DNA-based hydrogel for promoting bone repair as well as preparation method and application of DNA-based hydrogel

Abstract

The invention provides DNA-based hydrogel for promoting bone repair as well as a preparation method and application of the DNA-based hydrogel. The DNA-based hydrogel is prepared from 5% of PEG-NB, MMP degradable peptide, RGD peptide, a nucleic acid aptamer, VEGF and LAP. The DNA hydrogel provided by the invention has good controllable photo-crosslinking property, slow release property, biomineralization ability, biocompatibility and the like; meanwhile, according to the DNA-based hydrogel, the biological function of VEGF, the selectivity and specificity of the nucleic acid aptamer and the unique property of PEG-NB are combined together by utilizing the synergistic effect that VEGF protein promotes angiogenesis of a bone defect part and the nucleic acid aptamer degrades and releases phosphate radicals to promote mineralization, and the effect of promoting bone repair and regeneration of the DNA-based hydrogel is remarkably enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical material preparation, and in particular relates to a DNA-based hydrogel for promoting bone repair, and a preparation method and application thereof. Background Art

[0002] With the continuous development of biomedicine and the continuous improvement of human health awareness, tissue engineering, as an effective means of repairing diseased tissues and organs, has gradually attracted widespread attention. Tissue bioengineering materials must not only support, connect, trigger, and guide, but also provide clues for tissue structural repair and restore physiological status, and be able to make corresponding adjustments and changes through various components and signals emitted from within the body. Therefore, the suitability of a material as a tissue engineering scaffold requires consideration of multiple factors, including biocompatibility, mechanical properties, permeability, stability, degradability, and ability to recognize biological signals. In recent years, the rapid development of DNA nanotechnology has provided a new approach for the preparation of high-performance tissue engineering scaffold materials, which has become one of the most active hotspots in the field of nanobiotechnology and biomaterials.

[0003] DNA-based hydrogels are three-dimensional network structures composed of DNA molecules, with the characteristics of hydration and high water content. They are commonly used in biomedicine, sensors, drug delivery and other fields. DNA-based hydrogels have become a typical representative of DNA construction materials due to their various characteristics and advantages. These characteristics include sequence programmability, precise molecular recognition, ease of functional modification, stimulus responsiveness, biocompatibility and biodegradability. Through sequence design and condition control, such as light, pH, biomolecule response, etc., the DNA assembly structure can be artificially manipulated. The main methods for synthesizing DNA-based hydrogels include self-assembly, chemical cross-linking and physical cross-linking. By designing specific DNA sequences and structures or modifying DNA, the performance and stability of the hydrogel can be enhanced, thereby improving the application effect of DNA-based hydrogels in vivo.

[0004] Patent document CN118767211A discloses an MMP-responsive degradable hydrogel, its preparation method, and application. It was found that the degradation rates of different MMP-responsive degradable hydrogels were significantly correlated with the mechanism of promoting bone regeneration. When L-hydrogel with a faster degradation rate was selected, it could significantly promote cell migration and matrix formation in the bone regeneration tissue of mice, thereby promoting bone repair and regeneration. However, the L-hydrogel prepared in this application is not specific for repairing the damaged site, and its ability to promote bone repair and regeneration is limited, and further improvement and enhancement are needed.

[0005] Therefore, providing a DNA-based hydrogel that specifically targets and repairs bone tissue and further improving the repair effect of DNA-based hydrogels is of great significance for clinical research on promoting bone tissue repair and regeneration. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention provides a DNA-based hydrogel that promotes bone repair, as well as a preparation method and application thereof. Vascular endothelial growth factor (VEGF) and nucleic acid aptamers are innovatively added to the raw materials for preparing the DNA-based hydrogel, and the DNA-based hydrogel is jointly constructed with PEG-NB (8-arm polyethylene glycol-norbornene). The nucleic acid aptamer is degraded to release phosphate to promote mineralization, VEGF protein promotes angiogenesis, and PEG-NB promotes osteogenic differentiation, significantly improving the effect of the DNA-based hydrogel in promoting bone repair and regeneration.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides a DNA-based hydrogel precursor solution, comprising PEG-NB and a nucleic acid aptamer.

[0009] The preparation and application of existing DNA hydrogels are not only complex and time-consuming, but also have limited repair and therapeutic effects. Therefore, to enhance the repair effect of hydrogels on bone tissue, the present invention innovatively uses nucleic acid aptamers and vascular endothelial growth factor (VEGF) to prepare DNA-based hydrogels.

[0010] Based on the characteristics of nucleic acid aptamers that can specifically recognize and bind to target molecules, the present invention innovatively introduces nucleic acid aptamers into the preparation of DNA-based hydrogels. In some embodiments, the present invention uses bivalent nucleic acid aptamers obtained by screening with existing SELEX technology for design and modification. On the one hand, multiple adenine bases are modified at one end of the nucleic acid aptamer, which increases the phosphate released by the degradation of the nucleic acid aptamer and promotes the role of the phosphate in promoting mineralization during the degradation of the DNA-based hydrogel; on the other hand, a thiol group is modified at the other end of the nucleic acid aptamer. The present invention has demonstrated through experiments that the nucleic acid aptamer is grafted with PEG-NB through a thiol-ene click reaction after being modified with a thiol group. This method is not only efficient, but also ensures that the nucleic acid aptamer is stably grafted to the hydrogel body to achieve its functionality.

[0011] Furthermore, the DNA-based hydrogel precursor solution also includes VEGF, and the molar ratio of the VEGF to the nucleic acid aptamer is (1-5):20.

[0012] Vascular endothelial growth factor (VEGF), an important factor that specifically promotes endothelial cell growth and induces angiogenesis, plays a crucial role in bone formation and metabolism, primarily by promoting angiogenesis and participating in bone development. VEGF not only specifically promotes endothelial cell growth and angiogenesis, but also induces endothelial cell division and proliferation. Therefore, addressing the limited effectiveness of existing hydrogels in promoting bone regeneration and repair, the present invention creatively incorporates VEGF into the raw materials used to prepare DNA-based hydrogels and combines VEGF with nucleic acid aptamers to enhance VEGF's ability to bind to its target. Combining the biological functions of VEGF, the selectivity and specificity of nucleic acid aptamers, and the unique properties of PEG-NB, the synergistic effect of these three factors significantly enhances the effectiveness of DNA-based hydrogels in promoting bone regeneration and repair.

[0013] Furthermore, the nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO.1, and one end is modified with a thiol group; the PEG-NB includes an 8-arm PEG modified with an NB group, and the nucleic acid aptamer is grafted to the PEG-NB through a click reaction.

[0014] The nucleic acid aptamer provided by the present invention is modified with a thiol group at one end and then firmly grafted to PEG-NB through a thiol-ene click reaction. By undergoing a thiol-ene click reaction with the NB group on PEG, it is firmly bonded to the PEG backbone in a chemical bond manner, significantly improving the stability of the connection and preventing premature loss of the nucleic acid aptamer. At the same time, the nucleic acid aptamer binds to VEGF with high affinity, thereby ensuring the stable release of VEGF and enhancing the effect of VEGF in promoting angiogenesis and bone regeneration. If only a physical mixing method is used, the nucleic acid aptamer and the targeted VEGF may quickly diffuse into the surrounding environment as the hydrogel swells, resulting in unstable or excessively rapid VEGF release, and failing to achieve the expected sustained-release effect. Therefore, the chemical grafting method provided by the present invention not only ensures the long-term release of VEGF, but also further enhances the functional performance and bone repair effect of the hydrogel, showing significant advantages.

[0015] In some embodiments, the present invention uses 8-arm PEG modified with NB groups, combined with vascular endothelial growth factor and nucleic acid aptamers to construct DNA-based hydrogels. At the same thiol-ene ratio, the 8-arm hydrogels exhibit enhanced mechanical properties. Furthermore, the inclusion of NB groups in the hydrogels allows for spatiotemporally controllable photocrosslinking, facilitating the hydrogel formation process. Therefore, the use of 8-arm PEG to construct hydrogels further enhances the osteogenic differentiation-promoting effect of DNA-based hydrogels.

[0016] Furthermore, the DNA-based hydrogel precursor solution further comprises an MMP-degradable peptide and an RGD peptide, wherein the amino acid sequence of the MMP-degradable peptide is Ac-GCRDGPQGIWGQDRCG-NH2, and the amino acid sequence of the RGD peptide is CGRGDSG.

[0017] Furthermore, the ratio of the PEG-NB is 5%, the PEG-NB and the MMP-degradable peptide are mixed at a thiol-ene ratio of 8:5, and the added amount of the RGD peptide is 2 mM.

[0018] Furthermore, the DNA-based hydrogel precursor solution further includes a photosensitizer LAP, and the proportion of the photosensitizer LAP is 0.05%.

[0019] In another aspect, the present invention provides a DNA-based hydrogel formed by irradiating the DNA-based hydrogel precursor solution described above with ultraviolet light.

[0020] In some embodiments, the present invention demonstrates through experiments the significant advantages of the aforementioned DNA-based hydrogels over existing hydrogels. Results show that a hydrogel constructed from 5% PEG-NB, an MMP-degradable peptide, an RGD peptide, a nucleic acid aptamer, VEGF165, and LAP exhibits excellent controllable photocrosslinking, mechanical properties, and biocompatibility. Furthermore, compared to PV hydrogels, PAV hydrogels (DNA-based hydrogels) are able to achieve sustained VEGF release; compared to P hydrogels, PAV hydrogels also exhibit stronger biomineralization capacity. Comparative experiments on hydrogel-induced bone regeneration also demonstrate that PAV hydrogels significantly promote bone regeneration and repair compared to the other three hydrogels. This may be due to the synergistic effect of PAV hydrogels, which utilizes the localized sustained release of VEGF to promote angiogenesis at the bone defect site, and the release of phosphate groups from the degradation of nucleic acid aptamers to promote mineralization.

[0021] Therefore, in order to reveal the mechanism of DNA-based hydrogels affecting osteogenesis (promoting bone repair and regeneration), the present invention further performed RNA sequencing on bone regeneration tissues and performed GO enrichment analysis and KEGG analysis on the differentially expressed genes in PAV hydrogels. GO classification showed the enrichment of differentially expressed genes in three major GO categories: biological process (BP), cellular component (CC), and molecular function (MF): (1) In biological process, genes were mainly enriched in metabolic process, cellular process, developmental process, and stimulus response, indicating that DNA-based hydrogels may promote tissue regeneration by regulating cell metabolism and repair processes; (2) In cellular components, target genes were related to cell anatomical structures and protein complexes, suggesting that DNA-based hydrogels may affect the physical structure of cells and the stability of complexes; (3) In molecular function, genes with catalytic activity and binding activity were most enriched, indicating that DNA-based hydrogels may promote metabolic reactions and molecular transport by regulating key enzyme activities and molecular binding processes. KEGG pathway analysis revealed a significant increase in gene enrichment in signal transduction, immune system, and metabolism-related pathways in the DNA-based hydrogel compared to the control group, suggesting that the DNA-based hydrogel may promote cell repair and tissue regeneration by regulating signal transduction, metabolic balance, and immune regulation. These results provide reliable molecular evidence for the biological function and mechanism of action of DNA-based hydrogels in promoting bone tissue regeneration, suggesting promising application prospects.

[0022] In another aspect, the present invention provides a use of a hydrogel for preparing an agent for promoting bone repair, wherein the hydrogel comprises a nucleic acid aptamer and PEG-NB, wherein the nucleic acid aptamer is grafted to the PEG-NB through a click reaction.

[0023] Furthermore, the hydrogel further comprises VEGF, MMP-degradable peptide, RGD peptide and photosensitizer LAP.

[0024] In another aspect, the present invention provides a use of a nucleic acid aptamer for preparing a DNA-based hydrogel precursor solution, wherein the DNA-based hydrogel precursor solution consists of PEG-NB, a nucleic acid aptamer, VEGF, an MMP-degradable peptide, and an RGD peptide.

[0025] Furthermore, the nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO.1, and one end is modified with a thiol group.

[0026] In another aspect, the present invention provides the use of nucleic acid aptamers for preparing DNA-based hydrogels for sustained release of VEGF, wherein the DNA-based hydrogel is formed by irradiating a precursor solution consisting of PEG-NB, nucleic acid aptamers, VEGF, MMP-degradable peptides and RGD peptides with ultraviolet light.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention innovatively adds vascular endothelial growth factor (VEGF) to the raw materials for preparing DNA hydrogel, and utilizes VEGF to promote angiogenesis at the bone defect site to enhance the effect of hydrogel in promoting bone repair.

[0029] 2. VEGF binds to nucleic acid aptamers to improve VEGF's ability to bind to targets. At the same time, the phosphate released by the degradation of nucleic acid aptamers is used to promote mineralization. VEGF and nucleic acid aptamers significantly enhance the hydrogel's ability to promote bone repair and regeneration through synergistic enhancement.

[0030] 3. The hydrogel main body PEG-NB is grafted with nucleic acid aptamers through a thiol-ene click reaction, and the nucleic acid aptamers then specifically bind to VEGF, achieving stable connection of the nucleic acid aptamer to PEG-NB and continuous release of VEGF. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the H NMR spectrum of 8-arm PEG-NB;

[0032] Figure 2 The injectability and photocrosslinking properties of the four hydrogels of the present invention;

[0033] Figure 3 These are cryo-scanning electron microscopy images of four hydrogels of the present invention;

[0034] Figure 4 is the rheological property of PEG-NB hydrogel;

[0035] Figure 5A is the surface stiffness of P hydrogel;

[0036] Figure 5B is the mechanical properties of PA hydrogel;

[0037] Figure 5C The mechanical properties of PV hydrogels;

[0038] Figure 5D is the mechanical properties of PAV hydrogel;

[0039] Figure 5E Mechanical properties analysis of four types of hydrogels;

[0040] Figure 6 DNA staining images of P hydrogel and PAV hydrogel;

[0041] Figure 7 is the swelling properties of the four hydrogels of the present invention;

[0042] Figure 8 The degradation properties of the four hydrogels of the present invention;

[0043] Figure 9 is the VEGF release rate of PV hydrogel and PAV hydrogel;

[0044] Figure 10 TEM images of P hydrogel and PAV hydrogel after mineralization;

[0045] Figure 11 Live / dead staining of BMSC cells after co-culture with the four hydrogels of the present invention;

[0046] Figure 12 CCK-8 assay was used to quantitatively analyze BMSC cell activity after co-culture with four hydrogels;

[0047] Figure 13 Live / dead staining of HUVEC cells after co-culture with the four hydrogels of the present invention;

[0048] Figure 14 For CCK-8 assay, quantitative analysis of HUVEC cell activity after co-culture with four hydrogels was performed;

[0049] Figure 15 The staining results of the four hydrogels of the present invention and alkaline phosphatase (ALP);

[0050] Figure 16 The staining results of the four hydrogels of the present invention and Alizarin Red (ARS);

[0051] Figure 17 Results of the analysis of the osteogenic effects of four hydrogels in vivo;

[0052] Figure 18 GO enrichment of RNA sequencing results of in vivo regenerated bone tissue of PAV hydrogel and Control hydrogel;

[0053] Figure 19 KEGG analysis of RNA sequencing results of in vivo regenerated bone tissue of PAV hydrogel and Control hydrogel. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below with reference to the examples. It should be noted that the examples described below are intended to facilitate understanding of the present invention and do not have any limiting effect on the present invention.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0056] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0057] Example 1. Preparation of DNA-based hydrogel (PAV hydrogel) provided by the present invention

[0058] (1) Preparation of 8-arm PEG-NB

[0059] 200 mg of 8-arm PEG-COOH (8-arm carboxyl polyethylene glycol), 153.4 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide iodide), and 92.07 mg of NHS (N-hydroxysuccinimide) were dissolved in 15 ml of DCM and magnetically stirred at room temperature for 3 hours. Then, 98.56 mg of NB-NH2 (β-naphthylamine) was added to the mixture, and magnetic stirring was continued for 72 hours. After the reaction, the DCM solvent was removed by rotary evaporation. The resulting solution was dissolved in 20% THF (tetrahydrofuran) and placed in a dialysis bag (MWCO: 3000 Da). Excess impurities were removed by gradient dialysis: dialyzed against 20%, 15%, 10%, and 5% THF solutions for 6 hours, followed by dialysis against deionized water for 3 days, with the water changed regularly. The purified solution was then filtered through a 0.8 μm filter to obtain a purified PEG-NB solution. The solution was placed in liquid nitrogen for quick freezing and then freeze-dried at -80°C. The resulting polymer (8-arm PEG-NB) was then 1 H-NMR identification ( Figure 1 ).

[0060] (2) Design and synthesis of nucleic acid aptamers

[0061] The bivalent nucleic acid aptamer sequence obtained by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) screening was designed and modified with reference to the method in Son J, Kim J, Lee K, et al. DNA aptamerimmobilized hydroxyapatite for enhancing angiogenesis and bone regeneration. Acta Biomater. 2019(99), 469-478. A 3′-terminal thiol-modified nucleic acid aptamer was synthesized. The sequence (SEQ ID NO. 1) is shown below:

[0062] 5′-AAAAAAAAAAAAAAAAAAAAAAAA-TGTGGGGGTGGACTGGGTGGGTACC TTTTTTTTTTTGTGGGGGTGGACTGGGTGGGTACC-3′, where the 3′ sulfhydryl group is modified.

[0063] (3) Preparation of PAV hydrogel precursor solution

[0064] The PAV hydrogel precursor solution consisted of 5% 8-arm PEG-NB, MMP-degradable peptide (Ac-GCRDGPQGIWGQDRCG-NH2), RGD peptide (CGRGDSG), nucleic acid aptamer, VEGF165 and LAP.

[0065] An 8-arm PEG-NB and an MMP-degradable peptide were mixed at a thiol-ene ratio of 8:5 to form a solution. 2 mM RGD peptide, 0.05% LAP, and 50 ng / ml VEGF were then added to the solution at a 1:20 molar ratio of VEGF to aptamer to form a PAV hydrogel precursor solution. The PAV hydrogel precursor solution was then irradiated with 365 nm ultraviolet light for 30 seconds to form a PAV hydrogel.

[0066] Example 2. Preparation of P hydrogel

[0067] The P hydrogel precursor solution consisted of an 8-arm PEG-NB and an MMP-degradable peptide mixed at a thiol-ene ratio of 8:5, followed by the addition of 2 mM RGD peptide and 0.05% LAP. The 8-arm PEG-NB was prepared using the same method as in Example 1. The P hydrogel precursor solution was irradiated with 365 nm ultraviolet light for 30 seconds to form a P hydrogel.

[0068] Example 3. Preparation of PA hydrogel

[0069] A PA hydrogel precursor solution was prepared by mixing 8-arm PEG-NB and an MMP-degradable peptide at a thiol-ene ratio of 8:5, followed by the addition of 2 mM RGD peptide, 0.05% LAP, and a nucleic acid aptamer. The amount of the nucleic acid aptamer added was the same as in Example 1. The preparation method of the 8-arm PEG-NB and the sequence of the nucleic acid aptamer were the same as in Example 1. The PA hydrogel precursor solution was irradiated with 365 nm ultraviolet light for 30 seconds to form a PA hydrogel.

[0070] Example 4. Preparation of PV hydrogel

[0071] A PV hydrogel precursor solution was prepared by mixing 8-arm PEG-NB and an MMP-degradable peptide at a thiol-ene ratio of 8:5, followed by the addition of 2 mM RGD peptide, 0.05% LAP, and 50 ng / ml VEGF. The 8-arm PEG-NB was prepared using the same method as in Example 1. The PV hydrogel precursor solution was irradiated with 365 nm ultraviolet light for 30 seconds to form a PV hydrogel.

[0072] Example 5: Characterization of hydrogel properties

[0073] The present invention verifies the material properties of the four hydrogels prepared in Examples 1-4, including injectability, porous structure, rheological behavior, degradation performance and biocompatibility.

[0074] (1) The hydrogel has good controllable photocrosslinking, injectability and porous structure

[0075] A portion of the precursor solution was taken up with a pipette, irradiated with 365 nm UV light for 30 s, and then the mold was removed. It can be seen that the hydrogel has been formed ( Figure 2 ), indicating that the four hydrogels have good controllable photocrosslinking and injectability.

[0076] In order to evaluate the internal microstructure of the hydrogel, scanning electron microscopy (SEM) was used to observe its morphology and pore structure. After the hydrogel sample was freeze-dried, a gold film was coated on its surface using an ion sputtering device to enhance conductivity. The sample was imaged by cryo-SEM, and the results showed that ( Figure 3 ), all four hydrogels exhibited a typical three-dimensional porous network structure with uniform pore size distribution and similar pore sizes, with an average pore size range of approximately 15 μm. This uniform pore size distribution facilitates the diffusion of nutrients and cell penetration, providing a favorable microenvironment for cell adhesion and proliferation. This demonstrates that the four hydrogels prepared in Examples 1-4 have good internal structural controllability and biocompatibility.

[0077] (2) Hydrogel has good rheological and mechanical properties

[0078] The precursor solution of the hydrogel was placed on a rheometer for scanning to determine the rheological properties of the hydrogel, and after scanning, 365 nm ultraviolet light was applied for 30 seconds to crosslink the hydrogel. Figure 4 It can be seen from the rheological behavior curves of the four hydrogels that there is no obvious difference in the storage modulus and loss modulus, and the storage modulus increases rapidly and exceeds the loss modulus after the UV is turned on. Both can remain stable after gelation.

[0079] The rheometer can observe the cross-linking process of the hydrogel. As can be seen from the figure, before the UV light is turned on, the precursor solution is in a liquid state. When the UV light is turned on around 3 seconds later, the storage modulus increases rapidly and intersects with the loss modulus. This indicates that the hydrogel has transformed into a solid state, which takes about 1 second after the UV light is turned on. After about 20 seconds, the storage modulus and loss modulus of the hydrogel remain stable, indicating that the hydrogel has entered a solid state.

[0080] The Young's modulus of the surfaces of different hydrogels was detected using a nanoindenter and further plotted. As shown in Figure 5 , the surface stiffness of the four hydrogels was different, and there was no obvious difference in the mechanical properties of the four hydrogels.

[0081] (3) Hydrogel has good loading and dispersion capabilities

[0082] To evaluate the distribution of aptamers in DNA-based hydrogels, aptamers in PAV hydrogels were labeled with SYBR Green dye and imaged using confocal laser scanning microscopy (CLSM). The hydrogels were incubated with SYBR Green solution for 30 minutes and then washed three times with PBS to remove unbound dye. The stained hydrogel samples were placed on confocal microscope slides and imaged using a confocal laser scanning microscope at an excitation wavelength of 488 nm.

[0083] like Figure 6 As shown in the results, the SYBR Green-labeled nucleic acid aptamer exhibited a uniform green fluorescence distribution inside the DNA-based hydrogel, with no obvious agglomeration or uneven deposition, indicating that the nucleic acid aptamer was evenly distributed in the DNA-based hydrogel, confirming the good DNA loading and dispersion capabilities of the PAV hydrogel.

[0084] (4) Hydrogel has good swelling and degradation properties

[0085] In order to clarify the swelling and degradation properties of the four hydrogels, the four hydrogels were freeze-dried and added to PBS and different concentrations of type I collagenase solution (0.1U / ml and 0.01U / ml), and placed in a constant temperature shaker. The swelling properties of the four hydrogels ( Figure 7 ) and degradation rate ( Figure 8 ) had no significant difference.

[0086] (5) Hydrogel has good biocompatibility

[0087] In order to verify that the four hydrogels have good biocompatibility, the proliferation ability and activity of BMSCs and HUVEC cells after co-culture with the four hydrogels were detected using CCK-8 kit and AM / PI staining reagent. Figure 9 and Figure 10 As shown in Figure 3, compared with the control group, the morphology of BMSCs was normal after being co-cultured with the hydrogels in each group for 2 days, and the cell activity at 450 nm had no significant difference compared with the control group; Figure 11 、 12 As shown in the figure, HUVECs maintained good cell activity and normal morphology after being co-cultured with each group of hydrogels for 2 days, indicating that the four hydrogels were non-cytotoxic and could maintain cell activity and proliferation.

[0088] Example 6: Characterization of sustained-release properties and biomineralization ability of PAV hydrogel

[0089] To measure the release profile of VEGF after binding to the aptamer, the PAV hydrogel from Example 1 and the PV hydrogel from Example 4 were placed in separate 50-ml centrifuge tubes, sealed, and placed in a release medium containing PBS (pH 7.4). The tubes were then gently shaken in a 37°C constant-temperature shaker. 2 mL of the release medium was sampled at time points 1, 2, 3, 5, 7, 9, and 14 days and replenished with an equal volume of fresh PBS. The VEGF concentration in the release medium was determined using a VEGF ELISA kit. The released amount was calculated based on a standard curve, and the cumulative release rate was expressed as a percentage of the initial VEGF loading. Finally, a VEGF release curve was plotted with release time as the abscissa and cumulative release rate as the ordinate to evaluate its release kinetics.

[0090] like Figure 13 As shown in the data, the VEGF release rate in PV hydrogel was faster, and the VEGF in the hydrogel was completely released on the 9th day, while the VEGF release rate in PAV hydrogel was slower and the release time was longer. The reason may be that in PAV hydrogel, after the nucleic acid aptamer specifically binds to VEGF, the nucleic acid aptamer is stably grafted to the hydrogel body through the thiol-ene click reaction, avoiding the premature loss of VEGF and thus achieving the effect of sustained sustained release.

[0091] The present invention further evaluated the mineralization ability of PAV and P hydrogels in simulated body fluid (SBF). The PAV hydrogel from Example 1 and the P hydrogel from Example 2 were immersed in SBF at 37°C for three days. After immersion, the samples were removed, gently rinsed three times with deionized water, and then freeze-dried at room temperature to remove moisture. The dried hydrogel samples were then observed using transmission electron microscopy (TEM).

[0092] TEM imaging results show that ( Figure 14 ), the PAV hydrogel showed a large number of mineralized particles with irregular spherical or lamellar shapes, similar in morphology and crystal structure to hydroxyapatite (HAp). In contrast, the P hydrogel showed only a small number of amorphous particles with no obvious HAp characteristics. This result indicates that the PAV hydrogel exhibited a stronger biomineralization ability in SBF. Furthermore, during the experiment, the present inventors found that the PV hydrogel, due to the lack of nucleic acid aptamers, did not promote mineralization.

[0093] Example 7: Effect of hydrogel on bone regeneration

[0094] In order to evaluate the promoting effect of the materials on osteogenic differentiation, alkaline phosphatase (ALP) activity detection experiments were used to analyze the differences in the effects of the four hydrogels prepared in Examples 1-4. Bone marrow mesenchymal stem cells (BMSCs) and HUVECs were seeded in 24-well plates containing the materials at a seeding density of 5×10 4 Cells were plated at 400 nmol / well and cultured in a medium containing osteogenic induction solution for 7 and 14 days. At the designated time points, the medium was discarded, and the cells were washed three times with PBS. An ALP activity assay kit was used, and the reaction was allowed to proceed for 30 minutes. The cells were then photographed using a microplate reader. The activity and differentiation status of the osteoblasts were assessed based on the staining results of the BMSCs.

[0095] like Figure 15 As shown, the cells in the PAV group were the darkest in color, reflecting the strongest cell differentiation and bone formation abilities, indicating that the PAV hydrogel had a better effect in promoting bone differentiation.

[0096] Alizarin red (ARS) staining was further used to evaluate the promoting effect of the four hydrogels on mineralization ability. BMSCs were seeded in a 12-well plate containing the materials at a cell seeding density of 5×10 4 Cells were placed in a PBS-containing culture medium for 21 days. After culture, the culture medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes and then washed three more times with PBS. The cells were stained with 0.1% Alizarin Red S for 20 minutes. After removing excess dye, the cells were repeatedly washed with PBS until no free dye remained. The cells were then photographed using a microplate reader. The BMSC staining results were used to assess the formation of mineralized nodules by osteoblasts.

[0097] like Figure 16 The results showed that the cells in the PAV group were the darkest in color, reflecting the strongest mineralization ability, indicating that PAV hydrogel has a good mineralization-promoting effect.

[0098] To further evaluate the in vivo osteogenic effects of the four hydrogels, this example further examined skull defect modeling in SD rats. A 5 mm diameter skull defect was drilled into the rat skull. The hydrogel was then injected into the defect. Once the hydrogel filled the defect area, it was irradiated with 365 nm UV light to form a gel. Three rats from each group were sacrificed by cervical dislocation at 6 and 12 weeks post-surgery. Skull specimens were removed and fixed in 4% paraformaldehyde for 48 hours. Micro-CT scans and reconstructions were then performed.

[0099] from Figure 17It can be seen that compared with the control group, PAV hydrogel can significantly promote bone regeneration, while the P and PA groups have no obvious promoting effect. The effect of PV hydrogel in promoting bone repair and regeneration is not as good as that of the PAV group. The reason is that PAV hydrogel contains both VEGF and nucleic acid aptamers, and the local sustained release of VEGF and the phosphate released by the degradation of nucleic acid aptamers jointly promote mineralization. The two enhance the bone regeneration effect through synergistic action. In particular, the sustained release of VEGF can significantly promote angiogenesis at the bone defect site, providing the necessary nutrient supply for bone repair. Combined with the mineralization effect of nucleic acid aptamers, a significant bone regeneration effect is achieved. In summary, the DNA-based hydrogel (PAV hydrogel) provided by the present invention has significant advantages in promoting bone repair and regeneration.

[0100] Example 8: Study on the mechanism of hydrogel-promoting osteogenesis

[0101] To further reveal the mechanism of PAV hydrogel's effect on osteogenesis, we performed RNA sequencing on bone regeneration tissues and performed GO enrichment analysis and KEGG analysis on differentially expressed genes in DNA-based hydrogels. The GO classification of target genes showed the enrichment of differentially expressed genes in three GO categories: biological process (BP), cellular component (CC), and molecular function (MF). Figure 18 ). In biological processes, genes were mainly enriched in metabolic processes, cellular processes, developmental processes, and stimulus responses, indicating that DNA-based hydrogels may promote tissue regeneration by regulating cellular metabolism and repair processes. Among cellular components, target genes were related to cellular anatomical structures and protein complexes, suggesting that DNA-based hydrogels may affect the physical structure of cells and the stability of complexes. Among molecular functions, genes with catalytic activity and binding activity were the most enriched, indicating that DNA-based hydrogels may promote metabolic reactions and molecular transport by regulating key enzyme activities and molecular binding processes. These results reveal the potential molecular mechanisms of DNA-based hydrogels in promoting cellular function, metabolic activity, and tissue regeneration.

[0102] The enrichment results of KEGG pathways showed that ( Figure 19 ), with the main pathways including metabolic pathways (such as lipid metabolism, amino acid metabolism, and glycosylation biosynthesis), cellular processes (such as cell migration and cell colonies), signal transduction (such as signal molecules and interactions, signal transduction), and disease-related pathways (such as infectious diseases, neurodegenerative diseases, and endocrine and metabolic diseases). The bar graph shows that compared with the control group, the number of genes enriched in signal transduction, immune system, and metabolic-related pathways in DNA-based hydrogels increased significantly, indicating that DNA-based hydrogels may promote cell repair and tissue regeneration by regulating signal transduction, metabolic balance, and immune regulation. These results provide reliable molecular evidence for the biological function and mechanism of action of DNA-based hydrogels in promoting bone tissue regeneration and have potential application value.

[0103] Example 9: Design and screening of nucleic acid aptamer sequences

[0104] According to a 3R02 bivalent aptamer reported in existing literature, the sequence is 5′-TGTGGGGGTGGACTGGTGGGTACCTTTTTTTTTTTGTGGGGGTGGACTGGTGGG TACC-3′, which shows high binding affinity for VEGF. The present invention designs and modifies this aptamer based on its sequence and evaluates the differences in sustained-release properties between DNA-based hydrogels prepared after binding VEGF with aptamers of different sequences. In this example, the four aptamers shown in Table 1 were used to prepare DNA-based hydrogels. Other preparation methods and steps were essentially the same as in Example 1. The hydrogels were compared with the PAV hydrogels of Example 1. The VEGF release from the two hydrogels was recorded and the release rate was calculated according to the method of Example 6. The test results are shown in Table 1.

[0105] Table 1 VEGF release rate in hydrogel ① and hydrogel ② (%)

[0106]

[0107] As can be seen in Table 1, the VEGF release rate in hydrogels ② and ③ is faster than that in hydrogel ① (PAV hydrogel). In the same period of time, hydrogels ② and ③ release more VEGF. It is speculated that this may be because the improved nucleic acid aptamer sequence of the present invention contains multiple adenine bases, which gives it a higher affinity and stronger binding force with VEGF, ensuring the stable release of VEGF. Therefore, the use of the nucleic acid aptamer designed by the present invention to construct DNA-based hydrogels can further enhance the role of VEGF in promoting angiogenesis and bone regeneration, and the effect is better.

[0108] Example 10, Screening and Optimization of Grafting Methods

[0109] During the research process, the present inventors discovered that because VEGF itself forms disulfide bonds and cannot react with the double bond of the NB group on the 8-arm PEG, only nucleic acid aptamers can be grafted onto the 8-arm PEG-NB. The grafting of nucleic acid aptamers onto the hydrogel host 8-arm PEG-NB has a significant impact on the stability of the nucleic acid aptamer and the sustained release of VEGF therein. Therefore, the present inventors further evaluated the effects of different grafting methods on the stability of the nucleic acid aptamer. Two different hydrogels were prepared according to the method of Example 1, using chemical grafting using a thiol click reaction and physical mixing, respectively. The VEGF release rate of the two hydrogels was analyzed using the method of Example 6. The analysis results are shown in Table 2.

[0110] Table 2 Effect of different grafting methods on VEGF release rate

[0111]

[0112] As shown in Table 2, the VEGF release rate from the DNA-based hydrogel prepared by Method 1 was slower than that from Method 2. This may be because the thiol-ene click reaction used to graft the aptamer to the 8-arm PEG-NB firmly secures the aptamer to the hydrogel, preventing premature loss. Furthermore, the high-affinity binding between the aptamer and VEGF forms a single entity, resulting in sustained VEGF release. However, using only physical mixing (Method 2) would result in the aptamer and targeted VEGF rapidly diffusing into the surrounding environment as the hydrogel swells, leading to unstable or excessively rapid VEGF release and failure to achieve the desired sustained release effect. In summary, only chemical grafting can ensure the stable release of VEGF, thereby further enhancing its angiogenesis-promoting and bone regeneration effects.

[0113] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0114]

Claims

1. A DNA-based hydrogel precursor solution, characterized in that: The precursor solution includes PEG-NB and nucleic acid aptamer.

2. The DNA-based hydrogel precursor solution according to claim 1, wherein It also includes VEGF, and the molar ratio of VEGF to nucleic acid aptamer is (1-5):

20.

3. The DNA-based hydrogel precursor solution according to claim 2, wherein The nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO.1, and one end is modified with a thiol group; the PEG-NB includes an 8-arm PEG modified with an NB group, and the nucleic acid aptamer is grafted to the PEG-NB through a click reaction.

4. The DNA-based hydrogel precursor solution according to claim 3, wherein It also includes an MMP-degradable peptide and an RGD peptide. The amino acid sequence of the MMP-degradable peptide is Ac-GCRDGPQGIWGQDRCG-NH2, and the amino acid sequence of the RGD peptide is CGRGDSG.

5. The DNA-based hydrogel precursor solution according to claim 4, wherein The ratio of PEG-NB is 5%, The PEG-NB and the MMP-degradable peptide were mixed at a thiol-ene ratio of (2-7):5, and the added amount of the RGD peptide was 2 mM.

6. The DNA-based hydrogel precursor solution according to claim 5, wherein The composition further includes a photosensitizer LAP, and the proportion of the photosensitizer LAP is 0.05%.

7. A DNA-based hydrogel, characterized in that The DNA-based hydrogel precursor solution according to any one of claims 1 to 6 is irradiated with ultraviolet light to form the hydrogel.

8. Use of a hydrogel for preparing an agent for promoting bone repair, characterized in that: The hydrogel comprises a nucleic acid aptamer and PEG-NB, and the nucleic acid aptamer is grafted to the PEG-NB through a click reaction.

9. Use of nucleic acid aptamers for preparing DNA-based hydrogel precursor solutions, characterized in that: The DNA-based hydrogel precursor solution consists of PEG-NB, nucleic acid aptamer, VEGF, MMP-degradable peptide and RGD peptide.

10. Use of nucleic acid aptamers for preparing DNA-based hydrogels for sustained release of VEGF, characterized in that: The DNA-based hydrogel is formed by irradiating a precursor solution consisting of PEG-NB, nucleic acid aptamer, VEGF, MMP-degradable peptide and RGD peptide with ultraviolet light.

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