliver proinflammatory factors and inflammation-inhibiting factors so as to regulate and control a macrophage-mediated osseointegration process and obviously improve the osseointegration performance of the titanium implant.
The embodiments described above are merely preferred embodiments of the present invention, and should not be considered as limitations of the present invention, and features in the embodiments and examples in the present application may be arbitrarily combined with each other without conflict. The protection scope of the present invention is defined by the claims, and includes equivalents of technical features of the claims. I.e., equivalent alterations and modifications within the scope hereof, are also intended to be within the scope of the invention.
Claims (10)
1. A method for preparing a macromolecular micelle coating for sequentially delivering an immunomodulatory factor on a nanorod-arrayed surface is characterized by comprising the following steps of:
step 1: micro-arc oxidation is carried out on a pure titanium or titanium alloy matrix in electrolyte containing phosphorus ions and calcium ions, so that a microporous titanium dioxide coating is formed on the surface of the matrix;
step 2: preparing a hydroxyapatite nanorod-structured coating on the microporous titanium dioxide coating obtained in the step 1 by adopting a hydrothermal treatment method;
and step 3: dissolving saturated fatty acid, N-hydroxysuccinimide as a catalyst and excessive dicyclohexylcarbodiimide in an anhydrous dimethyl sulfoxide solution to obtain a mixed solution, wherein the concentration of the saturated fatty acid in the mixed solution is 0.05-0.1 mol/L, the concentration of the N-hydroxysuccinimide in the mixed solution is 0.05-0.1 mol/L, and the concentration of the dicyclohexylcarbodiimide in the mixed solution is 0.075-0.2 mol/L, and then stirring at normal temperature for 12 hours to obtain a solution containing saturated fatty acid active ester;
and 4, step 4: dissolving gelatin in deionized water, heating to 50-65 ℃ for dissolving, cooling to room temperature to obtain a gelatin aqueous solution with the concentration of 10-20 g/L, adding a dimethyl sulfoxide solution, fully stirring for reaction to obtain a gelatin solution, adding 0.2-0.4 time of the volume of the dimethyl sulfoxide to the gelatin aqueous solution, dropwise adding the solution containing the saturated fatty acid active ester obtained in the step (3) into the gelatin solution, wherein the volume ratio of the gelatin solution to the solution containing the saturated fatty acid active ester is 5: 1-10: 1, adjusting the pH to 8-8.5 by using sodium hydroxide or hydrochloric acid, reacting for 12-24 h, centrifuging the obtained solution to remove precipitates, collecting supernatant, dialyzing for 3 days, and freeze-drying for later use to obtain a gelatin-g-saturated fatty acid copolymer;
and 5: loading the gelatin-g-saturated fatty acid copolymer prepared in the step 4 with an anti-inflammatory factor S1P by a coprecipitation method, dissolving in water, and uniformly mixing with the chitosan hydrochloride aqueous solution for later use;
step 6: loading the proinflammatory factor LPS on the gelatin-g-saturated fatty acid copolymer prepared in the step 4 in a self-assembly mode, dissolving the copolymer in water, and uniformly mixing the copolymer with the chitosan hydrochloride aqueous solution for later use;
and 7: and (3) taking the mixed solution prepared in the steps (5) and (6) as an electrolyte, and sequentially depositing the mixed solution on the hydroxyapatite nanorod-structured coating prepared in the step (2) in an electrodeposition mode to form a nanorod-arrayed polymer micelle coating with the surface sequentially delivering the immune regulatory factors.
2. The method for preparing the polymeric micelle coating with the nanorod-arrayed surface for sequential delivery of the immunomodulatory factors according to claim 1, wherein the step 1 specifically comprises:
taking a pure titanium or titanium alloy matrix as an anode and a stainless steel electrolytic tank as a cathode, performing micro-arc oxidation at the temperature of 295-305K, washing with deionized water, and drying to form a microporous titanium dioxide coating on the surface of the pure titanium or titanium alloy matrix; the parameters of the micro-arc oxidation are as follows: the arc frequency is 100-120 Hz, the positive voltage is 350-450V, and the duty ratio is 15-25%; the concentration of sodium hydroxide in the electrolyte is 0.01-0.02 mol/L, the concentration of calcium acetate is 0.3-0.5 mol/L, and the concentration of beta-phosphoglyceride disodium salt pentahydrate is 0.03-0.05 mol/L.
3. The method for preparing the polymer micelle coating with the nanorod-arrayed surface for sequential delivery of the immunomodulatory factors according to claim 1, wherein the step 2 specifically comprises:
step 2.1: putting the substrate of the microporous titanium dioxide coating obtained in the step 1 into a sodium hydroxide aqueous solution with the concentration of 0.02-0.04 mol/L, and sealing for primary hydrothermal treatment;
step 2.2: removing liquid in the product obtained in the step 2.1, adding uniformly stirred aqueous solutions of calcium sodium ethylenediaminetetraacetate, beta-glycerol phosphate disodium salt pentahydrate and sodium hydroxide, and sealing for secondary hydrothermal treatment, wherein the concentration of the calcium sodium ethylenediaminetetraacetate in the uniformly stirred aqueous solutions of the calcium sodium ethylenediaminetetraacetate, the beta-glycerol phosphate disodium salt pentahydrate and the sodium hydroxide is 0.1-0.2 mol/L, the concentration of the beta-glycerol phosphate disodium salt pentahydrate is 0.02-0.04 mol/L, and the concentration of the sodium hydroxide is 0.2-0.4 mol/L; and after the secondary hydrothermal treatment is finished, cleaning and drying the product for later use.
4. The method for preparing the polymeric micelle coating with the nanorod-arrayed surface for sequential delivery of the immunomodulatory factors according to claim 3, wherein the temperature of the primary hydrothermal treatment is 365-395K, and the time is 2-3 h.
5. The method for preparing the polymer micelle coating with the nanorod-arrayed surface for sequential delivery of the immunomodulatory factors according to claim 3, wherein the temperature of the secondary hydrothermal treatment is 385-415K, and the time is 22-24 h.
6. The method for preparing the polymeric micelle coating with the nanorod-arrayed surface for sequential delivery of the immunomodulatory factors according to claim 1, wherein the step 5 specifically comprises: dissolving the anti-inflammatory factor S1P and the gelatin-g-saturated fatty acid copolymer in glacial acetic acid, volatilizing a solvent to obtain a blend of the anti-inflammatory factor S1P and the gelatin-g-saturated fatty acid copolymer at a molecular level, then dissolving the blend in deionized water, encapsulating the anti-inflammatory factor S1P to a hydrophobic core of a micelle in a dissolving self-assembly process of the gelatin-g-saturated fatty acid copolymer to obtain a micelle solution carrying the anti-inflammatory factor S1P, wherein the concentration of the micelle solution carrying the anti-inflammatory factor S1P is 10-20 mg/mL, in the step 5, the concentration of a chitosan hydrochloride aqueous solution is 10-20 mg/mL, and mixing the micelle solution carrying the anti-inflammatory factor S1P and the chitosan hydrochloride aqueous solution according to a volume ratio of 2: 1-1: 2.
7. The method for preparing the polymer micelle coating with the nanorod-arrayed surface for sequential delivery of the immunomodulatory factors according to claim 1, wherein the step 6 specifically comprises: the method comprises the steps of simultaneously dissolving the proinflammatory factor LPS and the gelatin-g-saturated fatty acid copolymer in deionized water, self-assembling the proinflammatory factor LPS and the gelatin-g-saturated fatty acid copolymer into micelles together based on hydrophilic and hydrophobic effects and electrostatic effects to obtain a micellar solution carrying the proinflammatory factor LPS, wherein the concentration of the micellar solution carrying the proinflammatory factor LPS is 10-20 mg/mL, the concentration of a chitosan hydrochloride aqueous solution in the step 6 is 10-20 mg/mL, and the micellar solution carrying the proinflammatory factor LPS is mixed with a chitosan hydrochloride aqueous solution according to the volume ratio of 2: 1-1: 2.
8. The method for preparing the polymer micelle coating with the nanorod-arrayed surface for sequential delivery of the immunomodulatory factors according to claim 1, wherein the step 7 specifically comprises: and (3) sequentially depositing the micelles loaded with the anti-inflammatory factor S1P and the micelles loaded with the pro-inflammatory factor LPS on the surface of the hydroxyapatite nanorod-structured coating by using a constant voltage electrodeposition process and taking the mixed solution in the steps 5 and 6 as an electrolyte, wherein the deposition voltage is 2V-8V, and the deposition time is 5S-10 min.
9. A high molecular micelle coating for sequentially delivering an immunomodulatory factor on a nanorod-arrayed surface, which is prepared by the preparation method of any one of claims 1-7.
10. The use of the nanorod-arrayed surface-sequential polymeric micelle coating of claim 9 for the delivery of immunomodulatory factors as an implant coating material.
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