Antioxidant gelatin sponge and its preparation method

Abstract

The present application relates to the technical field of biology, and particularly relates to an anti-oxidation gelatin sponge and a preparation method thereof. The preparation method comprises the following steps: preparing a lipid nanoparticle containing three nucleic acids of Oct4, Sox2 and Klf4; grafting phenylboronic acid to a gelatin skeleton to obtain a phenylboronic acid gelatin; dissolving the phenylboronic acid gelatin in a lipid nanoparticle solution to form a mixed solution, then adding a tannic acid solution and a hyaluronic acid solution into the mixed solution in sequence to obtain the anti-oxidation gelatin sponge. The anti-oxidation gelatin sponge can be directly implanted or injected into a high fracture site (such as a vertebral body or a femoral neck) of an osteoporosis patient, and can achieve the purposes of treating osteoporosis and preventing first or secondary fracture by long-term regulation of local bone metabolism, reduction of bone resorption and increase of bone density.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antioxidant gelatin sponge and its preparation method. Background Technology

[0002] Osteoporosis is a systemic metabolic bone disease characterized by low bone mass, bone microarchitectural deterioration, and increased bone fragility. It is the leading cause of significantly increased fracture risk in the elderly, especially postmenopausal women. Osteoporotic fractures (also known as fragility fractures) most commonly occur in the vertebrae, hip, and distal radius, and are characterized by high morbidity, high disability rate, and high mortality rate. Statistics show that approximately one-third of women and one-fifth of men over the age of 50 will experience at least one osteoporotic fracture in their lifetime, placing a heavy burden on patients and the social healthcare system.

[0003] Currently, the main strategies for treating osteoporosis in clinical practice fall into two categories: anti-resorption drugs (such as bisphosphonates, denosumab, and selective estrogen receptor modulators) and bone-forming drugs (such as teriparatide and romosomab). These drugs are usually administered systemically (orally or by injection). While they effectively reduce fracture risk, they share several common problems: long-term use of bisphosphonates may lead to atypical femoral fractures and osteonecrosis of the mandible; rebound bone loss occurs after discontinuation of denosumab; teriparatide requires daily subcutaneous injections and has a limited treatment duration (not exceeding 24 months); systemic administration may cause gastrointestinal reactions and flu-like symptoms. Furthermore, systemic administration makes it difficult to achieve effective drug concentrations in high-risk fracture areas (such as the vertebral body and femoral neck), and patient compliance is poor.

[0004] Local intervention strategies represent a new direction for preventing osteoporotic fractures. For example, while injecting bone cement into the vertebral body during percutaneous vertebroplasty (PVP) and kyphoplasty (PKP) can relieve pain and stabilize fractures, bone cement lacks bioactivity, cannot improve local bone metabolism, and carries the risk of leakage. In recent years, injectable biomaterials have shown potential as drug-releasing carriers or bone metabolism modulators in the local treatment of osteoporosis. Ideal materials should possess: good biocompatibility and biodegradability; the ability to modulate local oxidative stress and the inflammatory microenvironment; inhibition of osteoclast activity; promotion of osteoblast differentiation; and the ability to provide mechanical support and long-term drug release at high-fracture sites.

[0005] Therefore, existing technologies need to be improved. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide an antioxidant gelatin sponge and its preparation method, aiming to solve at least one of the following problems: existing anti-osteoporosis drugs have large systemic side effects, poor local targeting, lack of active bone metabolism regulation ability of materials, and inability to simultaneously treat and prevent fractures.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing an antioxidant gelatin sponge, comprising the following steps: S1. Preparation of lipid nanoparticles containing three nucleic acids: Oct4, Sox2, and Klf4; S2. Benzylboronic acid is grafted onto the gelatin backbone to prepare phenylboronic acid gelatin. S3. Dissolve the phenylboronic acid gelatin in a lipid nanoparticle solution to form a mixed solution, and then add tannic acid solution and hyaluronic acid solution to the mixed solution in sequence to obtain the antioxidant gelatin sponge.

[0008] Optionally, the preparation of lipid nanoparticles containing the three nucleic acids Oct4, Sox2, and Klf4 includes the following steps: S11. Prepare lipid organic phase solution; S12. Dissolve the three nucleic acids Oct4, Sox2, and Klf4 in a buffer solution (such as sodium citrate buffer) to form an aqueous solution of the three nucleic acids. S13. Mix the lipid organic phase solution and the trinucleotide aqueous phase solution to obtain a preliminary mixture of lipid nanoparticles loaded with trinucleotides; S14. The initial mixture of lipid nanoparticles loaded with three nucleic acids is filtered, concentrated, and impurities are removed to obtain the lipid nanoparticles loaded with the three nucleic acids.

[0009] Optionally, the mass ratio of Oct4, Sox2, and Klf4 is 1-3:1:1.

[0010] Optionally, the preparation of the lipid organic phase solution includes: dissolving ionizable lipids (cationic lipid DLin-MC3-DMA), auxiliary lipids (DSPC), cholesterol, and polyethylene glycol-modified lipids (DSPE-PEG2000) together in anhydrous ethanol, and dissolving them thoroughly by vortexing and water bath to obtain the lipid organic phase solution.

[0011] Optionally, the molar ratio of ionizable lipids (cationic lipid DLin-MC3-DMA), cofactor lipids (DSPC), cholesterol, and PEGylated lipids (DSPE-PEG2000) is 55:5:38.5:1.5.

[0012] Optionally, the lipid organic phase solution and the trinucleotide aqueous phase solution can be mixed using a microfluidic method for precise mixing. The flow rate ratio of the two is 3:1 (aqueous phase:organic phase) and injected into the Y-type microfluidic chip, with the total flow rate set to 9-12 mL / min.

[0013] Optionally, the filtration, concentration, and impurity removal include: diluting the initial mixture of lipid nanoparticles loaded with three nucleic acids 20 times with PBS and transferring it to a pretreated 5kDa molecular weight cutoff ultrafiltration tube, and continuously ultrafiltration and concentration at 4°C to completely remove residual ethanol, free mRNA, and unencapsulated lipids.

[0014] Optionally, phenylboronic acid is grafted onto a gelatin backbone to prepare phenylboronic acid gelatin, comprising the following steps: S21. Dissolve gelatin in buffer solution, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and activate at room temperature for 30-60 min to obtain activated gelatin. S22. Add aminophenylboronic acid to the activated gelatin and react at room temperature for 12-24 hours. S23, filtration, dialysis, and freeze-drying yield phenylboronic acid gelatin.

[0015] Specifically, gelatin is dissolved in deionized water before being dissolved in the buffer solution.

[0016] Specifically, the buffer solution is 2-morpholinoethanesulfonic acid (MES).

[0017] Optionally, the mass ratio of phenylboronic acid gelatin in the mixed solution is 1-2%.

[0018] Optionally, the volume ratio of tannic acid solution to mixed solution is 1-2:1, and the volume ratio of hyaluronic acid solution to the total volume of mixed solution and tannic acid solution is 2-5:5-8.

[0019] Secondly, the present invention provides an antioxidant gelatin sponge, which is prepared by the aforementioned preparation method.

[0020] Beneficial Effects: This invention provides an antioxidant gelatin sponge and its preparation method. First, lipid nanoparticles containing Oct4, Sox2, and Klf4 nucleic acids are prepared. These lipid nanoparticles (LNPs) encapsulate the nucleic acids (mRNA) of Oct4, Sox2, and Klf4. The LNPs help the negatively charged mRNA cross the equally negatively charged cell membrane, allowing for efficient uptake by the cell. Oct4, Sox2, and Klf4 temporarily and controllably "partially reprogram" senescent cells in bone tissue, restoring their gene expression patterns to a younger, healthier state. Gelatin, as a natural biomolecule, is widely used as a drug delivery carrier and tissue engineering scaffold due to its RGD sequence, degradability, and low immunogenicity. Tannic acid is a plant-derived natural polyphenol with strong antioxidant and anti-inflammatory activities. Phenylboronic acid and its derivatives can form reversible dynamic borate ester bonds with the catechol groups in tannic acid. By grafting phenylboronic acid onto a gelatin backbone and dynamically covalently cross-linking it with tannic acid, a sponge material is formed. This material not only possesses a porous structure and excellent wet adhesion, but also releases active ingredients on demand in the osteoporotic microenvironment (acidic, high oxidative stress) through the pH / ROS responsiveness of borate ester bonds. This antioxidant gelatin sponge can be directly implanted or injected into high-fracture sites (such as the vertebral body and femoral neck) in osteoporosis patients. By long-term regulation of local bone metabolism, reducing bone resorption, and increasing bone density, it aims to treat osteoporosis and prevent first or recurrent fractures. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of antioxidant gelatin sponge according to an embodiment of the present invention.

[0022] Figure 2 The flowchart illustrates the preparation process of lipid nanoparticles containing three nucleic acids, Oct4, Sox2, and Klf4, in an embodiment of the present invention.

[0023] Figure 3 This is a flowchart illustrating the preparation process of grafting phenylboronic acid onto a gelatin matrix according to an embodiment of the present invention.

[0024] Figure 4 Physical images of DPPH radical scavenging at different ratios (Gel-PBA / TA:HA).

[0025] Figure 5 The DPPH radical scavenging rate is represented by different ratios (Gel-PBA / TA:HA).

[0026] Figure 6 Comparison of microCT images of rats treated with different materials for 4 weeks.

[0027] Figure 7Comparison of β-Gal staining results after OSK@LNP treatment of young and senescent osteoblasts. Detailed Implementation

[0028] This invention provides an antioxidant gelatin sponge and its preparation method. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0029] Current local treatment strategies still face several key scientific challenges that need to be overcome.

[0030] First, the complexity of the local microenvironment in osteoporosis. Osteoporotic sites typically exhibit chronic low-grade inflammation, elevated oxidative stress levels, accumulation of acidic metabolites, and abnormal accumulation of senescent cells. Senescent cells release large amounts of pro-inflammatory factors (such as IL-6, TNF-α, and IL-1β), matrix metalloproteinases, and chemokines through the secretion of the senescence-associated secretory phenotype (SASP). This not only directly stimulates osteoclast activation but also inhibits osteoblast differentiation, creating a vicious cycle of imbalance between bone resorption and bone formation. Traditional anti-resorption drugs can only temporarily block osteoclast activity and cannot reverse the deterioration of the microenvironment driven by senescent cells; while bone-forming drugs have a short duration of action, with their effects rapidly disappearing after discontinuation. Therefore, a novel intervention strategy that can fundamentally "reset" the local cellular senescence state is urgently needed.

[0031] Second, existing drug delivery systems struggle to achieve spatiotemporally controlled release. Locally implanted or injected materials, lacking responsive release mechanisms, often result in either burst release or insufficient release. The characteristic changes in the osteoporotic microenvironment—decreased pH (pH can drop to 4.5-5.0 in bone resorption lacunae) and increased reactive oxygen species (ROS) levels—provide endogenous triggering signals for designing smart responsive materials. Utilizing these pathological characteristics to achieve on-demand drug release could improve efficacy and reduce systemic side effects.

[0032] Third, the potential and challenges of inducing pluripotency factors in bone regeneration. Transcription factors such as Oct4, Sox2, and Klf4 are core factors in maintaining cell pluripotency and reprogramming. Recent studies have found that transient and controllable "partial reprogramming" of senescent cells can restore their youthful phenotype, improve mitochondrial function, and eliminate aging-related secretory phenotypes without inducing tumors. Applying this strategy to bone tissue holds promise for reversing localized age-related bone metabolic abnormalities at the cellular level. However, efficiently delivering the nucleic acids of these transcription factors into target cells within bone tissue (such as bone marrow mesenchymal stem cells, osteoblast progenitor cells, and osteocytes) and achieving transient and safe expression still requires efficient nucleic acid delivery vectors.

[0033] Fourth, modification strategies for natural biomacromolecule-based materials. Gelatin, as a natural biomacromolecule, has been widely used as a drug delivery carrier and tissue engineering scaffold due to its inherent RGD sequence, good biocompatibility, degradability, and low immunogenicity. However, gelatin itself has weak mechanical properties and degrades too quickly in vivo, making it difficult to meet the long-term support requirements of bone defects. Simultaneously, gelatin lacks responsiveness to reactive oxygen species or pH changes, making it difficult to achieve intelligent release triggered by the pathological microenvironment. Tannic acid is a plant-derived natural polyphenol rich in catechol groups, possessing strong antioxidant and anti-inflammatory activities. However, when used directly as a local implant material, it suffers from rapid release and lack of targeting. Phenylboronic acid and its derivatives can reversibly form borate ester bonds with the catechol groups in tannic acid at physiological pH. These bonds break under acidic or high oxidative stress conditions, thereby endowing the material with responsive release properties.

[0034] Based on this, this embodiment provides a method for preparing an antioxidant gelatin sponge, such as... Figure 1 As shown, it includes the following steps: S1. Preparation of lipid nanoparticles containing three nucleic acids: Oct4, Sox2, and Klf4; S2. Benzylboronic acid is grafted onto the gelatin backbone to prepare phenylboronic acid gelatin. S3. Dissolve the phenylboronic acid gelatin in a lipid nanoparticle solution to form a mixed solution, and then add tannic acid solution and hyaluronic acid solution to the mixed solution in sequence to obtain the antioxidant gelatin sponge.

[0035] It should be noted that in this embodiment, lipid nanoparticles containing Oct4, Sox2, and Klf4 nucleic acids were first prepared. These lipid nanoparticles (LNPs) were then used to encapsulate the nucleic acids (mRNA) of Oct4, Sox2, and Klf4. LNPs facilitate the passage of negatively charged mRNA across the equally negatively charged cell membrane, allowing for efficient uptake by the cell. Partial cell reprogramming using Oct4, Sox2, Klf4, and c-Myc can enhance tissue plasticity and repair capabilities. However, while c-Myc can improve efficiency, it is also a potent proto-oncogene, posing a tumorigenic risk. The combination of Oct4, Sox2, and Klf4 (hereinafter referred to as OSK) has regenerative potential while avoiding the cancer-causing risks associated with c-Myc. Oct4 is an indispensable core dominant factor; Sox2 synergistically binds with Oct4 and activates the pluripotency gene network; and Klf4 drives metabolic conversion. Together, these three constitute the minimum necessary combination to initiate somatic cell reprogramming. Therefore, the three factors Oct4, Sox2, and Klf4 can temporarily and controllably "partially reprogram" senescent cells in bone tissue, restoring their gene expression patterns to a younger, healthier state. Gelatin, as a natural biomolecule, is widely used as a drug delivery carrier and tissue engineering scaffold due to its naturally preserved RGD bioactive sequence, degradability, and low immunogenicity. Tannic acid is a plant-derived natural polyphenol with strong antioxidant and anti-inflammatory activities. Phenylboronic acid and its derivatives can form reversible dynamic borate ester bonds with the catechol groups in tannic acid. A sponge material formed by grafting phenylboronic acid onto a gelatin backbone and then dynamically covalently cross-linking it with tannic acid not only possesses a porous structure and good wet adhesion but also releases active ingredients on demand in the osteoporotic microenvironment (acidic, high oxidative stress) through the pH / ROS responsiveness of the borate ester bonds. This material can be directly implanted or injected into high-risk fracture sites (such as the vertebral body and femoral neck) in osteoporosis patients. By regulating local bone metabolism, reducing bone resorption, and increasing bone density over the long term, it aims to treat osteoporosis and prevent first or recurrent fractures. Hyaluronic acid can dilute the cross-linked network and disrupt rigid hydrogen bonds, making Gel-PBA / TA more flexible and giving it better flexibility and biofunctionality.

[0036] In some embodiments, lipid nanoparticles containing the three nucleic acids Oct4, Sox2, and Klf4 are prepared, such as... Figure 2 As shown, it includes the following steps: S11. Prepare lipid organic phase solution; S12. Dissolve the three nucleic acids Oct4, Sox2, and Klf4 in a buffer solution (such as sodium citrate buffer) to form an aqueous solution of the three nucleic acids. S13. Mix the lipid organic phase solution and the trinucleotide aqueous phase solution to obtain a preliminary mixture of lipid nanoparticles loaded with trinucleotides; S14. The initial mixture of lipid nanoparticles loaded with three nucleic acids is filtered, concentrated, and impurities are removed to obtain the lipid nanoparticles loaded with the three nucleic acids.

[0037] It should be noted that in this embodiment, when the lipid organic phase solution is mixed with an aqueous phase solution containing Oct4, Sox2, and Klf4 trinucleotides, the lipid molecules self-assemble into nanoparticles in the aqueous environment. Simultaneously, negatively charged nucleic acids are encapsulated within the particles through electrostatic or hydrophobic interactions, thus obtaining lipid nanoparticles loaded with the three nucleic acids. Subsequent filtration, concentration, and impurity removal processes (including dialysis or ultrafiltration) remove organic solvents and unencapsulated free nucleic acids, resulting in uniform nanoparticle size. The lipid nanoparticles of this embodiment effectively protect nucleic acids from nuclease degradation, significantly improving the in vitro and in vivo stability of the three factor mRNAs (Oct4, Sox2, and Klf4 trinucleotides). The nanoscale particles can be efficiently taken up by senescent cells in bone tissue, promoting the co-delivery of the three factors and transient, controllable partial reprogramming, thereby reversing age-related secretory phenotypes and restoring local bone metabolic balance. Furthermore, this method is simple to prepare and highly reproducible. The resulting lipid nanoparticles can be further compounded with gelatin-tannic acid sponges to achieve a synergistic sustained-release effect in minimally invasive local treatment of osteoporosis.

[0038] In one embodiment, the mass ratio of Oct4, Sox2, and Klf4 is 1-3:1:1. Among the three factors, Oct4 is the key factor that initiates and dominates the reprogramming process, and the preferred mass ratio of Oct4, Sox2, and Klf4 is 3:1:1.

[0039] In some embodiments, the preparation of the lipid organic phase solution includes: dissolving an ionizable lipid (cationic lipid DLin-MC3-DMA), an auxiliary lipid (DSPC), cholesterol, and polyethylene glycol-modified lipid (DSPE-PEG2000) together in anhydrous ethanol, and dissolving them thoroughly by vortexing and water bath to obtain a lipid organic phase solution.

[0040] In one embodiment, the molar ratio of ionizable lipid (cationic lipid DLin-MC3-DMA), cofactor lipid (DSPC), cholesterol, and polyethylene glycol-modified lipid (DSPE-PEG2000) is 55:5:38.5:1.5. This is the gold standard ratio for the preparation of lipid nanoparticles.

[0041] In one embodiment, the lipid organic phase solution and the trinucleotide aqueous phase solution are mixed using a microfluidic method for precise mixing. The flow rate ratio of the two is 3:1 (aqueous phase:organic phase), and the mixture is injected into a Y-type microfluidic chip with a total flow rate set to 9-12 mL / min.

[0042] In one embodiment, the filtration, concentration, and impurity removal include: diluting the initial mixture of lipid nanoparticles loaded with three nucleic acids 20 times with PBS and transferring it to a pretreated 5kDa molecular weight cutoff ultrafiltration tube, and continuously ultrafiltration and concentration at 4°C to completely remove residual ethanol, free mRNA, and unencapsulated lipids.

[0043] In some embodiments, phenylboronic acid is grafted onto a gelatin backbone to prepare phenylboronic acid gelatin, such as... Figure 3 As shown, it includes the following steps: S21. Dissolve gelatin in buffer solution, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and activate at room temperature for 30-60 min to obtain activated gelatin. S22. Add aminophenylboronic acid to the activated gelatin and react at room temperature for 12-24 hours. S23, filtration, dialysis, and freeze-drying yield phenylboronic acid gelatin.

[0044] It should be noted that in this embodiment, gelatin is dissolved in buffer solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added. The mixture is activated at room temperature for 30-60 minutes. In this step, the EDC / NHS system is used to activate the carboxyl groups on the side chains of the gelatin molecules. Subsequently, aminophenylboronic acid is added, and the reaction is carried out at room temperature for 12-24 hours. This allows the primary amine groups in the aminophenylboronic acid to undergo an amide condensation reaction with the activated carboxyl groups, thereby covalently grafting phenylboronic acid onto the gelatin backbone. Finally, insoluble matter is removed by filtration, unreacted small molecules and byproducts are removed by dialysis, and the purified phenylboronic acid gelatin is obtained by lyophilization. The key to this embodiment is the use of an EDC / NHS-mediated amidation reaction to introduce phenylboronic acid groups into gelatin under mild conditions, preserving the original biocompatibility and RGD sequence of the gelatin. The phenylboronic acid groups introduced into phenylboronic acid gelatin can form reversible dynamic borate ester bonds with the catechol groups in tannic acid, thereby constructing a cross-linked sponge with pH / reactive oxygen species responsiveness. This method is simple to operate, has mild conditions, can achieve different grafting ratios, and the product is easy to store and process, providing a key functional intermediate for the subsequent preparation of antioxidant gelatin sponges.

[0045] In some implementations, the gelatin is dissolved in deionized water before being dissolved in the buffer solution.

[0046] In some embodiments, the buffer solution is 2-morpholinoethanesulfonic acid (MES).

[0047] In some embodiments, the mass ratio of phenylboronic acid gelatin in the mixed solution is 1-2%.

[0048] In some embodiments, the volume ratio of tannic acid solution to mixed solution is 1-2:1. The tannic acid solution is a phosphate-buffered saline (PBS) solution of tannic acid, wherein the concentration of tannic acid is 1 mg / ml. Excessive tannic acid concentration will cause excessive cross-linking with phenylboronic acid gelatin, resulting in flocculent precipitation; insufficient concentration will lead to inadequate cross-linking.

[0049] In some embodiments, the volume ratio of the hyaluronic acid solution to the total volume of the mixed solution and the tannic acid solution is 2-5:5-8. The hyaluronic acid solution is a phosphate-buffered saline (PBS) solution of hyaluronic acid, wherein the mass concentration of hyaluronic acid is 1%. It should be noted that the gelatin sponge obtained by freeze-drying using the above ratio in this embodiment not only has a porous structure but also a sponge with good wet adhesion.

[0050] This embodiment also provides an antioxidant gelatin sponge, which is prepared by the preparation method described above.

[0051] The present invention will be further described below through specific embodiments.

[0052] Example 1 1. Preparation of lipid nanoparticles containing three nucleic acids: Oct4, Sox2, and Klf4 includes: Preparation of lipid organic phase solution: cationic lipid DLin-MC3-DMA, auxiliary lipid DSPC, cholesterol and polyethylene glycol-modified lipid (DSPE-PEG2000) were dissolved in anhydrous ethanol at a ratio of 10 mg / ml, and mixed in a molar ratio of 55:5:38.5:1.5 to form a homogeneous lipid organic phase solution. The solution was vortexed until completely dissolved. Preparation of aqueous nucleic acid (mRNA) solution: Dissolve Oct4, Sox2, and Klf4 mRNAs in sodium citrate buffer to prepare an aqueous nucleic acid solution. The total mRNA amount is 165 μg, with the mass ratio of Oct4, Sox2, and Klf4 being 3:1:1. Preparation of a preliminary mixture of lipid nanoparticles loaded with three nucleic acids: A Y-type microfluidic chip was used to achieve precise mixing of the lipid organic phase solution and the nucleic acid aqueous phase solution. The nucleic acid aqueous phase solution and the lipid organic phase solution were injected into the Y-type microfluidic chip at a flow rate ratio of 3:1 (aqueous phase:organic phase), with the total flow rate set at 12 mL / min, to obtain the preliminary mixture of lipid nanoparticles loaded with three nucleic acids (LNPs-mRNA preliminary mixture). The initial mixture of lipid nanoparticles loaded with three nucleic acids was diluted 20-fold with PBS and transferred to a pretreated 5kDa molecular weight cutoff ultrafiltration tube. The mixture was continuously ultrafiltered and concentrated at 4°C to completely remove residual ethanol, free mRNA and unencapsulated lipids, resulting in lipid nanoparticles encapsulating Oct4, Sox2 and Klf4 (denoted as OSK@LNP).

[0053] 2. Benzylboronic acid is grafted onto the gelatin backbone to prepare phenylboronic acid gelatin (denoted as Gel-PBA), which includes: Add 2g of type B gelatin to 200ml of deionized water and dissolve the type B gelatin at 37℃; then add 0.1M of 2-morpholinoethanesulfonic acid (MES). Next, 37 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to dissolve them and reacted for half an hour. Next, add 0.5g of 3-aminophenylboronic acid (PBA); After reacting overnight, the precipitate was removed by filtration. The precipitate was dialyzed with 7kDA for one week, and the resulting product was freeze-dried for 3 days to obtain the phenylboronic acid gelatin (denoted as Gel-PBA).

[0054] The synthesized Gel-PBA was mixed with OSK@LNP to prepare a 2% solution; Prepare a TA solution at a concentration of 1 mg / ml; Slowly add the TA solution dropwise into Gel-PBA at a 1:1 ratio; The solution from step 3 is mixed with hyaluronic acid (HA, 1%). HA can dilute the cross-linked network and break rigid hydrogen bonds, making Gel-PBA / TA more flexible and giving it better flexibility and biofunctionality. Freeze-dry for 48 hours.

[0055] 3. Preparation of antioxidant gelatin sponge: Lipid nanoparticles were dissolved in phosphate-buffered saline (PBS) to form a lipid nanoparticle solution. Then, the phenylboronic acid gelatin was dissolved in the lipid nanoparticle solution to form a mixed solution with a mass concentration of 2% of Gel-PBA in the mixed solution. Tannic acid (TA) was dissolved in phosphate-buffered saline (PBS) to prepare a TA solution of 1 mg / ml. The TA solution was then slowly added dropwise to the above mixed solution at a volume ratio of 1:1. Hyaluronic acid was dissolved in phosphate-buffered saline (PBS) to prepare a 1% hyaluronic acid solution (HA solution). The volume ratio of the HA solution to the above mixed solution (including Gel-PBA and TA) was 2:8. The solution was freeze-dried for 48 hours to obtain the antioxidant gelatin sponge.

[0056] Example 2 1. Preparation of lipid nanoparticles containing three nucleic acids: Oct4, Sox2, and Klf4 includes: Preparation of lipid organic phase solution: cationic lipid DLin-MC3-DMA, auxiliary lipid DSPC, cholesterol and polyethylene glycol-modified lipid (DSPE-PEG2000) were dissolved in anhydrous ethanol at a ratio of 10 mg / ml, and mixed in a molar ratio of 55:5:38.5:1.5 to form a homogeneous lipid organic phase solution. The solution was vortexed until completely dissolved. Preparation of aqueous nucleic acid (mRNA) solution: Dissolve Oct4, Sox2, and Klf4 mRNAs in sodium citrate buffer to prepare an aqueous nucleic acid solution. The total mRNA amount is 165 μg, with the mass ratio of Oct4, Sox2, and Klf4 being 3:1:1. Preparation of a preliminary mixture of lipid nanoparticles loaded with three nucleic acids: A Y-type microfluidic chip was used to achieve precise mixing of the lipid organic phase solution and the nucleic acid aqueous phase solution. The nucleic acid aqueous phase solution and the lipid organic phase solution were injected into the Y-type microfluidic chip at a flow rate ratio of 3:1 (aqueous phase:organic phase), with the total flow rate set at 12 mL / min, to obtain the preliminary mixture of lipid nanoparticles loaded with three nucleic acids (LNPs-mRNA preliminary mixture). The initial mixture of lipid nanoparticles loaded with three nucleic acids was diluted 20-fold with PBS and transferred to a pretreated 5kDa molecular weight cutoff ultrafiltration tube. The mixture was continuously ultrafiltered and concentrated at 4°C to completely remove residual ethanol, free mRNA and unencapsulated lipids, resulting in lipid nanoparticles encapsulating Oct4, Sox2 and Klf4 (denoted as OSK@LNP).

[0057] 2. Benzylboronic acid is grafted onto the gelatin backbone to prepare phenylboronic acid gelatin (denoted as Gel-PBA), which includes: Add 2g of type B gelatin to 200ml of deionized water and dissolve the type B gelatin at 37℃; then add 0.1M of 2-morpholinoethanesulfonic acid (MES). Next, 37 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to dissolve them and reacted for half an hour. Next, add 0.5g of 3-aminophenylboronic acid (PBA); After reacting overnight, the precipitate was removed by filtration. The precipitate was dialyzed with 7kDA for one week, and the resulting product was freeze-dried for 3 days to obtain the phenylboronic acid gelatin (denoted as Gel-PBA).

[0058] The synthesized Gel-PBA was mixed with OSK@LNP to prepare a 2% solution; Prepare a TA solution at a concentration of 1 mg / ml; Slowly add the TA solution dropwise into Gel-PBA at a 1:1 ratio; The solution from step 3 is mixed with hyaluronic acid (HA, 1%). HA can dilute the cross-linked network and break rigid hydrogen bonds, making Gel-PBA / TA more flexible and giving it better flexibility and biofunctionality. Freeze-dry for 48 hours.

[0059] 3. Preparation of antioxidant gelatin sponge: Lipid nanoparticles were dissolved in phosphate-buffered saline (PBS) to form a lipid nanoparticle solution. Then, the phenylboronic acid gelatin was dissolved in the lipid nanoparticle solution to form a mixed solution with a mass concentration of 2% of Gel-PBA in the mixed solution. Tannic acid (TA) was dissolved in phosphate-buffered saline (PBS) to prepare a TA solution of 1 mg / ml. The TA solution was then slowly added dropwise to the above mixed solution at a volume ratio of 1:1. Hyaluronic acid was dissolved in phosphate-buffered saline (PBS) to prepare a 1% hyaluronic acid solution (HA solution). The HA solution was slowly added dropwise to the above mixed solution, with a volume ratio of HA solution to the above mixed solution (including Gel-PBA and TA) of 3:7. The solution was freeze-dried for 48 hours to obtain the antioxidant gelatin sponge.

[0060] The difference between this embodiment and Embodiment 1 is that the volume ratio of the HA solution to the above-mentioned mixed solution (including Gel-PBA and TA) is different.

[0061] Example 3 1. Preparation of lipid nanoparticles containing three nucleic acids: Oct4, Sox2, and Klf4 includes: Preparation of lipid organic phase solution: cationic lipid DLin-MC3-DMA, auxiliary lipid DSPC, cholesterol and polyethylene glycol-modified lipid (DSPE-PEG2000) were dissolved in anhydrous ethanol at a ratio of 10 mg / ml, and mixed in a molar ratio of 55:5:38.5:1.5 to form a homogeneous lipid organic phase solution. The solution was vortexed until completely dissolved. Preparation of aqueous nucleic acid (mRNA) solution: Dissolve Oct4, Sox2, and Klf4 mRNAs in sodium citrate buffer to prepare an aqueous nucleic acid solution. The total mRNA amount is 165 μg, with the mass ratio of Oct4, Sox2, and Klf4 being 3:1:1. Preparation of a preliminary mixture of lipid nanoparticles loaded with three nucleic acids: A Y-type microfluidic chip was used to achieve precise mixing of the lipid organic phase solution and the nucleic acid aqueous phase solution. The nucleic acid aqueous phase solution and the lipid organic phase solution were injected into the Y-type microfluidic chip at a flow rate ratio of 3:1 (aqueous phase:organic phase), with the total flow rate set at 12 mL / min, to obtain the preliminary mixture of lipid nanoparticles loaded with three nucleic acids (LNPs-mRNA preliminary mixture). The initial mixture of lipid nanoparticles loaded with three nucleic acids was diluted 20-fold with PBS and transferred to a pretreated 5kDa molecular weight cutoff ultrafiltration tube. The mixture was continuously ultrafiltered and concentrated at 4°C to completely remove residual ethanol, free mRNA and unencapsulated lipids, resulting in lipid nanoparticles encapsulating Oct4, Sox2 and Klf4 (denoted as OSK@LNP).

[0062] 2. Benzylboronic acid is grafted onto the gelatin backbone to prepare phenylboronic acid gelatin (denoted as Gel-PBA), which includes: Add 2g of type B gelatin to 200ml of deionized water and dissolve the type B gelatin at 37℃; then add 0.1M of 2-morpholinoethanesulfonic acid (MES). Next, 37 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to dissolve them and reacted for half an hour. Next, add 0.5g of 3-aminophenylboronic acid (PBA); After reacting overnight, the precipitate was removed by filtration. The precipitate was dialyzed with 7kDA for one week, and the resulting product was freeze-dried for 3 days to obtain the phenylboronic acid gelatin (denoted as Gel-PBA).

[0063] The synthesized Gel-PBA was mixed with OSK@LNP to prepare a 2% solution; Prepare a TA solution at a concentration of 1 mg / ml; Slowly add the TA solution dropwise into Gel-PBA at a 1:1 ratio; The solution from step 3 is mixed with hyaluronic acid (HA, 1%). HA can dilute the cross-linked network and break rigid hydrogen bonds, making Gel-PBA / TA more flexible and giving it better flexibility and biofunctionality. Freeze-dry for 48 hours.

[0064] 3. Preparation of antioxidant gelatin sponge: Lipid nanoparticles were dissolved in phosphate-buffered saline (PBS) to form a lipid nanoparticle solution. Then, the phenylboronic acid gelatin was dissolved in the lipid nanoparticle solution to form a mixed solution with a mass concentration of 2% of Gel-PBA in the mixed solution. Tannic acid (TA) was dissolved in phosphate-buffered saline (PBS) to prepare a TA solution of 1 mg / ml. The TA solution was then slowly added dropwise to the above mixed solution at a volume ratio of 1:1. Hyaluronic acid was dissolved in phosphate-buffered saline (PBS) to prepare a 1% hyaluronic acid solution (HA solution). The volume ratio of the HA solution to the above mixed solution (including Gel-PBA and TA) was 4:6. The solution was freeze-dried for 48 hours to obtain the antioxidant gelatin sponge.

[0065] The difference between this embodiment and Embodiment 1 is that the volume ratio of the HA solution to the above-mentioned mixed solution (including Gel-PBA and TA) is different.

[0066] Example 4 1. Preparation of lipid nanoparticles containing three nucleic acids: Oct4, Sox2, and Klf4 includes: Preparation of lipid organic phase solution: cationic lipid DLin-MC3-DMA, auxiliary lipid DSPC, cholesterol and polyethylene glycol-modified lipid (DSPE-PEG2000) were dissolved in anhydrous ethanol at a ratio of 10 mg / ml, and mixed in a molar ratio of 55:5:38.5:1.5 to form a homogeneous lipid organic phase solution. The solution was vortexed until completely dissolved. Preparation of aqueous nucleic acid (mRNA) solution: Dissolve Oct4, Sox2, and Klf4 mRNAs in sodium citrate buffer to prepare an aqueous nucleic acid solution. The total mRNA amount is 165 μg, with the mass ratio of Oct4, Sox2, and Klf4 being 3:1:1. Preparation of a preliminary mixture of lipid nanoparticles loaded with three nucleic acids: A Y-type microfluidic chip was used to achieve precise mixing of the lipid organic phase solution and the nucleic acid aqueous phase solution. The nucleic acid aqueous phase solution and the lipid organic phase solution were injected into the Y-type microfluidic chip at a flow rate ratio of 3:1 (aqueous phase:organic phase), with the total flow rate set at 12 mL / min, to obtain the preliminary mixture of lipid nanoparticles loaded with three nucleic acids (LNPs-mRNA preliminary mixture). The initial mixture of lipid nanoparticles loaded with three nucleic acids was diluted 20-fold with PBS and transferred to a pretreated 5kDa molecular weight cutoff ultrafiltration tube. The mixture was continuously ultrafiltered and concentrated at 4°C to completely remove residual ethanol, free mRNA and unencapsulated lipids, resulting in lipid nanoparticles encapsulating Oct4, Sox2 and Klf4 (denoted as OSK@LNP).

[0067] 2. Benzylboronic acid is grafted onto the gelatin backbone to prepare phenylboronic acid gelatin (denoted as Gel-PBA), which includes: Add 2g of type B gelatin to 200ml of deionized water and dissolve the type B gelatin at 37℃; then add 0.1M of 2-morpholinoethanesulfonic acid (MES). Next, 37 mM of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added to dissolve them and reacted for half an hour. Next, add 0.5g of 3-aminophenylboronic acid (PBA); After reacting overnight, the precipitate was removed by filtration. The precipitate was dialyzed with 7kDA for one week, and the resulting product was freeze-dried for 3 days to obtain the phenylboronic acid gelatin (denoted as Gel-PBA).

[0068] The synthesized Gel-PBA was mixed with OSK@LNP to prepare a 2% solution; Prepare a TA solution at a concentration of 1 mg / ml; Slowly add the TA solution dropwise into Gel-PBA at a 1:1 ratio; The solution from step 3 is mixed with hyaluronic acid (HA, 1%). HA can dilute the cross-linked network and break rigid hydrogen bonds, making Gel-PBA / TA more flexible and giving it better flexibility and biofunctionality. Freeze-dry for 48 hours.

[0069] 3. Preparation of antioxidant gelatin sponge: Lipid nanoparticles were dissolved in phosphate-buffered saline (PBS) to form a lipid nanoparticle solution. Then, the phenylboronic acid gelatin was dissolved in the lipid nanoparticle solution to form a mixed solution with a mass concentration of 2% of Gel-PBA in the mixed solution. Tannic acid (TA) was dissolved in phosphate-buffered saline (PBS) to prepare a TA solution of 1 mg / ml. The TA solution was then slowly added dropwise to the above mixed solution at a volume ratio of 1:1. Hyaluronic acid was dissolved in phosphate-buffered saline (PBS) to prepare a 1% hyaluronic acid solution (HA solution). The volume ratio of the HA solution to the above mixed solution (including Gel-PBA and TA) was 5:5. The solution was freeze-dried for 48 hours to obtain the antioxidant gelatin sponge.

[0070] The difference between this embodiment and Embodiment 1 is that the volume ratio of the HA solution to the above-mentioned mixed solution (including Gel-PBA and TA) is different.

[0071] The antioxidant gelatin sponges prepared in Examples 1-4 above were used to prepare extracts (soaked in anhydrous ethanol DPPH solvent). Each of these extracts was then reacted with DPPH reagent, and the results are as follows: Figure 4 , Figure 5 As shown. From Figure 4 It can be seen that pure DPPH reagent is a characteristic deep purple color. As the volume ratio of HA increases from 2:8 to 5:5, the solution color gradually fades from deep purple to light yellow, which directly reflects the gradual removal of DPPH free radicals; correspondingly Figure 5 This trend was further verified. The DPPH free radical scavenging rate increased significantly with the increase of HA ratio. The scavenging rate of the 5:5 group was significantly higher than that of the 2:8, 3:7 and 4:6 groups. This indicates that the introduction of HA can effectively enhance the free radical scavenging ability of gelatin sponge. Moreover, the antioxidant activity is enhanced with the increase of Gel-PBA ratio, which provides experimental evidence for its ability to remove excess ROS and alleviate oxidative stress in the treatment of osteoporosis.

[0072] To demonstrate the therapeutic effect of the antioxidant gelatin sponge obtained in this embodiment on osteoporosis, an ovariectomized (OVX) induced osteoporosis model was constructed in female rats. After establishing a bone defect in the distal femur, different materials were implanted for intervention. The following groups were established: an OVX blank control group, a simple Gel group (implanted only type B gelatin), a Gel-PBA group (phenylboronic acid gelatin), and an OSK group (the antioxidant gelatin sponge group from Example 1). Four weeks post-surgery, micro-CT scans were used to observe changes in the bone microstructure of the bone defect area. The results are as follows: Figure 6 As shown, the OVX group exhibited sparse, fractured, and structurally disordered trabeculae, displaying typical osteoporosis characteristics; the Gel group showed a slight increase in trabecular density, but the improvement was limited; the Gel-PBA group showed an increase in the number of trabeculae and a more dense and orderly arrangement; the OSK group had the thickest and densest trabeculae with a complete and continuous network structure, demonstrating the best bone repair effect. This directly confirms that Example 1 of the invention can effectively improve the bone microstructure of osteoporotic bone and promote bone defect regeneration. Therefore, this example can be used in high-risk fracture sites (vertebral body, femoral neck, etc.) of osteoporosis patients, achieving the dual goals of targeted therapy and fracture prevention through local implantation. This addresses the problem that existing phenylboronic acid-polyphenol dynamic cross-linking systems are mostly used only for the repair of ordinary bone defects (i.e., existing bone defects), rarely used for "treatment of osteoporosis" itself (increasing bone density and improving bone microstructure) or "prevention of osteoporotic fractures," and that existing anti-osteoporosis methods are mainly based on systemic administration, lacking local treatment.

[0073] To demonstrate that the antioxidant gelatin sponge of this invention can be implanted into high-risk osteoporosis sites (such as the vertebral body and femoral neck) to scavenge free radicals and provide mechanical support, and to demonstrate that it can partially reprogram senescent osteoblasts (OBs) to restore their function to a youthful state and thus prevent osteoporotic fractures, β-galactosidase (β-Gal) staining was performed on young osteoblasts (Y-OB), young osteoblasts (Y-OSK) treated with OSK@LNP (lipid nanoparticles), senescent osteoblasts (A-OB), and senescent osteoblasts (A-OSK) treated with OSK@LNP (A-OSK) to reflect the cellular senescence level. The results are as follows: Figure 7 As shown, the A-OB group exhibited a significant increase in senescence-related β-Gal positive signals stained blue, displaying typical staining characteristics of senescent cells. In contrast, the A-OSK group treated with OSK@LNP showed a significant decrease in the number of blue-stained positive cells and a marked reduction in staining intensity, indicating a significant improvement in the senescence phenotype. Simultaneously, the blue staining signals in both the Y-OB and Y-OSK groups remained at low levels, with no significant difference between the two groups, suggesting that OSK@LNP has no significant adverse effects on young osteoblasts. This result directly confirms that OSK@LNP can effectively downregulate β-Gal activity in senescent osteoblasts, reverse their senescence characteristics, and achieve cell "rejuvenation," providing crucial cellular-level evidence for subsequently improving bone repair capacity in osteoporotic states.

[0074] In summary, this invention provides an antioxidant gelatin sponge and its preparation method. First, lipid nanoparticles containing Oct4, Sox2, and Klf4 nucleic acids are prepared. These lipid nanoparticles (LNPs) encapsulate the nucleic acids (mRNA) of Oct4, Sox2, and Klf4. The LNPs facilitate the passage of negatively charged mRNA across the equally negatively charged cell membrane, allowing for efficient uptake by the cell. Oct4, Sox2, and Klf4 temporarily and controllably "partially reprogram" senescent cells in bone tissue, restoring their gene expression patterns to a younger, healthier state. Gelatin, as a natural biomolecule, is widely used as a drug delivery carrier and tissue engineering scaffold due to its RGD sequence, degradability, and low immunogenicity. Tannic acid is a plant-derived natural polyphenol with potent antioxidant and anti-inflammatory activities. Phenylboronic acid and its derivatives can form reversible dynamic borate ester bonds with the catechol groups in tannic acid. By grafting phenylboronic acid onto a gelatin backbone and dynamically covalently cross-linking it with tannic acid, a sponge material is formed. This material not only possesses a porous structure and excellent wet adhesion but also enables localized sustained release of tannic acid. Furthermore, through the pH / ROS responsiveness of the borate ester bonds, it releases active ingredients on demand within the osteoporotic microenvironment (acidic, high oxidative stress). This antioxidant gelatin sponge can be directly implanted or injected into high-fracture sites in osteoporosis patients (such as the vertebral body and femoral neck). By long-term regulation of local bone metabolism, reducing bone resorption, and increasing bone density, it aims to treat osteoporosis and prevent first- or second-fracture events.

[0075] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing an antioxidant gelatin sponge, characterized in that, Includes the following steps: S1. Preparation of lipid nanoparticles containing three nucleic acids: Oct4, Sox2, and Klf4; S2. Benzylboronic acid is grafted onto the gelatin backbone to prepare phenylboronic acid gelatin. S3. Prepare the lipid nanoparticles into a lipid nanoparticle solution; dissolve the phenylboronic acid gelatin in the lipid nanoparticle solution to form a mixed solution, and then add tannic acid solution and hyaluronic acid solution to the mixed solution in sequence to obtain the antioxidant gelatin sponge.

2. The method for preparing an antioxidant gelatin sponge according to claim 1, characterized in that, The preparation of lipid nanoparticles containing Oct4, Sox2, and Klf4 nucleic acids includes the following steps: S11. Prepare lipid organic phase solution; S12. Dissolve Oct4, Sox2, and Klf4 nucleic acids in buffer solution to form an aqueous solution of the three nucleic acids; S13. Mix the lipid organic phase solution and the trinucleotide aqueous phase solution to obtain a preliminary mixture of lipid nanoparticles loaded with trinucleotides; S14. The initial mixture of lipid nanoparticles loaded with three nucleic acids is filtered, concentrated, and impurities are removed to obtain the lipid nanoparticles loaded with the three nucleic acids.

3. The method of claim 2, wherein the antioxidant gelatin sponge is prepared by adding the antioxidant to the gelatin solution before the gelatin solution is cast into a film. 5 The mass ratio of Oct4, Sox2, and Klf4 is 1-3:1:

1.

4. The method of claim 2, wherein the antioxidant gelatin sponge is prepared by adding the antioxidant to the gelatin solution before the gelatin solution is cast into a film. The preparation of the lipid organic phase solution includes: dissolving ionizable lipids, auxiliary lipids, cholesterol, and polyethylene glycol-modified lipids together in anhydrous ethanol until fully dissolved to obtain the lipid organic phase solution; The molar ratio of the ionizable lipids, auxiliary lipids, cholesterol, and PEGylated lipids is 55:5:38.5:1.

5.

5. The method of claim 2, wherein the antioxidant gelatin sponge is prepared by adding the antioxidant to the gelatin solution before the gelatin solution is cast into a film. 5 The lipid organic phase solution and the trinucleotide aqueous phase solution were mixed using a microfluidic method. During mixing, the flow rate ratio of the trinucleotide aqueous phase solution to the lipid organic phase solution was 3:

1. The mixture was injected into a Y-type microfluidic chip, and the total flow rate was set to 9-12 mL / min.

6. The method of claim 1, wherein the antioxidant gelatin sponge is prepared by the steps of: Benzylboronic acid gelatin is prepared by grafting phenylboronic acid onto a gelatin backbone, including the following steps: S21. Dissolve gelatin in buffer solution, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and activate at room temperature for 30-60 min to obtain activated gelatin. S22. Add aminophenylboronic acid to the activated gelatin and react at room temperature for 12-24 hours. S23, filtration, dialysis, and freeze-drying yield phenylboronic acid gelatin.

7. The method for preparing an antioxidant gelatin sponge according to claim 6, characterized in that, The buffer solution is 2-morpholinoethanesulfonic acid.

8. The method for preparing an antioxidant gelatin sponge according to claim 1, characterized in that, The mass ratio of phenylboronic acid gelatin in the mixed solution is 1-2%.

9. The method for preparing an antioxidant gelatin sponge according to claim 1, characterized in that, The volume ratio of tannic acid solution to mixed solution is 1-2:1, and the volume ratio of hyaluronic acid solution to the total volume of mixed solution and tannic acid solution is 2-5:5-8.

10. An antioxidant gelatin sponge, characterized in that, Prepared by the preparation method according to any one of claims 1-9.

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