Hydrogel material for promoting intracellular lipid reduction of DHA (docosahexaenoic acid) as well as preparation method and application of hydrogel material

Abstract

The invention belongs to the technical field of advanced materials, and relates to a hydrogel material for promoting DHA intracellular lipid reduction and a preparation method and application thereof. Hydrogel formed by polyethylene glycol diacrylate (PEG-DA) and dithiothreitol (DTT) is used as a support to load nanometer microspheres, the nanometer microspheres load docosahexaenoic acid (DHA), and the nanometer microspheres are composed of chitosan and fucoidin according to the mass ratio of 5: (1-10). The hydrogel provided by the invention can reduce the lipid droplet accumulation level in local tissue cells of a hyperlipidemia patient and promote in-situ bone regeneration.

Description

Technical Field

[0001] The invention belongs to the technical field of advanced materials and relates to a hydrogel material that promotes DHA intracellular lipid reduction and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The repair and regeneration of human bone tissue is a dynamic process that depends on the activity and function of bone marrow mesenchymal stem cells and osteoblasts. However, the accumulation of intracellular lipid droplets and the surge in reactive oxygen species (ROS) caused by a high-fat environment strongly inhibit the biological activity of stem cells and osteoblasts, thereby seriously impairing the repair and regeneration of local bone defects of various sizes. Therefore, reducing the accumulation of intracellular lipid droplets is an effective strategy to promote in situ bone regeneration.

[0004] Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid that has been shown to regulate local inflammation levels and promote local bone defect healing. Therefore, applying DHA to the site where local bone defects need to be repaired in patients with hyperlipidemia is a promising way to promote in situ bone regeneration in a high-fat environment. However, since DHA is a small molecule fatty acid with relatively active chemical properties, local application faces difficult problems such as long-term loss of activity. At the same time, the cell's intake of fatty acids such as DHA is significantly reduced in a high-fat environment, which will lead to a significant reduction in the efficiency of absorbing DHA through active cell uptake to promote the efflux of intracellular lipid droplets. Therefore, how to apply active DHA to the local bone defect for a long time and increase its effective concentration in the cell to exert the function of DHA in promoting lipid droplet efflux and promoting in situ bone regeneration is still a key issue that needs to be solved. Summary of the invention

[0005] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a hydrogel material that promotes intracellular lipid-lowering of DHA and a preparation method and application thereof. The hydrogel provided by the present invention can reduce the accumulation level of fat droplets in local tissue cells of patients with hyperlipidemia and promote in situ bone regeneration.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In the first aspect, a hydrogel material that promotes intracellular lipid-lowering of DHA uses a hydrogel formed by polyethylene glycol diacrylate (PEG-DA) and dithiothreitol (DTT) as a scaffold to load nanoparticles, wherein the nanoparticles are loaded with docosahexaenoic acid (DHA), and the nanoparticles are composed of chitosan and fucoidan in a mass ratio of 5:(1-10).

[0008] The present invention has found through research that in a high-fat environment, simply increasing the concentration of DHA cannot increase the content of DHA in cells. Further experiments unexpectedly found that the hydrogel constructed by PEG-DA and DTT has the effect of further increasing the content of DHA in cells. On this basis, the present invention loads DHA through the constructed chitosan and fucoidan nanospheres, and loads it on the hydrogel constructed by PEG-DA and DTT. The rate and concentration of DHA entering the cell can be unaffected by the feedback inhibition of high concentration DHA in the cell, so as to achieve the rapid and even reverse concentration gradient entry of active DHA into the cell, thereby achieving the beneficial effects of significantly promoting the continuous entry of active DHA into the cell, increasing the peak concentration of DHA in the cell, and extending the duration of the peak concentration of DHA in the cell, and finally achieving the hydrogel material under the condition of hyperlipidemia. It can significantly promote the entry of active DHA into the cell and promote the efflux of fat droplets for a long time, thereby significantly improving the local bone defect repair and regeneration effect of hyperlipidemia patients.

[0009] In a second aspect, a method for preparing the above-mentioned hydrogel material for promoting intracellular lipid reduction of DHA comprises the following steps:

[0010] Mixing chitosan and fucoidan in proportion to prepare nanospheres, and then mixing the nanospheres with DHA to obtain nanospheres loaded with DHA;

[0011] Under the condition of 4°C to 10°C, PEG-DA and DTT are fully mixed in a solution to obtain a precursor solution;

[0012] At 4°C to 10°C, the DHA-loaded nanoparticles are mixed with the precursor solution.

[0013] The present invention can effectively avoid the interference of the chemical bond cross-linking reaction in the formation process of the thermosensitive hydrogel material by the olefin group and carbonyl group inside the DHA molecule, thereby forming a hydrogel material with higher long-term stability by pre-loading the active DHA to the nano-microspheres. PEG-DA and DTT can spontaneously cross-link under the condition of 4°C to 10°C. The cross-linked system has the characteristics of temperature responsiveness, which is liquid under the condition of 4°C to 10°C and hydrogel under the condition of 25°C to 40°C. Under the condition of 4°C to 10°C, it is conducive to the uniform loading of the nano-microspheres loaded with DHA.

[0014] In a third aspect, a hydrogel material for promoting intracellular lipid reduction of DHA is used in the preparation of a product for promoting local bone regeneration.

[0015] The beneficial effects of the present invention are:

[0016] 1. Experiments have shown that the hydrogel material provided by the present invention is safe and non-toxic. It can promote the entry of active DHA into cells and promote the efflux of lipid droplets in the case of hyperlipidemia, thereby significantly improving the repair and regeneration of local bone defects in patients with hyperlipidemia.

[0017] 2. The nano-microspheres loaded with DHA in the hydrogel material provided by the present invention have a particle size and structure that can penetrate the cell membrane in a high-fat environment, and can ensure that the active DHA is delivered into the cells for a long time within 10 days without being inhibited by the feedback of the high concentration of DHA in the cells, thereby increasing the peak concentration of DHA in the cells and prolonging its duration, thereby continuously promoting the efflux of intracellular lipid droplets under hyperlipidemia conditions, thereby reducing the level of intracellular reactive oxygen species (ROS) and reshaping the biological activity of the cells, and finally promoting the in situ repair of local bone defects in patients with hyperlipidemia.

[0018] 3. The hydrogel material provided by the present invention can be completely degraded within 15 to 20 days, has good biocompatibility, and does not form unnecessary space occupation. While reshaping the early repair process of local bone defects in patients with hyperlipidemia, it does not affect the later bone remodeling process (evolution from woven bone to lamellar bone), thereby greatly improving the in situ bone regeneration effect of patients with hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 The physicochemical properties, stability and surface morphology of the nanospheres CxFy-DHA loaded with active DHA in the embodiments of the present invention are shown; wherein, A shows the encapsulation rate of DHA by the nanospheres CxFy-DHA; B shows the particle size of the nanospheres CxFy-DHA; C shows the surface potential of the nanospheres CxFy-DHA; D shows the confidence value of the surface potential analysis of the nanospheres CxFy-DHA; E shows the stability of the nanospheres CxFy-DHA in different buffer conditions; F shows the stability of the nanospheres CxFy-DHA under different temperature conditions; G shows the surface morphology and particle size of the nanospheres CxFy-DHA under transmission electron microscopy.

[0021] Figure 2The physicochemical properties of the hydrogel material (PEGD@CF-DHA) that promotes intracellular lipid-lowering of DHA in the embodiments of the present invention are characterized; wherein A shows the surface morphology of the hydrogel material under a scanning electron microscope; B shows the pore size of the hydrogel material; C shows the complete degradation time and stability of the hydrogel material under different buffer conditions; D shows the release rate of the hydrogel material under different buffer conditions.

[0022] Figure 3 These are the test results of the hydrogel material (PEGD@CF-DHA) that promotes intracellular lipid-lowering of DHA in the embodiments of the present invention, in terms of promoting the performance of active DHA entering cells and promoting the efflux of lipid droplets; wherein A shows the effects of different extracellular DHA concentrations on the intracellular DHA peak value, time to reach the peak value, and peak duration; B shows the effects of different culture materials on the intracellular DHA peak value, time to reach the peak value, and peak duration; C and D show the accumulation of lipid droplets in cells after 7 days of continuous culture and statistical analysis.

[0023] Figure 4 These are the test results of the biocompatibility and biological effects of the hydrogel material (PEGD@CF-DHA) that promotes intracellular lipid-lowering of DHA in the embodiments of the present invention; wherein, A and B show the adhesion ability and cell morphology of osteoblasts on the hydrogel material; C and D show the changes and statistical analysis of the accumulation of lipid droplets (green fluorescence) in osteoblasts cultured on the hydrogel material.

[0024] Figure 5 The present invention relates to the effect of a hydrogel material (PEGD@CF-DHA) that promotes intracellular lipid-lowering of DHA on the osteogenic activity of cells under a high-fat environment; wherein, A and B show the effect and statistical analysis of incubation on a hydrogel material on increasing the expression of intracellular alkaline phosphatase (ALP) under a high-fat culture environment; C and D show the effect and statistical analysis of incubation on a hydrogel material on increasing the mineralization ability of cells under high-fat culture conditions.

[0025] Figure 6 The present invention relates to the effect of the hydrogel material (PEGD@CF-DHA) that promotes intracellular lipid-lowering of DHA on the bone repair and regeneration at the local defect caused by the prepared hole after implantation in hyperlipidemia; wherein, A and B show the effect and statistical analysis of the hydrogel material that promotes intracellular lipid-lowering of DHA on increasing the amount of new bone at the local defect caused by the prepared hole as identified by Micro-CT scanning. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0028] Considering that the cellular uptake of DHA is significantly reduced under a high-fat environment, resulting in low lipid droplet efflux efficiency and weak in situ bone regeneration function, the present invention proposes a hydrogel material that promotes intracellular lipid reduction of DHA, and a preparation method and application thereof.

[0029] A typical embodiment of the present invention provides a hydrogel material for promoting intracellular lipid-lowering of DHA, wherein a hydrogel formed by polyethylene glycol diacrylate (PEG-DA) and dithiothreitol (DTT) is used as a scaffold to load nanoparticles, wherein the nanoparticles load docosahexaenoic acid (DHA), and the nanoparticles are composed of chitosan and fucoidan in a mass ratio of 5:(1-10).

[0030] First, DHA is easily oxidized. The present invention uses hydrogel as a scaffold to load nano-microspheres as a loading system for DHA, which can effectively prevent DHA from contacting the external environment, thereby preventing DHA from being oxidized and maintaining the long-term steady state and biological activity of DHA. Studies have shown that DHA is negatively charged in solution. Therefore, the present invention uses chitosan and fucoidan in a mass ratio of 5: (1-10) to form positively charged nano-microspheres, which can load DHA on the nano-microspheres through electrostatic adsorption, which is beneficial to improving the loading efficiency of DHA.

[0031] Secondly, since the nano-microspheres cannot be applied alone locally in the bone defect, and ordinary buffer solutions or the like as carriers cannot prevent the nano-microspheres from being rapidly diluted by blood and tissue fluid, the present invention uses hydrogel as a scaffold-loaded nano-microsphere as a loading system for DHA, and the hydrogel composed of PEG-DA and DTT has the characteristic of temperature response, that is, it is liquid in a low-temperature refrigeration environment (4°C to 10°C), and can be rapidly converted to a hydrogel state in a room temperature to body temperature environment (25°C to 40°C). By utilizing the characteristic of temperature response, the fluidity of the liquid is utilized for injection at low temperatures, and when injected into the target part of the human body, the human body is utilized to heat the material to convert it into a hydrogel state, which is beneficial to reducing surgical wounds and quickly filling local bone defects of different shapes, thereby playing its role in promoting in situ bone regeneration.

[0032] Another embodiment of the present invention provides a method for preparing the above-mentioned hydrogel material for promoting intracellular lipid reduction of DHA, comprising the following steps:

[0033] Mixing chitosan and fucoidan in proportion to prepare nanospheres, and then mixing the nanospheres with DHA to obtain nanospheres loaded with DHA;

[0034] Under the condition of 4°C to 10°C, PEG-DA and DTT are fully mixed in a solution to obtain a precursor solution;

[0035] At 4°C to 10°C, the DHA-loaded nanoparticles are mixed with the precursor solution.

[0036] In some embodiments, chitosan and fucoidan are fully mixed in proportion and then centrifuged at 4° C. to 10° C. to obtain nanospheres, thereby ensuring the stability of the nanospheres under low temperature conditions.

[0037] In some embodiments, the mass ratio of chitosan to fucoidan is 5:(1-6). The mass ratio of chitosan to fucoidan can effectively adjust the loading amount and particle size of the nanospheres for DHA. When the mass ratio is 5:(4.5-5.5), the loading amount of the nanospheres for DHA is higher, and the particle size is more conducive to effectively carrying DHA across the cell membrane.

[0038] In some embodiments, the nanospheres are mixed with a DHA solution, and the concentration of the DHA solution is 1-10 mM. This condition can ensure that the nanospheres are fully loaded with DHA, thereby ensuring the long-term sustained release and effective concentration of DHA.

[0039] In some embodiments, the concentration of the PEG-DA solution is 200-400 mg / mL. Studies have shown that this condition can increase the stiffness of the injectable hydrogel material, thereby further improving its long-term stability after injection into bone defects of different shapes.

[0040] In some embodiments, the concentration of the DTT solution is 30-100 mg / mL, which can ensure the cross-linking effect of the hydrogel and the molding rate of the hydrogel material.

[0041] In some embodiments, the mass ratio of PEG-DA to DTT is (200-400):(0.3-1.0).

[0042] In some embodiments, the mass ratio of nanospheres to PEG-DA is 100:(200-400).

[0043] A third embodiment of the present invention provides a use of the above-mentioned hydrogel material for promoting intracellular lipid-lowering of DHA in the preparation of a product for promoting local bone regeneration.

[0044] Specifically, the product can be a medicine or a medical device.

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0046] Example

[0047] Experimental materials and methods:

[0048] (I) Preparation and characterization of hydrogel materials that promote intracellular lipid-lowering with DHA

[0049] 1. Preparation of nanospheres loaded with active DHA: First, chitosan aqueous solution and fucoidan aqueous solution with a concentration of 1 mg / mL were prepared respectively, and the above two solutions were fully mixed (2 min) at a mass ratio of chitosan to fucoidan of 5:1 to 5:10 (5:1, 5:2, 5:5, 5:10); secondly, nanosphere particles formed by different ratios were obtained by low-temperature high-speed centrifugation (4°C, 14000 rpm, 30 min), and resuspended to an appropriate concentration (1 mg / mL) with triple-distilled water for standby use; the above resuspended nanosphere (CxFy) particle solution was taken and mixed with an equal volume of DHA solution with a concentration of 1 mM, and the nanospheres loaded with active DHA (CxFy-DHA) were obtained again by low-temperature high-speed centrifugation (4°C, 14000 rpm, 30 min); finally, the above CxFy-DHA nanospheres were stored at 4°C for material characterization and further preparation of hydrogel materials.

[0050] 2. Characterization of nanoparticles loaded with active DHA: First, high-performance liquid chromatography (HPLC) was used to detect the encapsulation efficiency of DHA in the nanoparticles (CxFy-DHA); secondly, the particle size, dispersion degree, surface potential and other properties of the nanoparticles (CxFy-DHA) were detected using a Malvern laser particle size analyzer; thirdly, HPLC was used to detect the stability of the nanoparticles (CxFy-DHA) in different buffer solutions and temperatures; finally, transmission electron microscope (TEM) was used to detect the surface morphology and particle size of the nanoparticles (CxFy and CxFy-DHA).

[0051] 3. Preparation of hydrogel materials that promote intracellular lipid-lowering of DHA: First, thoroughly mix 1 mL of 300 mg / mL polyethylene glycol diacrylate (PEG-DA) aqueous solution and 10 μL of 50 mg / mL dithiothreitol (DTT) aqueous solution at 4°C (10 s); secondly, disperse 100 mg of the best-performing nanoparticles (CF-DHA) into the above mixed solution, which can be stored for a long time at 4°C; finally, when it is needed (material characterization, in vitro experiments, in vivo experiments), take an appropriate amount (5 μL) of the mixed solution and inject it into a mold or bone defect of the desired shape, heat it to 37°C, and it can be transformed from liquid to hydrogel within 1 minute.

[0052] 4. Characterization of hydrogel materials that promote intracellular lipid-lowering of DHA: First, the surface morphology and pore size of the hydrogel materials were detected using a scanning electron microscope (SEM); second, degradation experiments were used to detect the time required for complete degradation of the hydrogel materials; finally, HPLC was used to detect the ability of the hydrogel materials to release DHA in different buffers.

[0053] (II) In vitro experimental testing of the biological activity of hydrogel materials that promote intracellular lipid reduction by DHA

[0054] 1. Biocompatibility testing of hydrogel materials that promote intracellular lipid-lowering by DHA: First, osteoblasts were cultured on tissue culture plates (TCP) or hydrogel materials, and given ordinary culture medium or high-fat culture medium. Second, after incubation at 37°C for 3 days, the culture medium was discarded and the cells were fixed with paraformaldehyde. Third, the cytoskeleton (red) and focal adhesion protein (green) of the cells were marked by immunofluorescence staining, and the cell nucleus was counterstained with DAPI (blue). Finally, the cell morphology and adhesion of the cells under different culture conditions were observed under a fluorescence microscope, and further statistical analysis was performed.

[0055] 2. Performance test of hydrogel materials that promote intracellular lipid-lowering of DHA to continuously promote the entry of DHA into cells and reduce the accumulation of lipid droplets: First, osteoblasts were cultured on TCP and incubated in high-fat medium (HF), and DHA solutions were added to different wells to reach final concentrations of 100, 200, 500, and 1000 μM, respectively. Then, HPLC was used to detect the intracellular DHA concentration at different time points (1, 3, 5, and 7 days); secondly, osteoblasts were cultured on TCP, hydrogel PEGD@DHA, and hydrogel PEGD@CF-DHA, respectively, and cultured continuously for 13 days using HF+200 μM DHA culture medium, HF culture medium, and HF culture medium, respectively. Then, HPLC was used to detect the intracellular DHA concentration at different time points (1, 3, 5, 7, 9, and 11 days); finally, the osteoblasts cultured on the above-mentioned different materials were digested with trypsin, and the intracellular lipid droplet accumulation was observed using a transmission electron microscope (TEM).

[0056] 3. Detection of biological activity of hydrogel materials that promote intracellular lipid-lowering by DHA: First, osteoblasts were cultured on tissue culture plates (TCP) or hydrogel materials, given ordinary culture medium or high-fat culture medium, and Bodipy staining was used to detect changes in the accumulation of lipid droplets in osteoblasts cultured on hydrogel materials, and images were collected under a fluorescence microscope for statistical analysis; secondly, osteoblasts were cultured on tissue culture plates (TCP) or hydrogel materials, given ordinary culture medium or high-fat culture medium, and ALP staining was used to detect changes in the expression of ALP in cells, and images were collected under an optical microscope for statistical analysis; finally, osteoblasts were cultured on tissue culture plates (TCP) or hydrogel materials, given ordinary culture medium or high-fat culture medium, and Alizarin red staining was used to detect changes in cell mineralization ability, and images were collected under an optical microscope for statistical analysis.

[0057] (III) In vivo experiments to test the effect of hydrogel materials that promote intracellular lipid-lowering of DHA on bone repair and regeneration at local bone defects in hyperlipidemic mice

[0058] 1. The animal experiments were divided into four groups: the distal femur of normolipidemic mice was prepared with a hole (1.5 mm in diameter and 4 mm in depth), and a titanium implant (1 mm in diameter and 3 mm in length) was implanted, and PBS solution was injected around it, which was the NC group; the distal femur of hyperlipidemia mice was prepared with a hole (1.5 mm in diameter and 4 mm in depth), and a titanium implant (1 mm in diameter and 3 mm in length) was implanted, and PBS solution was injected around it, which was the HF group; the distal femur of hyperlipidemia mice was prepared with a hole (1.5 mm in diameter and 4 mm in depth), and a titanium implant (1 mm in diameter and 3 mm in length) was implanted, and PEGD@CF hydrogel material was injected around it, which was the PEGD@CF group; the distal femur of hyperlipidemia mice was prepared with a hole (1.5 mm in diameter and 4 mm in depth), and a titanium implant (1 mm in diameter and 3 mm in length) was implanted, and PEGD@CF-DHA hydrogel material was injected around it, which was the PEGD@CF-DHA group.

[0059] 2. After killing the mice, microcomputed tomography (Micro CT) was performed on the femoral samples of different groups to detect the local bone defect (peri-implant gap) caused by the prepared hole. After local injection of PEGD@CF-DHA hydrogel material, the effect of long-term sustained release of active DHA on improving the quality of local bone regeneration under hyperlipidemia was investigated.

[0060] Experimental results:

[0061] (I) Preparation and characterization of hydrogel materials that promote intracellular lipid-lowering with DHA

[0062] 1. As Figure 1 As shown in Figure 2, the encapsulation efficiency of DHA in the nanoparticles C5F5-DHA with the best performance can reach 52±3.3% ( Figure 1 A); the particle size of the microspheres is 188±7.7nm ( Figure 1 B), can effectively carry DHA through the cell membrane; its surface potential is 21.4±2.2 and the confidence value meets the detection accuracy requirements of Malvern nanoparticle size analyzer ( Figure 1 C, 2D).

[0063] 2.HPLC test results show that nanoparticles C5F5-DHA have the highest stability in both α-MEM and HF buffers ( Figure 1 E), and its stability is strongest at 25°C and 37°C ( Figure 1 F). The TEM test results show that the particle size of the nanospheres C5F5-DHA is consistent with the results of the Malvern nanoparticle size analyzer, and it shows a granular structure ( Figure 1 G).

[0064] 3. SEM test results show that the surface of the hydrogel material PEGD@CF-DHA that promotes intracellular lipid-lowering of DHA is loose and porous ( Figure 2 A), the pore size is about 137.25±11.1μm ( Figure 2 B), the pore size is suitable for cell migration, growth and differentiation; the degradation experiment results show that the complete degradation time of PEGD@CF-DHA in PBS, α-MEM, HF and other buffers is not significantly different, and all are within 15 to 18 days ( Figure 2 C), which meets the time course requirements of bone metabolism and bone repair at the local bone defect; HPLC test results show that compared with the experimental group using hydrogel material PEGD to load DHA alone, the sustained release time of PEGD@CF-DHA is significantly improved, and it can continue to release active DHA within 10 days in different buffers ( Figure 2 D), showing the superiority of using nanoparticles to preload DHA and then dispersing CF-DHA in PEGD hydrogel in terms of long-term sustained release of active DHA. (II) In vitro experimental detection of the biological activity of hydrogel materials that promote intracellular lipid-lowering with DHA

[0065] 1. The results of immunofluorescence staining showed that the cytoskeleton of osteoblasts cultured in HF medium was clearer and the actin filaments were more orderly when they were cultured on PEGE@CF-DHA, a hydrogel material that promotes intracellular lipid-lowering by DHA, than when they were cultured on TCP alone ( Figure 4 A); The statistical analysis results of the expression of focal adhesion proteins in osteoblasts showed that the expression of focal adhesion proteins in cells incubated on PEGD@CF-DHA was higher, which was 1.3 times and 2.1 times that of the NC group and HF group, respectively ( Figure 4 B), which shows the powerful effect of hydrogel materials in enhancing cell adhesion and maintaining normal cell morphology. At the same time, the above results also show that the hydrogel material PEGD@CF-DHA is non-toxic to cells, that is, it has good biocompatibility.

[0066] 2. If Figure 3 As shown in A, the HPLC test results show that when the extracellular DHA concentration is between 200 and 1000 μM, increasing the extracellular DHA concentration can only accelerate the time for the intracellular DHA concentration to reach its peak, but cannot further increase the intracellular DHA content or prolong the duration of the intracellular DHA peak. Figure 3As shown in Figure B, when the total DHA loading in the culture environment is consistent (200 μM), the hydrogel material PEGD@CF-DHA greatly increases the peak DHA concentration in osteoblasts by long-term sustained release of active DHA and promoting its entry into cells in the form of nanospheres. Compared with the experimental group using only hydrogel PEGD scaffold material to load DHA, the peak DHA concentration in cells increased by 33.3%, and the duration of the peak DHA concentration in cells increased by 4 times. Figure 3 As shown in Figure C, the accumulation of lipid droplets in osteoblasts cultured on different materials on the 7th day was significantly different. Compared with the HF group and the PEGD@DHA group, the accumulation of lipid droplets in osteoblasts incubated on the PEGD@CF-DHA hydrogel material decreased by 80% and 69%. The above experimental results show that the hydrogel material provided by the present invention can promote the entry of active DHA into cells and significantly promote the efflux of lipid droplets in a long-term manner under hyperlipidemia.

[0067] 3. Bodipy fluorescence staining results showed that the accumulation of intracellular lipid droplets in osteoblasts cultured in HF medium was significantly reduced when cultured in PEGE@CF-DHA, a hydrogel material that promotes intracellular lipid-lowering of DHA, compared with osteoblasts cultured in TCP alone. That is, the accumulation of intracellular lipid droplets in the PEGE@CF-DHA group was approximately 23.4% of that in the HF group ( Figure 4 C. Figure 4 D); and the simple use of hydrogel PEGD loaded nanoparticles CF without DHA has no significant effect on the downregulation of intracellular lipid droplet accumulation ( Figure 4 D), which indicates that long-acting sustained-release DHA can effectively promote the efflux of intracellular lipid droplets.

[0068] 4. ALP staining results showed that the expression of ALP in osteoblasts cultured in HF medium was significantly increased when cultured in PEGE@CF-DHA, a hydrogel material that promotes intracellular lipid-lowering of DHA, compared with osteoblasts cultured in TCP alone ( Figure 5 A), that is, the long-term sustained release of active DHA can effectively reshape the osteogenic activity of fruit cells in a high-fat environment ( Figure 5 B); Alizarin red staining results showed that the mineralization level of osteoblasts cultured in HF medium was significantly higher when cultured on PEGE@CF-DHA, a hydrogel material that promotes intracellular lipid-lowering of DHA, than when cultured on TCP alone ( Figure 5 C), and there was no significant difference in cell mineralization levels between the PEGD@CF-DHA group and the NC group ( Figure 5 D), that is, the long-acting sustained-release active DHA effectively reshapes the biological activity of osteoblasts.

[0069] (III) In vivo experiments to test the effect of hydrogel materials that promote intracellular lipid-lowering of DHA on bone repair and regeneration at local bone defects in hyperlipidemic mice

[0070] like Figure 6 As shown in the figure, compared with simply injecting PBS buffer into the peri-implant pores, i.e., the bone defect, injecting the hydrogel PEGD@CF-DHA into the bone defect area significantly increased the amount of new bone ( Figure 6 A); Further comparison of the new bone mass between the NC group and the PEGD@CF-DHA group showed that the injection of hydrogel material into the bone defect of hyperlipidemic mice greatly promoted local bone regeneration, and there was no significant difference in the bone regeneration effect at the local bone defect of mice with normal blood lipids ( Figure 6 B). The above in vivo experimental results show that the hydrogel material PEGD@CF-DHA, which promotes intracellular lipid-lowering of DHA, has good biocompatibility and can significantly improve the in situ bone tissue regeneration effect at the local bone defect of hyperlipidemic mice.

[0071] In summary, the hydrogel material PEGD@CF-DHA that promotes intracellular lipid-lowering of DHA successfully prepared in this embodiment has good biocompatibility, can reduce the accumulation of lipid droplets and the level of reactive oxygen free radicals in local tissue cells under the environment of hyperlipidemia, and improve the in situ bone tissue regeneration effect at the local bone defect.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hydrogel material that promotes DHA intracellular lipid reduction, characterized in that: The hydrogel formed by PEG-DA and DTT is used as a scaffold to load nano-microspheres, the nano-microspheres are loaded with DHA, and the nano-microspheres are composed of chitosan and fucoidan in a mass ratio of 5: (1-10).

2. A method for preparing a hydrogel material for promoting intracellular lipid reduction of DHA according to claim 1, characterized in that: The steps include: Mixing chitosan and fucoidan in proportion to prepare nanospheres, and then mixing the nanospheres with DHA to obtain nanospheres loaded with DHA; Under the condition of 4°C to 10°C, PEG-DA and DTT are fully mixed in a solution to obtain a precursor solution; At 4°C to 10°C, the DHA-loaded nanoparticles are mixed with the precursor solution.

3. The preparation method according to claim 2, characterized in that: After chitosan and fucoidan are fully mixed according to a certain proportion, the mixture is centrifuged at 4°C to 10°C to obtain nano-microspheres.

4. The preparation method according to claim 2, characterized in that: The mass ratio of chitosan to fucoidan is 5:(1-6).

5. The preparation method according to claim 2, characterized in that: The mass ratio of chitosan to fucoidan is 5:(4.5~5.5).

6. The preparation method according to claim 2, characterized in that: The nano-microspheres are mixed with a DHA solution, and the concentration of the DHA solution is 1-10 mM.

7. The preparation method according to claim 2, characterized in that: The concentration of PEG-DA solution is 200-400 mg / mL; Alternatively, the concentration of the DTT solution is 30 to 100 mg / mL.

8. The preparation method according to claim 2, characterized in that: The mass ratio of PEG-DA to DTT is (200-400):(0.3-1.0); Or, the mass ratio of nanospheres to PEG-DA is 100:(200-400).

9. Use of the hydrogel material for promoting intracellular lipid reduction of DHA according to claim 1 in the preparation of a product for promoting local bone regeneration.

10. The use according to claim 9, wherein the product is a medicine or a medical device.

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