CN113827772B - Biological antioxidant nano coating with redox response drug release function and preparation method and application thereof - Google Patents

Abstract

The invention discloses a biological anti-oxidation nano-coating with redox-responsive drug release function and a preparation method and application thereof. The coating comprises a nanotube drug-carrying system constructed on the surface of the substrate and in-situ polymerization on the surface of the nanotube to form a polydopamine modified by a redox-responsive functional group, and at least part of the orifice of the nanotube is fully or partially encapsulated Three-dimensional network structure of nanocomposite coatings.

Description

A kind of biological antioxidant nano-coating with redox response drug release function and preparation method and application thereof

technical field

The invention relates to a biological anti-oxidation nano-coating with good biological anti-oxidation function and redox-responsive drug release function, a preparation method and application thereof, and belongs to the technical field of biomedicine.

Background technique

With the acceleration of the aging process of the population and the increase of bone injury accidents caused by traffic accidents, diseases, natural disasters, etc., the demand for artificial implant materials is increasing day by day. The traditional solution to promote the repair of bone tissue around the implant is oral or intravenous injection of corresponding drugs, but this method will cause serious damage to the normal tissues and organs of the human body. Local administration can avoid the above-mentioned side effects while creating a better regenerative microenvironment for bone tissue repair.

The traditional drug release only combines the drug with the material in the form of simple van der Waals force or covalent bond. , causing severe damage to surrounding normal cells. Compared with patients with bone defects in normal physiological state, in patients with fractures or bone defects with systemic diseases (such as diabetes, hypertension, osteoporosis, etc.), reactive oxygen species (mainly including hydrogen peroxide molecules, superoxide radicals, etc.) The high level of ions and hydroxyl radicals) causes the body's oxidative capacity to exceed the antioxidant capacity, easily causing oxidative stress damage to the tissue around the bone implant material, inhibiting the activity of osteoblasts, and seriously affecting the repair of postoperative bone tissue. Therefore, combined with the characteristics of high levels of reactive oxygen species in patients and their special pathological microenvironment, the development of bone implant materials with redox-responsive drug release function and good biological antioxidant properties is important for promoting bone repair under oxidative stress. And improving bone quality has important clinical significance.

Due to the special structure of mussel-like adhesion proteins with catechol group and amino functional group, dopamine can undergo spontaneous oxidative polymerization-crosslinking reaction in aqueous solution, and can be formed on the surface of almost any solid material. The tightly attached composite layer has good biocompatibility, which is conducive to the adhesion and proliferation of osteoblasts on its surface. In addition, its surface is rich in _-NH2 groups, which can also carry out secondary reactions to introduce functional molecules into the material surface to achieve further functionalization of the material surface.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned defects in the prior art, the present invention provides a biological coating material with redox-responsive biological drug release function and good biological anti-oxidation performance, and a preparation method and application thereof.

In a first aspect, the present invention provides a biological antioxidant nanocoating with redox-responsive drug release function. The coating comprises a nanotube drug-carrying system constructed on the surface of the substrate and in-situ polymerization on the surface of the nanotube to form polydopamine modified by a redox-responsive functional group, so that at least part of the orifice of the nanotube is fully or partially encapsulated The three-dimensional network structure of nanocomposite coatings.

The redox-responsive functional group-modified polydopamine is a polydopamine modified with ferrocene formate and β-CD@CeO ₂ nanoparticles (composite nanoparticles composed of beta-cyclodextrin and CeO ₂ ). The "β-CD@CeO ₂ nanoparticles" can also be referred to as "β-CD functionalized nano CeO ₂ particles".

The biocoating can effectively avoid drug burst release, improve the intelligence and responsiveness of the substrate (also called "implant" or "implant material"), and at the same time endow the coating with good biological antioxidant properties . Compared with the direct loading of drugs on the surface of the material, in the case of a relatively high level of reactive oxygen species, the hydrophobic end of the ferrocene formate functional group on the polydopamine molecular chain is oxidized to a hydrophilic end, and the ferrocene functional group is positively charged. There is a large electrostatic repulsion between the chains, the three-dimensional network structure is relaxed, the encapsulated orifice is opened, and the coating can release the drug according to the surrounding oxidative stress level, effectively avoiding the burst release of the drug and avoiding the local drug around the material. Excessive concentrations can cause damage to normal tissues. In addition, the coating has good biocompatibility and biological antioxidant properties, and can effectively promote bone tissue repair in an oxidative stress environment. It is a potential biomedical material that can be used for hard tissue repair and replacement of biomaterials. and development.

In the above coatings, β-CD@CeO ₂ nanoparticles and ferrocene formate together constitute a redox-responsive switch. Nano _CeO particles in the coating can be used to eliminate excess reactive oxygen species. The hydrophobic end of ferrocene formate and the hydrophobic inner cavity of β-CD can be combined through host-guest recognition, and have a redox-responsive switch effect. When the level of active oxygen increases, the divalent ferrous ions on ferrocene formate are rapidly oxidized to positively charged ferric ions, and the hydrophobic end becomes the hydrophilic end. Due to the electrostatic repulsion between the molecular chains, The relaxation of the three-dimensional network structure is accompanied by the release of drugs (or _nanozymes ) and nano CeO2 particles in the tube, and the coating can release drugs (or nanozymes) that promote bone tissue repair according to the level of oxidative stress around the implant. , while effectively avoiding the burst release of drugs (or nano-enzymes), it has good biological antioxidant properties.

CeO ₂ nanoparticles in the present invention should be understood as cerium oxide nanoparticles. In some embodiments, the CeO ₂ nanoparticles may also be referred to as "CeO _2-x nanoparticles." The CeO ₂ nanoparticles (CeO _2-x nanoparticles) coexist with trivalent cerium ions and tetravalent cerium ions. The fitting analysis of the fine spectrum of Ce 3d orbital shows that the content of Ce ³⁺ is about 35%-39%, and the content of Ce ⁴⁺ is about 65% ^{-61 %} . The mass percent sum is 100%. When the content of Ce ⁴⁺ is large, it mainly exerts catalase mimetic activity, which can effectively decompose the excess H ₂ O ₂ around the implant, reduce the release rate of the drug, and achieve the purpose of sustained drug release.

Preferably, the substrate can be made of common medical metals or medical alloy materials including pure titanium, titanium alloy, stainless steel or cobalt-chromium-molybdenum alloy, and metal oxide materials such as titanium dioxide, zirconium oxide, aluminum oxide, and tantalum oxide.

Preferably, the length of the nanotube is 50-800 nm, the diameter is 20-150 nm, and the wall thickness is 5-20 nm. The larger the diameter and the longer the length of the nanotube, the greater the drug loading capacity.

Preferably, the nanotubes are metal or metal oxide nanotubes grown in situ on the surface of the substrate as the drug reservoir. The opening structure is perpendicular to the surface of the substrate, which is more conducive to the loading of the drug in the early stage and the responsive release of the drug in the later stage.

Preferably, the thickness of the nano-coating is 20-50 nm. The thickness of the nanocoating is related to the self-polymerization time of dopamine on the surface of the material. The longer the self-polymerization time is, the thicker the coating is. When the thickness of the nano-coating is lower than the limited thickness, the mouth of the nanotube cannot be completely encapsulated, and the drug burst is prone to occur. .

The nanotube drug-carrying system can be loaded with drugs or nanozymes. The drug may be a drug that promotes bone tissue repair. The drugs for promoting bone tissue repair include but are not limited to calcitonin, alendronate sodium, strontium ranelate and the like.

In a second aspect, the present invention provides a method for preparing the above-mentioned biological antioxidant nano-coating with redox-responsive drug release function. The preparation method comprises the following steps:

Step (1), forming a tubular nanostructure on the surface of the substrate by anodization;

In step (2), the nanotube-like structure obtained in step (1) is loaded with a drug or a nano-enzyme by a vacuum negative pressure method to obtain a substrate for constructing a nanotube drug-carrying system on the surface;

In step (3), the substrate on which the nanotube drug-carrying system is constructed on the surface is placed in a buffer solution containing a dopamine monomer, and a three-dimensional network structure nanocomposite coating is obtained through an oxidative polymerization reaction.

The preparation method has the advantages of low cost, simple operation, good repeatability, wide application range and the like.

The concentration of dopamine monomer in the above buffer solution is 1 mg/mL-3 mg/mL. Since free dopamine is unstable and easily oxidized, dopamine hydrochloride can be used as a dopamine monomer instead of free dopamine.

Preferably, the buffer solution further comprises ferrocene formate with a concentration of 0.5-1 mg/mL and β-CD@CeO ₂ nanoparticles with a concentration of 0.5-1 mg/mL.

In an optional embodiment, the -COOH functional group of ferrocene formate should be activated before use, for example, a Tris-HCl buffer solution prepared with EDC and NHS is used for activation for 12-24 hours.

Preferably, the voltage of the anodic oxidation is 10-30V, and the oxidation time is 15-60 minutes.

Preferably, the specific process of loading the drug or nanozyme in the nanotubular structure obtained in step (1) by the vacuum negative pressure method is as follows: immersing the substrate on which the surface of the tubular nanostructure is formed is immersed in the drug solution and vacuum drying. In an optional embodiment, the concentration of the drug solution is 15-30 mg/L.

Preferably, the dopamine monomer undergoes oxidative polymerization by ultraviolet irradiation.

The invention adopts the solution reaction method with simple operation and large-scale production, and in-situ oxidative polymerization on the surface of the substrate obtains a nano-biological coating with redox-responsive drug release, thereby obtaining a bone implant material with good biological anti-oxidation function. , will be expected to be applied to bone repair in various pathological microenvironments.

In a third aspect, the present invention provides the application of the above-mentioned anti-oxidative nano-biological coating with redox-responsive drug release function in the preparation of hard tissue repair and replacement biological materials.

Description of drawings

a in Figure 1 is Ti substrate, direct drug release nanocoating (TiO ₂ -Sr), polydopamine surface modified drug release nanocoating (TiO ₂ -Sr-PDA), redox responsive nanocoating XRD patterns of (TiO ₂ -PDA-CeO ₂ ) and redox-responsive drug release nanocoatings (TiO ₂ -Sr-PDA-CeO ₂ ); b in Fig. 1 is TiO ₂ -PDA-CeO ₂ and TiO ₂ -Enlarged image of XRD of Sr-PDA _- CeO coating.

a in Figure 2 is monomeric dopamine (DA), direct drug release nanocoating (TiO ₂ -Sr), modified drug release nanocoating with polydopamine (TiO ₂ -Sr-PDA), redox responsive _{Figure 2 _ _ _} b and c in it are the fine spectra of the nano-Ce 3d orbitals on the surface of two redox-responsive nanocoatings (TiO ₂ ^-PDA -CeO ₂ and TiO ₂ -Sr-PDA-CeO ₂ ), respectively, and the Orbital fitting analysis of Ce ⁴⁺ content.

Figure 3 shows direct drug release nanocoatings (TiO ₂ -Sr), polydopamine modified drug release coatings (TiO ₂ -Sr-PDA), and redox responsive nano coatings (TiO ₂ -PDA-CeO ₂ ) And the SEM image of the redox-responsive drug release nanocoating (TiO ₂ -Sr-PDA-CeO ₂ ).

Figure 4 shows the change curves of the concentration of strontium ions in the solution before and after stimulation by H ₂ O ₂ for three nano-drug-loaded coatings of TiO ₂ -Sr, TiO ₂ -Sr-PDA and TiO ₂ -Sr-PDA-CeO ₂ .

Figure 5 shows the direct drug release nanocoating (TiO ₂ -Sr), polydopamine modified drug release coating (TiO ₂ -Sr-PDA), redox responsive nano coating (TiO ₂ -PDA-CeO ₂ coating) Layer) and redox-responsive drug release nanocoatings (TiO ₂ -Sr-PDA-CeO ₂ coating) before and after oxidative stress stimulation, the change of the coating contact angle and the corresponding schematic diagram.

Figure 6 is the confocal fluorescence staining observation pictures of the reactive oxygen species in osteoblasts on the surface of different nano-coating materials under normal and oxidative stress states.

Detailed ways

The present invention is further described by the following embodiments, and it should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

In the present invention, a tubular nanostructure is constructed on the surface of the bone implant material and the drug is loaded; the monomer dopamine is modified with functionalized functional groups to make it have a special function of redox response; the functionalized dopamine monomer is implanted in the above-mentioned implant. The surface of the bulk material undergoes rapid oxidative polymerization and cross-links to form a nanocomposite coating with a three-dimensional network structure of polydopamine.

In the present invention, a three-dimensional network structure can be generated by spontaneous polymerization between dopamine monomers through catechol bond and -NH ₂ , and -NH ₂ on some dopamine monomers and carboxylic acid on ferrocene formate undergo an amidation reaction An amide bond is generated, and ferrocene formate is modified on the molecular chain of polydopamine. The ferrocene group in ferrocene formate is a hydrophobic end formed by a ferrous ion bonding two benzene rings, negatively charged, beta cyclodextrin (β-CD) is an inner hydrophobic, outer hydrophilic hole Therefore, ferrocene formate can be phase-recognized by the hydrophilic-hydrophobic interaction and beta-cyclodextrin-functionalized CeO ₂ nanoparticles, while the CeO ₂ nanoparticles are modified on the three-dimensional network structure. When the level of reactive oxygen species around the material increases, the ferrous iron on ferrocene formate is oxidized to ferric onium ions, which are positively charged, and the hydrophobic end becomes hydrophilic, beta cyclodextrin _- functionalized CeO nanoparticles It cannot be combined with ferrocene formate, the charged functional groups are exposed, there is a large electrostatic repulsion between the molecular chains, and the molecular chains are relaxed, which is more conducive to the response release of CeO ₂ nanoparticles.

Preferably, ferrocene formate and (beta cyclodextrin) β-CD modified nano-CeO ₂ particles are modified on dopamine monomers by amidation reaction, so that they have the function of redox response. For example, beta-cyclodextrin (β-CD) was modified _on the surface of CeO2 nanoparticles by precipitation method, so that it could recognize the functional group ferrocene formate on dopamine through host-guest interaction.

β-CD is a special hole-hole structure with a hydrophilic outer cavity and a hydrophobic inner cavity. The hydrophobic end of ferrocene formate and the hydrophobic inner cavity of β-CD can be combined through host-guest interaction recognition to form a redox response. function switch. When the level of reactive oxygen species increased, the divalent ferrous ions on ferrocene formate were rapidly oxidized to positively charged ferric ions, the hydrophobic end became hydrophilic, and the β-CD was stripped.

During the formation of nano-CeO ₂ particles, due to lattice distortion, oxygen atoms are prone to come out and form a large number of oxygen vacancies on the surface of the nano-particles, while the two tetravalent cerium ions around the oxygen atoms are respectively reduced to positive three ions by the remaining two electrons. Therefore, the cerium ions in the nano CeO ₂ particles exist in the form of positive trivalent (Ce ³⁺ ) and positive tetravalent (Ce ⁴⁺ ) at the same time. When the content of Ce ³⁺ is high, it mainly exerts superoxide dismutase-mimicking activity, and Ce ³⁺ is oxidized; while when the content of Ce ⁴⁺ is high, it mainly exerts catalase-mimicking activity, and Ce ⁴⁺ is reduced. The coexistence of cerium ions in mixed valence states (Ce ³⁺ /Ce ⁴⁺ ) in nano-CeO ₂ particles endows them with biological antioxidant properties, enabling them to catalyze the decomposition of excess reactive oxygen species in organisms, and to reduce the effects of various oxidative stress states. Cells, tissues and organs exhibit antioxidant and damage-resistant effects. In addition, the rare earth metal ion Ce ³⁺ also has a strong electrostatic coupling effect with the abundant -OH on the outer cavity of β-CD, and β-CD can be firmly modified on the surface of nano-CeO ₂ particles.

Therefore, the modification of ferrocene formate and β-CD functionalized nano _- CeO particles in the three-dimensional network structure of polydopamine at the same time as dopamine cross-linking and self-polymerization can endow it with the ability of redox response and eliminate excessive surrounding activities. Oxygen, provides antioxidant protection.

The following exemplifies the preparation method of the coating of the present invention. It can be prepared by anodizing method, vacuum negative pressure drug loading method, ultraviolet radiation oxidation method and the like in turn.

A nanotube-like structure is constructed on the surface of the substrate to obtain a substrate whose surface is covered with the nanotube-like structure. The nanotube-like structure can serve as a drug reservoir for the coating. By building nanostructures on the surface of the implant (also referred to as "substrate") to increase the specific surface area, the drug loading can be increased. For example, using titanium metal commonly used in medicine as the base material, metal platinum sheet as the cathode, titanium or titanium alloy as the anode, and a mixed solution composed of ammonium fluoride solution and glycerol as the electrolyte solution to carry out the anodic oxidation on the Ti base _TiO nanotube-like structures were prepared on the surface of the coated drug reservoirs. The voltage of the anodization is 10-30V, and the oxidation time is 15-60 minutes. The concentration of ammonium fluoride in the anodic oxidation electrolyte is 0.001-0.1 mol/L.

Drugs or nanozymes are loaded on substrates covered with nanotubular structures on the surface. For example, strontium ranelate is selected as a drug release model for promoting bone tissue repair, and alcohol is used as a solvent to prepare a solution with a concentration of 15-30 mg/L, and the anodized Ti substrate is immersed by vacuum negative pressure method. In the alcoholic solution of strontium ranelate, vacuum dried for 24h.

An activation solution including ferrocene formate, β-CD functionalized nano _- CeO particles, and dopamine hydrochloride was formulated.

Preparation of β-CD@CeO ₂ nanoparticles. Prepare 5mL of 0.5-1.5M cerium nitrate solution and 10mL of 0.5-1.5M β-CD aqueous solution. After mixing the above solutions uniformly, add dropwise to 30mL of ammonium hydroxide solution, and stir at 25°C for 24h. Centrifuge at 4000 rpm for 30 minutes, repeat twice, take the supernatant, carry out dialysis with 2000MCO dialysis membrane, and store at 4°C for later use.

The configuration of Tris-HCl buffer solution, a mixed solution of 50 mL of 0.1 mol/L tris (Tris) and 50 mL of 0.1 mol/L hydrochloric acid, adjusted the pH of the solution with 0.1 mol/L HCl and 0.1 mol/L NaOH Adjust to 7~9.2.

Use Tris-HCl buffer solution to prepare 15 mL of 10 mg/mL mixed solution of EDC and NHS respectively, add 10 mg of ferrocene formate, continue stirring, and activate carboxylic acid for 12-24 h to obtain an activated ferrocene formate solution.

Take 10 mL of activated ferrocene formate solution and 5 mL of β-CD@CeO ₂ solution, add 30 mg of dopamine hydrochloride, and adjust the pH of the solution to 8.5 with 0.1 mol/L NaOH.

The drug-loaded substrate is immersed in an activation solution, and a three-dimensional network structure nanocomposite coating is obtained by oxidative polymerization. The method of ultraviolet irradiation can make the dopamine monomer rapidly oxidatively polymerize on the surface of the substrate. For example, when the drug-loaded Ti substrate is immersed in the above solution, under the irradiation of an ultraviolet lamp, dopamine is rapidly oxidized and polymerized on the surface of the material. The power of the ultraviolet lamp is 48W, and the lamp source is about 5cm away from the liquid surface of the mixed solution. Light irradiation for 10 to 30 minutes.

The nano-coating of the invention has the function of releasing drugs or nano-enzymes in response to redox, and can release drugs that promote osteogenesis or nano-enzymes with biological anti-oxidation functions as the surrounding active oxygen content increases in a solution environment, reducing the activity Oxidative damage to osteoblasts by oxygen can improve the activity of osteoblasts and promote the regeneration of surrounding bone tissue. In some embodiments, the sustained release period of the drug responsive to oxidative stress can be as long as 27-30 days.

The following further examples are given to illustrate the present invention in detail. The present invention is further illustrated by the following specific examples below. It should be understood that the following examples are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the content of the present invention belong to the protection scope of the present invention. The specific process parameters and the like in the following embodiments are only an example in the suitable range, that is, those skilled in the art can make selections within the suitable range through the description herein, rather than being limited to the specific numerical values exemplified below.

Example

A. Preparation of direct drug release nanocoatings

First, the titanium sheet was cut into small pieces of 20mm×10mm×0.2mm, and the cut pieces were cleaned (ethanol-water-ethanol-deionized water, 15min each time); Platinum is used as cathode, medical titanium sheet is used as anode, the electrolyte solution is a mixed solution containing ammonium fluoride aqueous solution and glycerol, the mass fraction of ammonium fluoride is about 0.015%, and the volume ratio of glycerol to water is about 1: 1. Using constant voltage mode, anodized at 20V for 20min, cleaned the anodized titanium sheet with deionized water, and vacuum-dried for 24h. Using alcohol as a solvent to prepare a solution with a concentration of 20 mg/L, the anodized Ti substrate was immersed in an alcohol solution of strontium ranelate by vacuum negative pressure method, and the direct drug release was obtained after vacuum drying for 24 hours. type of drug-loaded coating, abbreviated as TiO ₂ -Sr.

It can be seen from Figure 1 that the TiO ₂ nanotubes on the surface of the titanium implant after anodization are in an amorphous state, therefore, only the XRD diffraction peaks of metallic titanium can be detected.

It can be seen from the FTIR diagram in Fig. 2 that there are very obvious absorption peaks in the range of 600-700 cm ^-1 , which are the mixed peaks of TiO ₂ -Sr surface and Ti-O-Ti, Ti-OH and metal Sr ⁺ respectively.

From the SEM image in Fig. 3, we can clearly see the nanotube-like structure of _TiO2 with regular openings upward, which can be used as a drug reservoir, which is beneficial to increase the loading of strontium ranelate.

B. Preparation of Polydopamine Modified Drug Release Nanocoatings

First, the titanium sheet was cut into small pieces of 20mm×10mm×0.2mm, and the cut pieces were cleaned (ethanol-water-ethanol-deionized water, 15min each time); Platinum is used as cathode, medical titanium sheet is used as anode, the electrolyte solution is a mixed solution containing ammonium fluoride aqueous solution and glycerol, the mass fraction of ammonium fluoride is about 0.015%, and the volume ratio of glycerol to water is about 1: 1. Using constant voltage mode, anodized at 20V for 20min, cleaned the anodized titanium sheet with deionized water, and vacuum-dried for 24h. Using alcohol as a solvent to prepare a solution with a concentration of 20 mg/L, the anodized Ti substrate was immersed in an alcohol solution of strontium ranelate by vacuum negative pressure method, and the direct drug release was obtained after vacuum drying for 24 hours. type of biocoating.

Next, a Tris-HCl buffer solution was prepared, and 50 mL of 0.1 mol/L tris (Tris) was mixed with 50 mL of 0.1 mol/L hydrochloric acid solution. The pH of the solution was adjusted to the range of 7-9.2 with 0.1 mol/L HCl and 0.1 mol/L NaOH. Take 15 mL of Tris-HCl buffer solution, add 30 mg of dopamine hydrochloride, adjust the pH of the solution to 8.5 with 0.1 mol/L NaOH, and immerse the drug-loaded coating in the above solution. Rapidly oxidize and polymerize on the surface of the material, the power of the ultraviolet lamp is 48W, the distance between the lamp source is about 5cm from the liquid surface of the mixed solution, and the ultraviolet light irradiation time is 20min, to obtain a nano drug-releasing coating modified by polydopamine, abbreviated as TiO ₂ - Sr-PDA.

It can be seen from Figure 2 that there is a very obvious absorption peak of catechol-OH functional group on the surface of TiO ₂ -Sr-PDA at 3437cm ^-1 , an absorption peak of -OH conjugated aromatic hydrocarbons at 1580cm ^-1 , and 600cm ^-1 position The absorption peak at is associated with the coupling of Sr ⁺ on strontium ranelate with polydopamine.

It can be clearly seen from Figure 3 that a very obvious organic film-like structure is formed on the surface of the TiO ₂ -Sr-PDA nanotubes, and the diameter of the nanotubes decreases, indicating that dopamine polymerizes to form a film on the surface of the material.

C. Preparation of redox-responsive nanocoatings

First, the titanium sheet was cut into small pieces of 20mm×10mm×0.2mm, and the cut pieces were cleaned (ethanol-water-ethanol-deionized water, 15min each time); Platinum is used as cathode, medical titanium sheet is used as anode, the electrolyte solution is a mixed solution containing ammonium fluoride aqueous solution and glycerol, the mass fraction of ammonium fluoride is about 0.015%, and the volume ratio of glycerol to water is about 1: 1. Using constant voltage mode, anodized at 20V for 20min, cleaned the anodized titanium sheet with deionized water, and vacuum-dried for 24h.

Prepare 5mL of 1.0M cerium nitrate solution and 10mL of 1.0M β-CD aqueous solution, mix the above solutions evenly, add dropwise to 30mL of ammonium hydroxide solution, and stir at 25°C for 24h. Centrifuge at 4000 rpm for 30 minutes, repeat twice, take the supernatant, and dialyze it with a 2000MCO dialysis membrane to obtain an aqueous solution of β-CD-modified CeO ₂ nanoparticles, which is stored at 4°C for later use.

Prepare Tris-HCl buffer solution, mix 50 mL of 0.1 mol/L tris (Tris) and 50 mL of 0.1 mol/L hydrochloric acid solution. The pH of the solution was adjusted to the range of 7-9.2 with 0.1 mol/L HCl and 0.1 mol/L NaOH. Prepare 15 mL of 10 mg/mL mixed solution of EDC and NHS respectively, add 10 mg of ferrocene formate, continue stirring, and activate the carboxylic acid for 12 h.

Take 10 mL of activated ferrocene formate solution, 5 mL of β-CD@CeO ₂ solution, add 30 mg of dopamine hydrochloride, adjust the pH of the solution to 8.5 with 0.1 mol/L NaOH, and immerse the anodized Ti sheet in the above solution Under the irradiation of ultraviolet lamp, dopamine rapidly oxidized and polymerized on the surface of the material. The power of the ultraviolet lamp was 48W, the distance between the lamp source and the liquid surface of the mixed solution was about 5cm, and the ultraviolet light was irradiated for 20min to obtain an intelligent oxidative stress responsive coating. , abbreviated as TiO ₂ -PDA-CeO ₂ .

From the analysis of XRD diffraction peaks in Figure 1, comparing with the CeO ₂ standard PDF card PDF#34-0394, we can see that the (111), (220) and (311) planes of the nano-CeO ₂ particles are very obvious on the coating surface. The crystal diffraction peaks are broadened to a certain extent by the nano-size effect of CeO ₂ .

In Figure 2, we can see that when dopamine polymerizes on the surface of the material to form polydopamine, the coating has an absorption peak of NH single bond at the position of 1520cm ^-1 , indicating that a three-dimensional network structure is formed between dopamine monomers. Iron reacts with the terminal amine bond in polydopamine by amidation to graft dicocene formate in the network. The valence state fitting of the Ce 3d orbitals of CeO ₂ nanoparticles on the surface of the material shows that the content of Ce ⁴⁺ on the surface of the TiO ₂ -PDA-CeO ₂ coating is 64.13%, the content of Ce ³⁺ is 35.87%, and the content of TiO ₂ -Sr The content of Ce ⁴⁺ on the surface of the -PDA-CeO ₂ coating is 61.84%, and the content of Ce ³⁺ is 38.16%.

It can be seen from the SEM image in Figure 3 that the addition of nano CeO ₂ particles is beneficial to increase the thickness of polydopamine polymerized on the surface of the material, and the TiO ₂ nanotube orifice is very tightly sealed, which will facilitate the sustained release of the drug in the tube.

D. Preparation of redox-responsive drug-releasing nanocoatings

First, the titanium sheet was cut into small pieces of 20mm×10mm×0.2mm, and the cut pieces were cleaned (ethanol-water-ethanol-deionized water, 15min each time); Platinum is used as cathode, medical titanium sheet is used as anode, the electrolyte solution is a mixed solution containing ammonium fluoride aqueous solution and glycerol, the mass fraction of ammonium fluoride is about 0.015%, and the volume ratio of glycerol to water is about 1: 1. Using constant voltage mode, anodized at 20V for 20min, cleaned the anodized titanium sheet with deionized water, and vacuum-dried for 24h. Using alcohol as a solvent, a solution with a concentration of 20 mg/L was prepared, and the anodized Ti substrate was immersed in an alcohol solution of strontium ranelate by vacuum negative pressure method, and dried in vacuum for 24 hours before use.

Prepare 5mL of 1.0M cerium nitrate solution and 10mL of 1.0M β-CD aqueous solution, mix the above solutions evenly, add dropwise to 30mL of ammonium hydroxide solution, and stir at 25°C for 24h. Centrifuge at 4000 rpm for 30 minutes, repeat twice, take the supernatant, and dialyze it with a 2000MCO dialysis membrane to obtain an aqueous solution of β-CD-modified CeO ₂ nanoparticles, which is stored at 4°C until use.

Prepare Tris-HCl buffer solution, mix 50 mL of 0.1 mol/L tris (Tris) and 50 mL of 0.1 mol/L hydrochloric acid solution. The pH of the solution was adjusted to the range of 7-9.2 with 0.1 mol/L HCl and 0.1 mol/L NaOH. Prepare 15 mL of 10 mg/mL mixed solution of EDC and NHS respectively, add 10 mg of ferrocene formate, continue stirring, and activate the carboxylic acid for 12 h.

Take 10 mL of activated ferrocene formate solution, 5 mL of β-CD@CeO ₂ solution, add 30 mg of dopamine hydrochloride, adjust the pH of the solution to 8.5 with 0.1 mol/L NaOH, and immerse the anodized Ti sheet loaded with the drug in In the above solution, under the irradiation of ultraviolet lamp, dopamine rapidly oxidized and polymerized on the surface of the material. The power of the ultraviolet lamp was 48W, the distance between the lamp source and the liquid surface of the mixed solution was about 5cm, and the ultraviolet light was irradiated for 20min to obtain an intelligent oxidative stress response type. Coating, abbreviated as TiO ₂ -Sr-PDA-CeO ₂ .

The XRD diffraction peaks are analyzed from Fig. 1, and the partial enlarged view of the diffraction peaks in the right figure shows that the surface of the TiO ₂ -Sr-PDA-CeO ₂ coating has very obvious nanometer CeO ₂ particles (111), (220) As well as the crystal diffraction peak of the (311) plane, compared with the CeO ₂ standard PDF card PDF#34-0394, it proves that the nano CeO ₂ particles are successfully modified on the surface of the coating.

In Fig. 2, we can see that when dopamine is polymerized on the surface of the material to form polydopamine, the coating also has an absorption peak of NH single bond at the position of 1520cm ^-1 . The orbital fitting of the Ce 3d orbital of CeO ₂ nanoparticles on the surface of the material shows that the content of Ce ⁴⁺ on the surface of the TiO ₂ -PDA-CeO ₂ coating is 64.13%, the content of Ce ³⁺ is 35.87%, and the content of TiO ₂ -Sr- The content of Ce ⁴⁺ on the surface of the PDA-CeO ₂ coating is 61.84%, and the content of Ce ³⁺ is 38.16%.

It can be seen from the SEM image in Fig. 3 that the addition of nano CeO ₂ particles is beneficial to increase the thickness of polydopamine polymerized on the surface of the material. The TiO ₂ -PDA-CeO ₂ and TiO ₂ -Sr-PDA-CeO ₂ coatings have similar morphologies. It shows that the loading of the drug will not have a serious impact on the polymerization reaction.

E. Detection of redox-responsive drug release ability

The three materials TiO ₂ -Sr, TiO ₂ -Sr-PDA and TiO ₂ -Sr-PDA-CeO ₂ loaded with the drug strontium ranelate were cut into small squares of 10mm×10mm×0.2mm. Three parallel samples were soaked in 4 mL of PBS solution respectively, the first supernatant was collected after 3 h, and the fresh PBS solution was replaced, and the second PBS solution was replaced after 6 h. After 24h, replace the PBS solution for the third time. To simulate oxidative stress, PBS solution with H ₂ O ₂ concentration of 0.3 mmol/L was added and the solution was collected after 3 h and supplemented with fresh PBS solution without H ₂ O ₂ , and so on, on the 7th day, respectively. and 14 days to repeat this operation. For the rest of the time, just replace with fresh PBS solution. The concentration of strontium ions in the solution was detected by inductively coupled plasma emission spectrometer to evaluate the release of the coating drug.

It can be seen from the concentration curve of strontium ions in Fig. 4 that TiO ₂ nanotubes as drug reservoirs have very serious drug bursting phenomenon. Modification of polydopamine on the surface of TiO ₂ nanotubes can delay the release rate of the drug to a certain extent, but there is still a short burst of drug release. The intelligent drug-releasing coating TiO ₂ -Sr-PDA-CeO ₂ with oxidative stress response can release the drug only under the stimulation of oxidative stress (H ₂ O ₂ ), and then the release rate of the drug decreases.

F: Detection of hydrophilicity of coating surface under oxidative stress

The contact angle changes of the materials under normal and oxidative stress states were detected using a contact angle meter, respectively. In the normal group, deionized water was used; in the oxidative stress group, the H ₂ O ₂ solution with a concentration of 0.3 mmol/L was used for simulation.

It can be seen from Figure 5 that under normal and oxidative stress conditions, the surface of TiO ₂ -Sr nanotubes exhibits good hydrophilicity, and the polymerization of dopamine on the surface of nanotubes makes the surface hydrophilicity worse. The introduction of _2-x particles resulted in an increase in the thickness of the dopamine membrane, which became less hydrophilic. However, under the oxidative stress state, the hydrophilicity of the two coatings TiO ₂ -PDA-CeO ₂ and TiO ₂ -Sr-PDA-CeO ₂ with intelligent oxidative stress response function was significantly improved, which is in agreement with the polymer The functional group ferrocene formate on the dopamine network structure is positively charged by oxidation, and the hydrophobic end becomes the hydrophilic end. At the same time, due to the electrostatic repulsion between the molecular network structures, the molecular network structure becomes loose. Therefore, The contact angle on the surface of the material becomes smaller and the hydrophilicity becomes better, which is more conducive to the release of the drug in the tube.

G: Detection of intracellular reactive oxygen species in osteoblasts under normal and oxidative stress

MC3T3-E1 cells in good growth state were collected, digested and the concentration of cell suspension was adjusted. 1 mL of cell suspension (20000 cells/mL) was planted on the surface of each well material, and according to the experimental protocol, some wells were added with a final concentration of 0.3 mM H ₂ O ₂ , and incubated in a 37° C., 5% CO ₂ cell incubator for 24 h. The supernatant was discarded, the cells were washed twice with PBS, and a final concentration of 10 μM DCFH-DA solution was added to each well. Incubate at 37°C for 20min. The supernatant was discarded and the cells were washed 3 times with PBS. Observation using a laser confocal microscope (using a 488nm laser tube to excite and receive a fluorescence signal at 500-550nm).

It can be seen from Figure 6 that under normal circumstances, the fluorescence signals of reactive oxygen species in osteoblasts on the surface of TiO ₂ -PDA-CeO ₂ and TiO ₂ -Sr-PDA-CeO ₂ materials are relatively weak, indicating that the existence of nano CeO ₂ particles to a certain degree of antioxidant protection. Under the condition of oxidative stress, the oxidative stress signal in osteoblasts on Ti surface and TiO ₂ -Sr surface is stronger. H ₂ O ₂ can oxidize the catechol group of dopamine, therefore, polydopamine shows a certain effect of antioxidant protection, and the existence of nano-CeO ₂ further improves the antioxidant protection ability of the coating.

The biological antioxidant nano-coating with redox response drug release function provided by the invention has the characteristics of intelligent responsiveness, can effectively avoid drug burst release, and has good biocompatibility and biological antioxidant properties, which can effectively reduce Oxidative stress injury of osteocytes, and promotes the repair of bone tissue under oxidative stress environment.

Claims (8)

1. A biological anti-oxidation nano coating with a redox response drug release function is characterized in that the coating comprises a nano tube drug loading system constructed on the surface of a substrate and a three-dimensional reticular structure nano composite coating which is formed by polydopamine modified by a redox response functional group and enables at least part of tube orifices of the nano tube to be completely or partially encapsulated and is formed by in-situ polymerization on the surface of the nano tube; the polydopamine modified by the redox response functional group is modified ferrocene formate and beta-CD @ CeO ₂ Nanoparticulate polydopamine, beta-CD @ CeO ₂ The nano-particles and the ferrocene formate jointly form a response switch of oxidation reduction.

2. The biological oxidation resistant nano-coating according to claim 1, wherein the length of the nano-tube is 50 to 800nm, the tube diameter is 20 to 150nm, and the wall thickness is 5 to 20 nm.

3. The biological oxidation-resistant nanocoating of claim 1, wherein said nanotubes are metal or metal oxide nanotubes grown in situ on the substrate surface as drug reservoirs, the open structure of the nanotubes being perpendicular to the substrate surface.

4. The bio-antioxidant nanocoating as defined in claim 1, wherein the substrate comprises a medical metal material or a metal oxide material, wherein the medical metal material comprises pure titanium, a titanium alloy, stainless steel, or a cobalt-chromium-molybdenum alloy; the metal oxide material comprises titania, zirconia, alumina, or tantalum oxide.

5. The preparation method of the biological oxidation resistant nano coating with the redox response drug release function according to any one of claims 1 to 4, characterized in that the preparation method comprises the following steps:

step (1), forming a tubular nano structure on the surface of a base material by an anodic oxidation method;

step (2), loading a drug or nanoenzyme in the nanotube-shaped structure obtained in the step (1) by a vacuum negative pressure method to obtain a substrate with a nanotube drug-loading system constructed on the surface;

step (3), placing the base material with the surface provided with the nano-tube drug-loading system in a buffer solution containing dopamine monomer, and obtaining the three-dimensional network structure nano-composite coating through oxidative polymerization; the buffer solution also comprises 0.5-1 mg/mL of ferrocene formate and 0.5-1 mg/mL of beta-CD @ CeO ₂ And (3) nanoparticles.

6. The preparation method according to claim 5, wherein the anodic oxidation voltage is 10-30V, and the oxidation time is 15-60 minutes.

7. The method according to claim 5 or 6, wherein the dopamine monomer is oxidatively polymerized by UV irradiation.

8. Use of the redox-responsive drug-releasing functional bio-antioxidant nanocoating of any one of claims 1 to 4 for the preparation of hard tissue repair and replacement biomaterials.

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