Schiff base-sulfonated hyaluronic acid light-cured microsphere, preparation method and application

Abstract

The invention relates to the technical field of biomedical materials. The invention discloses a Schiff base-sulfonated hyaluronic acid light-cured microsphere as well as a preparation method and application thereof. The microspheres are prepared by condensing DL-lysine and paeonol to generate Schiff base, then conjugating the Schiff base with sulfonated hyaluronic acid through a carbodiimide chemical method, and finally molding by adopting a microfluidic technology and carrying out ultraviolet curing. The metal chelation characteristic of Schiff base is combined with the precise structure regulation advantage of the microspheres, and the prepared microspheres show an excellent synergistic effect when applied to the surfaces of implant materials such as degradable magnesium alloy, iron alloy and zinc alloy: the corrosion resistance of a base material can be remarkably improved, an effective protective layer is formed, and the service life of the microspheres is prolonged. Meanwhile, the compound has multiple biological functions of promoting endothelial cell proliferation, inhibiting excessive proliferation of smooth muscle cells and regulating and controlling macrophages to be polarized to anti-inflammatory phenotypes, and has a wide application prospect in the biomedical fields of intravascular stents, bone fixing instruments and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to Schiff base-sulfonated hyaluronic acid photocurable microspheres, their preparation method, and their applications. Background Technology

[0002] Biodegradable metal alloys, which can gradually degrade and be absorbed in the human body, avoid the pain of secondary surgery for removal and show great promise in implantable medical fields such as vascular stents and bone fixation devices. However, the degradation rate of these materials in the physiological environment is often too fast, exceeding the tissue healing cycle, leading to premature loss of their mechanical integrity. They may also cause inflammatory reactions due to local alkalization, hydrogen accumulation, and excessive release of metal ions, ultimately resulting in implantation failure.

[0003] To control degradation rates and improve biocompatibility, surface coating technology has been a commonly used approach. Currently, various coating systems have been reported, such as single-functional coatings like fluorinated coatings and polymer coatings. These primarily provide physical protective barriers to delay corrosion, but often lack bioactivity for tissue repair and regeneration, and their long-term stability is insufficient. For example, biofunctional coatings, such as the composite coating of Schiff base (SB) and sulfonated hyaluronic acid (S-HA) nanoparticles constructed on magnesium alloy surfaces using electrostatic spraying, can, to some extent, inhibit degradation and promote endothelial function. However, the uniformity and density of the coating formed by the electrostatic spraying process need improvement, and the specific surface area and drug loading capacity of the nanoparticles are limited. Another approach involves preparing photosensitive tyramine-sulfonated hyaluronic acid (S-HA-Tyr) microspheres using microfluidic technology. While this technology achieves structural uniformity in the coating, the tyramine used primarily acts as a photocrosslinking agent and lacks the ability to specifically bind to metal ions, thus contributing limitedly to the corrosion resistance of the substrate and failing to fully utilize the microsphere structure to maximize the synergistic effect of corrosion protection and biofunctionality.

[0004] Therefore, existing technologies still face the following challenges: it is difficult to achieve efficient synergy and unity between corrosion resistance and multiple biological functions (such as endothelial promotion, anti-proliferation, and anti-inflammation) while rapidly constructing a uniform coating. Therefore, developing a novel multifunctional coating system that can simultaneously resolve the contradiction between degradation control and tissue repair has become an urgent technical challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to provide Schiff base-sulfonated hyaluronic acid photocurable microspheres, their preparation method, and their applications, in order to solve the technical problem of rapid mixing rate of corrosion and healing of biodegradable implant materials.

[0006] The technical solution of the present invention is as follows: A method for preparing Schiff base-sulfonated hyaluronic acid photocurable microspheres is disclosed. The microspheres are obtained by microfluidic molding and photocuring of a conjugate formed by covalent bonding of a Schiff base and sulfonated hyaluronic acid. The Schiff base is a lysine Schiff base. The specific preparation method of the microspheres is as follows: (1) Dissolve the Schiff base-sulfonated hyaluronic acid conjugate in PBS to prepare a solution with a concentration of [missing information]. 50 mg / ml prepolymer solution; (2) Using a microfluidic device, microsphere droplets are generated with the prepolymer solution in step (1) as the aqueous phase and the droplet-generating oil as the oil phase; (3) The microsphere droplets are irradiated with ultraviolet light to solidify and form microspheres.

[0007] Further optimization is achieved by preparing the Schiff base-sulfonated hyaluronic acid conjugate in step (1) as follows: activate the carboxyl group of sulfonated hyaluronic acid in MES buffer using EDC and NHS, add Schiff base, react at room temperature for 24 hours, with a pH value of 4.7, and adjust the pH value to 7 after the reaction is completed.

[0008] Further optimization involves synthesizing the Schiff base via a condensation reaction of DL-lysine and paeonol. The specific preparation method is as follows: DL-lysine is dissolved in anhydrous ethanol, and after ultrasonic reaction, sodium hydroxide is added and stirred until clear. Paeonol solution is then slowly added dropwise and reacted at 60°C for 2 hours. The mixture is then filtered and dried to obtain the lysine Schiff base.

[0009] Schiff base-sulfonated hyaluronic acid photocurable microspheres, wherein the microspheres have a particle size of 0.5-2 μm.

[0010] Application of Schiff base-sulfonated hyaluronic acid photocurable microspheres in surface modification of biodegradable plant metal materials.

[0011] Further optimization involves the inclusion of one of the following biodegradable plant-metal materials: biodegradable magnesium alloy, biodegradable iron alloy, and biodegradable zinc alloy, with its surface coated with Schiff base-sulfonated hyaluronic acid photocurable microspheres.

[0012] Further optimization includes the application of bone implant materials or vascular implant materials.

[0013] The beneficial effects of this technical solution are: (1) Combining lysine Schiff base with microfluidic photocurable microsphere technology improves the surface corrosion resistance of biodegradable plant metal materials. Its capacitive arc radius, charge transfer resistance and corrosion rate are all better than those of metal materials that have not been modified by lysine Schiff base. This effectively proves the strong chelating ability of lysine Schiff base for metal ions in the degradation products of metal materials. Combined with the dense physical barrier formed by microspheres, a dual corrosion mechanism is constructed, which solves the problem of biodegradable metal implants failing due to excessive degradation. (2) The microspheres prepared by this invention can significantly promote the proliferation and adhesion of endothelial cells, which is crucial for the rapid endothelialization of vascular stents and the prevention of late thrombosis. Furthermore, the microspheres can effectively inhibit the excessive proliferation of smooth muscle cells, reducing the risk of restenosis after stent implantation. (3) Co-culture results with macrophages showed that the microspheres could inhibit pro-inflammatory M1 macrophages (CD68). + It also promotes anti-inflammatory M2 macrophages (CD206) + Polarization has a good anti-inflammatory effect, creating a good healing microenvironment for the implant; (4) The spherical particles with uniform size and regular shape prepared by combining microfluidic technology with photopolymerization avoid the defects of wide particle size distribution and irregular morphology prepared by traditional methods. (5) The Schiff base-sulfonated hyaluronic acid microsphere system was successfully applied to the surface modification of biodegradable metal implants. Its application is not limited to magnesium alloys, but can be extended to biodegradable iron alloys, zinc alloys and other systems. The application scenarios cover a variety of implants such as vascular stents and bone fixation devices. It provides a new and efficient solution to the common technical bottleneck of mismatch between corrosion rate and healing cycle in various biodegradable implant materials. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the reaction process for preparing microspheres according to the present invention; Figure 2 The FTIR spectra of S-HA, lysine Schiff base, and S-HA@SB are shown. Figure 3 XPS spectra of S-HA, lysine Schiff base, and S-HA@SB; Figure 4 This is a schematic diagram illustrating the effect of S-HA@SB-2-microsphere-UV on endothelial cell proliferation. Figure 5 A schematic diagram showing the state of S-HA@SB-2-microsphere-UV cultured with endothelial cells for 24 hours. Figure 6A schematic diagram illustrating the inhibition of excessive proliferation of smooth muscle cells by S-HA@SB-2-microsphere-UV culture for 48 h. Figure 7 A schematic diagram illustrating how S-HA@SB-2microsphere-UV inhibits the excessive proliferation of inflammatory macrophages after 72 hours of macrophage culture; Figure 8 This is a schematic diagram of the coating preparation process; Figure 9 For the process Figure 8 After the process was completed, SEM / EDS images of S-HA@SB-2-microsphere-UV on the alloy surface were obtained. Figure 10 This is a schematic diagram comparing the corrosion resistance of various coatings. Figure 11 Electrochemical impedance spectroscopy for various coatings. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments. The components of the embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0016] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0017] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0018] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0019] Example 1

[0020] Preparation of lysine Schiff base 1492.1 mg of DL-lysine was dissolved in 50 mL of anhydrous ethanol and stirred for 5 min at 30-40 °C and 700-800 rpm. The suspension was then sonicated for 30 min and stirred for another 15 min. Next, 400 mg of sodium hydroxide was added, and the mixture was stirred until the solution became clear. In a three-necked flask equipped with a condenser, 1661.7 mg of paeonol was dissolved in 15 mL of ethanol and heated in a 60 °C water bath. The DL-lysine solution was slowly added dropwise to the paeonol solution while continuously stirring. The reaction mixture was kept at 60 °C for 2 h, then stirred for another 30 min at room temperature. The product was filtered, air-dried overnight, and then further dried in an oven. The yellow lysine Schiff base was collected and stored in a desiccator for later use.

[0021] Preparation of S-HA@SB-2, a conjugate of Schiff base-sulfonated hyaluronic acid 25 mg of sulfated hyaluronic acid (S-HA) was dissolved in 50 mL of 0.1 mol / L MES (2-morpholinoethanesulfonic acid) buffer (pH 5.5). 47.9 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 28.8 mg of NHS (N-hydroxysuccinimide) were added, and the solution was stirred at room temperature for 1 h to activate the carboxyl group of S-HA. After activation, 13.8 mg of lysine Schiff base was added to the solution, and the mixture was stirred at room temperature for 24 h. During the reaction, the pH of the reaction mixture was maintained at 4.7 using 0.1 mol / L sodium hydroxide, and adjusted to 7.0 after the reaction. The final product was purified by dialyzing in distilled water for 3 days. The purified Schiff base conjugated S-HA solution was freeze-dried to obtain the final S-HA-Schiff base conjugate, labeled S-HA@SB-2.

[0022] Preparation of cured microspheres S-HA@SB-2-microsphere-UV S-HA@SB-2 was dissolved in 50 mg / ml PBS (phosphate buffered saline) and cured by ultraviolet light to obtain Schiff base-sulfonated hyaluronic acid photocurable microspheres, labeled as S-HA@SB-2-microsphere-UV.

[0023] S-HA, lysine Schiff base, and S-HA@SB-2 were analyzed by FTIR spectroscopy. The results showed that the FTIR spectrum of S-HA was within the range of 3500 cm⁻¹. -1 A relatively wide absorption band is observed at 3200 cm⁻¹. -1 The OH stretching vibration corresponding to the hydroxyl group (-OH) in the hyaluronic acid backbone. 1600cm -1 The C=O stretching vibration at this point is attributed to the inherent carboxyl (-COOH) group in hyaluronic acid. The CO stretching vibration occurs at 1400 cm⁻¹. -1 This represents the ether (COC) bond in the polysaccharide structure. Most notably, at 1150 cm⁻¹... -1 The S=O stretching vibration at the point confirmed the introduction of sulfonic acid groups (-SO3) onto the hyaluronic acid side chain. - ).

[0024] In the Fourier transform infrared spectrum of lysine Schiff base, a value of 3400 cm⁻¹ was observed. -1 and 1650 cm -1 The key peak. 3400 cm. -1 The peak corresponds to the NH stretching vibration of the amino group (-NH2), while the 1650 cm⁻¹ peak corresponds to the NH stretching vibration of the amino group (-NH2). -1 The peak is attributed to the C=N stretching vibration, a hallmark of Schiff base formation. This confirms that an imine bond (C=N) is formed between the amino group of the lysine Schiff base and the carbonyl group of the aldehyde or ketone. At 1580 cm⁻¹ -1 The observed C–N stretching vibrations further confirm the presence of nitrogen in the lysine Schiff base.

[0025] S-HA@SB-2: Exists at 3500–3200 cm -1 The OH stretching vibrations between them indicate that the HA backbone... The hydroxyl T- group was retained, 1650 cm -1 The C=N stretching vibration at the site is more pronounced, which confirms that the lysine Schiff base has been successfully bound to S-HA.

[0026] XPS spectroscopy analysis was performed on S-HA, lysine Schiff base, and S-HA@SB-2 to confirm their chemical composition and successful coupling. For S-HA, characteristic peaks appeared in the C 1s region at 284.8 eV (C–C / C–H), 286.3 eV (C–OC), and 288.5 eV (O–C=O), while the O 1s peaks in the range of 531–533 eV corresponded to hydroxyl and carboxyl groups. A distinct S 2p doublet appeared at approximately 167–169 eV, confirming the presence of sulfonic acid groups (-SO3). - The components demonstrated the successful sulfonation of HA.

[0027] The C 1s spectrum shows contributions from C–C / C–H bonds in addition to C–N (~286.5–287 eV), while peaks appear in the N 1s region at approximately 399.0 eV (amine nitrogen) and 400.2 eV (imino nitrogen). These features confirm the formation of Schiff base bonds. The absence of the lysine Schiff base S signal further distinguishes this spectrum from the S-HA spectrum. For S-HA@SB-2, the combined chemical characteristics of the two components are readily apparent. The C 1s spectrum shows contributions from the polysaccharide carbon and imine groups, with slight shifts indicating changes in electronic properties. The superposition of two peaks at approximately 399 eV and 400–401 eV in the N 1s region, with intensities higher than those observed in pure S-HA, indicates the incorporation of the lysine Schiff base. The S 2p peak is still present, but slightly weakened relative to pure S-HA, suggesting partial coverage or interaction of the lysine Schiff base layer. This broadened O 1s peak reflects the overlapping oxygen signals from the carboxyl and hydroxyl groups of HA and the lysine functional group. Taken together, these results confirm that the L-lysine Schiff base was successfully incorporated into sulfonated HA without the loss of sulfonic acid groups.

[0028] Observation and analysis of S-HA@SB-2-microsphere-UV by EDS showed that the diameter of S-HA@SB-2-microsphere-UV is about 1 μm, exhibiting a stable spherical structure. The analysis confirmed the uniform distribution of key elements, thus verifying the successful preparation of S-HA@SB-2-microsphere-UV.

[0029] Example 2

[0030] The difference from Example 1 is that the amount of lysine Schiff base added is 6.9 mg, and the rest of the steps are the same as in Example 1, to obtain Schiff base-sulfonated hyaluronic acid photocurable microspheres, labeled as S-HA@SB-1- microsphere-UV.

[0031] Example 3

[0032] The difference from Example 1 is that the amount of lysine Schiff base added is 27.6 mg, and the rest of the steps are the same as in Example 1, to obtain Schiff base-sulfonated hyaluronic acid photocurable microspheres, labeled as S-HA@SB-3- microsphere-UV.

[0033] Example 4

[0034] The biocompatibility of S-HA@SB-1-microsphere-UV, S-HA@SB-2-microsphere-UV, and S-HA@SB-3-microsphere-UV was tested separately. The results showed that the materials could not only promote endothelial cell proliferation, but also inhibit the excessive proliferation of smooth muscle cells and inflammatory macrophages, thereby regulating the excessive proliferation and inflammatory response of tissues after material implantation.

[0035] Endothelial cell proliferation test Endothelial cells were grown in RMPI 1640 complete medium. To investigate the effects of each sample group on endothelial cells, cells were seeded at a concentration of 1 × 10⁶ cells / mL. 4 Cells were seeded at a density of 1 cell / mL at the bottom of a 96-well adherent plate. After adding 1 mg / mL of S-HA@SB-1-microsphere-UV, S-HA@SB-2-microsphere-UV, and S-HA@SB-3-microsphere-UV, respectively, the cells were incubated for 24 h. Cell viability was then tested using the CCK-8 kit.

[0036] The results showed that S-HA@SB-2-microsphere-UV had the best effect on promoting cell proliferation.

[0037] Endothelial cells were grown in RMPI 1640 complete medium. To investigate the effects of each sample group on endothelial cells, cells were cultured at a concentration of 1 × 10⁶ cells / mL. 4 Endothelial cells (ECs) were seeded at a density of 1 cell / mL at the bottom of 24-well adherent plates. After incubation for 24 h with 1 mg / mL S-HA@SB-2-microsphere-UV microspheres, the viability of ECs was observed. The extract incubation was detected using an acridine orange (AO) / ethidium bromide (EB) fluorescence staining kit manufactured by Solarbbio (China), and the EC cell status was observed using an inverted fluorescence microscope.

[0038] The results showed that after 24 hours of incubation, the S-HA@SB-2-microsphere-UV medium promoted the proliferation of endothelial cells without causing apoptosis.

[0039] Inhibit smooth muscle cell proliferation In the experiment, smooth muscle cells were cultured in DMEM complete medium (Gibco, USA). To investigate the effects of each sample group on smooth muscle cells, cells were cultured at 1.5 × 10⁶ cells / day. 4Smooth muscle cells were seeded at a density of 1 cell / mL at the bottom of 24-well adherent plates. After incubation for 48 h with 0.5 mg / mL S-HA@SB-2-microsphere-UV, the viability of smooth muscle cells was assessed. Cells were fixed and then incubated with a phalloidin fluorescent staining kit (Solarbbio, China). The smooth muscle cell status was then observed using super-resolution confocal fluorescence microscopy.

[0040] The results showed that after 48 hours of incubation, the S-HA@SB-2-microsphere-UV culture medium inhibited the spreading and proliferation of smooth muscle cells, caused significant apoptosis of smooth muscle cells, and reduced excessive narrowing of the implanted material surface.

[0041] Inhibit the excessive proliferation of inflammatory macrophages In the experiment, macrophages were cultured in DMEM basal medium. To investigate the effects of each sample group on endothelial cells, cells were seeded at a rate of 1 × 10⁶ cells / mL. 4 Cells were concentrated at a density of 1 cell / mL at the bottom of 24-well adherent plates. After incubation for 72 h with 0.2 mg / mL S-HA@SB-2-microsphere-UV, cell fixation, permeability testing, and blocking were performed. Anti-mannose receptor antibody (CD206) (1:100, Abcam, ab64693, England) and anti-CD68 (1:100, Abcam, ab955, England) were added at a concentration of 1:100 and incubated overnight at 4°C. Subsequently, goat anti-mouse (Alexa Fluorescence® 594) and goat anti-rat (Alexa Fluorescence® 488) IgG H&L were added at 1:200 and 1:200, respectively, and incubated at 4°C for 4 h. After adding DAPI (1:150, Solarbio, China) for 3 min, cell phenotype was observed using super-resolution confocal fluorescence microscopy.

[0042] The results showed that after 72 hours of incubation, the S-HA@SB-2-microsphere-UV medium reduced the proliferation rate of macrophages, accelerated macrophage apoptosis, and had a good anti-inflammatory effect.

[0043] Example 5

[0044] Coating preparation Step 1: Alloy Pretreatment Cut the alloy into cylinders with a diameter of 10 mm and a thickness of 3 mm. Polish them with sandpaper of 100 grit, 400 grit, 800 grit and 1000 grit in sequence until the surface is smooth and flat. Rinse them with deionized water and anhydrous ethanol in sequence, and dry them at 50°C for 10 min.

[0045] Step 2: Fluorination treatment of magnesium alloy surface The polished alloy sheet was placed in a 24-well plate (approximately 1.5 cm in diameter and 2 cm in depth, capable of holding approximately 3.3 mL of solution); 2 mL of 40 wt% hydrofluoric acid solution was added to the well containing the magnesium alloy sheet, and the 24-well plate was placed in a fume hood and left at room temperature for 48 h. Remove the fluorinated alloy sheet, rinse it sequentially with deionized water and anhydrous ethanol to remove residual hydrofluoric acid, and dry it at 50°C for 10 min for later use.

[0046] Step 3: Polydopamine deposition treatment Prepare a 10 mM aqueous solution of tris(hydroxymethyl)aminomethane, and adjust the pH to 8.5 by adding 0.5 mol / L dilute hydrochloric acid solution dropwise to obtain a tris(hydroxymethyl)aminomethane-hydrochloric acid solution. Dopamine hydrochloride was dissolved in the above tris(hydroxymethyl)aminomethane-hydrochloric acid solution to a concentration of 3.0 mg / mL, and the mixture was sealed and stirred for 30 min to obtain a dopamine solution. Fluorinated ZE21B magnesium alloy samples were placed in a 24-well plate, and 1.5 mL of the above dopamine solution was added to each well. The plate was then fixed on a constant temperature shaker at 60 rpm and deposited at 37°C for 6 h. The polydopamine-treated sample was taken out, rinsed sequentially with deionized water and anhydrous ethanol, and dried at 50°C for 10 min to obtain a sample with a MgF2 / PDA coating on the surface.

[0047] Step 4: Place the magnesium alloy sample coated with MgF2 / PDA into a 24-well plate, add 1.5 mL of PBS solution with a concentration of 2 mg / mL for S-HA@SB-2-microsphere-UV to each well, fix the plate on a constant temperature shaker at 60 rpm, and deposit at 37℃ for 7 h; take out the sample treated with S-HA@SB-2-microsphere-UV PBS solution, rinse with deionized water and anhydrous ethanol in sequence, and dry at 50℃ for 10 min to obtain the sample with a surface coated with MgF2 / PDA / S-HA@SB-2-microsphere-UV.

[0048] Comparing the samples with the MgF2 / PDA / S-HA-Tyr@microspheres coating prepared above and the MgF2 and MgF2 / PDA coatings on magnesium alloys, the graph shows that the coating prepared in this invention significantly shifts the horizontal position of the curve to the left, indicating that it greatly reduces the corrosion rate of the ZE21B magnesium alloy substrate. Compared with MgF2 and MgF2 / PDA, the coating with S-HA@SB-2-microsphere-UV exhibits superior performance, indicating that the S-HA@SB-2-microsphere-UV modified with "SB" achieves the best effect. This proves that SB functionalization is a key step in improving the corrosion resistance of biodegradable materials.

[0049] By combining electrochemical impedance spectroscopy, we conducted a more in-depth and quantitative analysis of the effect of S-HA@SB-2-microsphere-UV on improving corrosion resistance. The capacitive arc radius of S-HA@SB-2-microsphere-UV is significantly larger than all other groups. This is a very clear signal indicating that it has the highest charge transfer resistance. This means that corrosive media face the greatest difficulty and highest resistance in penetrating this coating to reach the magnesium alloy substrate surface for electrochemical reactions. The impedance arc radius of S-HA@SB-2-microsphere-UV is significantly larger than that of the unmodified amino acid Schiff base microsphere coating (S-HA@SB-2-microsphere-UV). This further demonstrates that amino acid Schiff base functionalization is a key step in significantly improving the corrosion resistance of the material.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.

Claims

1. A method for preparing Schiff base-sulfonated hyaluronic acid photocurable microspheres, characterized in that, The microspheres are obtained by microfluidic molding and photocuring of a conjugated compound formed by a Schiff base and sulfonated hyaluronic acid through covalent bonding. The Schiff base is a lysine Schiff base. The specific preparation method of the microspheres is as follows: (1) Dissolve the Schiff base-sulfonated hyaluronic acid conjugate in PBS to prepare a prepolymer solution with a concentration of 50 mg / ml; (2) Using a microfluidic device, microsphere droplets are generated with the prepolymer solution in step (1) as the aqueous phase and the droplet-generating oil as the oil phase; (3) The microsphere droplets are irradiated with ultraviolet light to solidify and form microspheres.

2. The method for preparing Schiff base-sulfonated hyaluronic acid photocurable microspheres according to claim 1, characterized in that, The preparation method of the Schiff base-sulfonated hyaluronic acid conjugate in step (1) is as follows: the carboxyl group of sulfonated hyaluronic acid is activated in MES buffer using EDC and NHS, Schiff base is added, and the reaction is carried out at room temperature for 24 hours with a pH value of 4.

7. After the reaction is completed, the pH value is adjusted to 7.

3. The method for preparing Schiff base-sulfonated hyaluronic acid photocurable microspheres according to claim 1, characterized in that, The Schiff base is synthesized by the condensation reaction of DL-lysine and paeonol. The specific preparation method is as follows: DL-lysine is dissolved in anhydrous ethanol, and after ultrasonic reaction, sodium hydroxide is added and stirred until clear. Paeonol solution is slowly added dropwise and reacted at 60°C for 2 hours. After filtration and drying, lysine Schiff base is obtained.

4. Schiff base-sulfonated hyaluronic acid photocurable microspheres prepared by any one of the preparation methods in claims 1-3, characterized in that, The microspheres have a particle size of 0.5-2 μm.

5. The application of the Schiff base-sulfonated hyaluronic acid photocurable microspheres as described in claim 4 in the surface modification of biodegradable plant metal materials.

6. The application according to claim 5, characterized in that, The biodegradable plant-metal material includes one of biodegradable magnesium alloy, biodegradable iron alloy, and biodegradable zinc alloy, and its surface is coated with a coating containing the Schiff base-sulfonated hyaluronic acid photocurable microspheres as described in claim 4.

7. The application according to any one of claims 5-6, characterized in that, The applications include bone implant materials or vascular implant materials.

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