Co-loaded with baicalin and Ca 2+ ROS-responsive in-situ photosensitive gel, its preparation method and application

Abstract

This invention discloses a method for co-loading baicalin and Ca 2+ This paper describes a ROS-responsive in-situ photosensitive gel, its preparation method, and its applications, including a phospholipid membrane-encapsulated composite of baicalin and calcium carbonate nanoparticles, and a ROS-responsive gel matrix. This photosensitive gel exhibits high adhesion, ROS-responsive drug release, in-situ photosensitized curing, and the ability to release baicalin and calcium carbonate nanoparticles. 2+ Its advantages, such as synergistic therapy, have significantly improved the treatment effect of periodontitis, providing new drug carriers and treatment options for the clinical treatment of periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a material co-loaded with baicalin and Ca. 2+ ROS-responsive in-situ photosensitive gels, their preparation methods, and applications. Background Technology

[0002] Periodontitis is a chronic infectious disease characterized by excessive gingival inflammation and alveolar bone loss. Clinical manifestations include chronic gingival bleeding and swelling, periodontal pocket formation, and alveolar bone destruction. Without timely intervention, it can lead to tooth loss and is a major cause of tooth loss in adults. Periodontitis is also an oral disease that harms overall health, and is a risk factor for diseases such as diabetes, rheumatoid arthritis, inflammatory bowel disease, and Alzheimer's disease.

[0003] Currently, the main clinical treatments for periodontitis are antibiotics, such as metronidazole, minocycline, and doxycycline. However, antibiotic treatment has limited efficacy, and long-term use can lead to dysbiosis and drug resistance. Therefore, patients with moderate to severe periodontitis often experience persistent tissue inflammation and alveolar bone destruction, resulting in a recurring problem in clinical treatment.

[0004] Regarding the pathogenesis of periodontitis, it is currently believed that pathogenic bacteria attached to the tooth surface are the initiating factor, inducing the rapid and massive recruitment of neutrophils, the first line of defense of the immune system. Neutrophils kill pathogens through phagocytosis and degranulation, and then induce apoptosis through mechanisms such as the caspase system. Because the host immune system in periodontitis tissues is out of control, apoptotic neutrophils cannot be efficiently and promptly cleared by the immune system. The accumulated large number of neutrophils induces a local "inflammatory storm" in the periodontal tissues, continuously releasing pro-inflammatory factors (IL-1β, IL-6, IL-8, TNF-α), reactive oxygen species (ROS), collagenases, etc., inducing macrophages to differentiate into osteoclasts, causing subsequent alveolar bone damage. The out-of-control host immune system in periodontitis tissues is the decisive factor in the persistence of inflammation and the prolonged healing of alveolar bone damage. Clearing apoptotic neutrophils is key to restoring the host immune balance in periodontal tissues; endocytosis is the process by which phagocytes clear and recycle programmed cell death cells (mainly apoptotic cells) in vivo. Therefore, enhancing the endodontic function is key to treating periodontitis.

[0005] Baicalin (BA), a natural flavonoid compound, possesses significant anti-inflammatory, antibacterial, and antioxidant activities. It can significantly reduce M1 characteristic proteins (iNOS, CD86) and increase M2 characteristic proteins (Arg-1, CD206), promoting the transformation of macrophages from M1 to M2 types and increasing the number of M2 macrophages. Importantly, baicalin can induce macrophage cytoskeleton remodeling by inhibiting RhoA protein and downregulating the RhoA-ROCK signaling pathway. Therefore, baicalin has the potential to increase the number of M2 macrophages and enhance their phagocytic function. However, baicalin has poor water solubility and rapid metabolism in vivo, resulting in low bioavailability when used alone, making it difficult to achieve ideal therapeutic effects.

[0006] Ca 2+ It is a fundamental ion that makes up bones and teeth, and is a type of ion that repairs bone defects. High concentrations of Ca 2+ It can negatively feedback inhibit osteoclast bone resorption and stimulate osteogenic differentiation of dental pulp stem cells, thus playing a therapeutic role in repairing alveolar bone. Recent studies have also found that after macrophages phagocytose neutrophils, the intracellular phagosome-lysosome fusion forms Ca... 2+ Dependency process, Ca 2+ The concentration is directly proportional to the digestive capacity of M2 macrophages. Therefore, Ca 2+ It also possesses the unique activity of enhancing the cytotoxicity of M2 macrophages. However, Ca... 2+ It is easy to deposit in non-target areas, leading to excessively high local concentrations and causing adverse reactions.

[0007] The local administration site for periodontal disease treatment agents is within the periodontal pocket. Chewing, vocalization, and frequent exchange of gingival crevicular fluid and saliva all contribute to the rapid loss of nanomedicines. Traditional gels struggle to achieve the "stable retention-controlled release" design target within the periodontal pocket. Therefore, developing a nanomedicine co-loaded with baicalin and Ca2+ is crucial for its strong targeting, controllable release, excellent adhesion, and significant therapeutic effect. 2+ The ROS-responsive in-situ photosensitizing gel has significant clinical application value. Summary of the Invention

[0008] Therefore, the primary objective of this invention is to provide a method for co-loading baicalin and Ca... 2+ A ROS-responsive in-situ photosensitive gel, which combines high adhesion, ROS-responsive drug release, in-situ photosensitized curing, and the release of baicalin and Ca. 2+ Its advantages, such as synergistic therapy, significantly improve the treatment effect of periodontitis.

[0009] Another object of the present invention is to provide the above-mentioned co-loaded baicalin and Ca 2+ A method for preparing ROS-responsive in-situ photosensitive gels is presented. This method is simple, has mild conditions, good reproducibility, and is suitable for industrial production.

[0010] Another object of the present invention is to provide the above-mentioned co-loaded baicalin and Ca 2+ The application of ROS-responsive in-situ photosensitive gel in the preparation of drugs for treating periodontitis provides a new drug carrier and treatment option for the clinical treatment of periodontitis.

[0011] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0012] A co-loaded baicalin and Ca 2+ The ROS-responsive in-situ photosensitive gel comprises a phospholipid membrane-encapsulated complex of baicalin and calcium carbonate nanoparticles and a ROS-responsive gel matrix.

[0013] In the phospholipid membrane-coated complex of baicalin and calcium carbonate nanoparticles, the components by weight are: baicalin 1-10 parts, calcium carbonate nanoparticles (CaNP) 1-20 parts, phospholipid material 30-50 parts, and cholesterol 10-30 parts.

[0014] The ROS-responsive gel matrix comprises the following components by weight: 10-30 parts of 4-hydroxyphenylboronic acid-modified methacrylic anhydride gelatin, 10-30 parts of PVA, and 0.01-0.05 parts of photosensitizer.

[0015] This invention achieves the loading of baicalin and CaNP through a phospholipid membrane (L-BA-CaNP). 2+ Highly efficient payload. Baicalin can transform macrophages from M1 to M2 type, increase the number of M2 macrophages, and induce macrophage cytoskeleton remodeling by inhibiting RhoA protein and downregulating the RhoA-ROCK signaling pathway. 2+ It not only inhibits osteoclast-mediated bone resorption and stimulates osteogenic differentiation of dental pulp stem cells, but also plays a therapeutic role in repairing alveolar bone, promoting intracellular phagosome-lysosome fusion, and enhancing the cytotoxic capacity of M2 macrophages. These two factors synergistically enhance the cytotoxic clearance of neutrophils by M2 macrophages, reshaping the host immune balance, alleviating excessive inflammatory responses, and repairing alveolar bone in the treatment of periodontitis. Furthermore, the lysophosphatidylcholine (LysoPC) in the phospholipid material induces macrophage aggregation at the site of neutrophil apoptosis, further enhancing the cytotoxic function.

[0016] This invention utilizes 4-hydroxyphenylboronic acid (PBA) to modify methacrylic anhydride gelatin (GelMA). PBA can specifically recognize the glycoprotein structure on the surface of periodontal pathogens, enabling active targeted drug delivery to the lesion site, reducing drug distribution in normal tissues, and improving drug bioavailability. The phenylboronic acid groups of 4-hydroxyphenylboronic acid-modified methacrylic anhydride gelatin (GM-B) can rapidly form ROS-responsive units with the hydroxyl groups of polyvinyl alcohol (PVA). Under ultraviolet light irradiation, GM-B / PVA assembles L-BA-CaNP into a gel "drug reservoir" in situ within the periodontal cavity. Compared to simple GelMA gel, this method increases the number of cross-linking sites within the gel, forming a reservoir-like structure that helps the drug remain stably within the periodontal pocket; simultaneously, it leverages the high ROS characteristics of the periodontal microenvironment to achieve intelligent controlled release of L-BA-CaNP.

[0017] Preferably, the phospholipid membrane-coated composite of baicalin and calcium carbonate nanoparticles has an average particle size of 160-170 nm and a potential of 40-45 mV, which is different from the particle size of the calcium carbonate nanoparticles and has the opposite potential, indicating that the phospholipid membrane has been successfully coated on the surface.

[0018] Preferably, the phospholipid material is selected from one or more of dipalmitoylcholine (DPPC), distearatelcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin phosphatidic acid, phosphatidylglycerol, dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS), lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LysoPC), dimyristoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylglycerol (DOPG), DOP-DEDA, DOTMA, Dlin-MC3-DMA, DODMA, or DOTAP, preferably one or more of dipalmitoylcholine (DPPC), lysophosphatidylcholine (LysoPC), or DOTAP.

[0019] Preferably, the photosensitizer is one or more of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), lithium phenyl 2,4,6-trimethylbenzoylphosphonate (LAP), 2-isopropylthioxanthone (ITX), and 2-hydroxy-2-methyl-1-phenyl-1-propanone.

[0020] This invention also provides co-loaded baicalin and Ca 2+ The preparation method of ROS-responsive in-situ photosensitizer includes the following steps:

[0021] (1) Baicalin and calcium carbonate nanoparticles were dissolved in ethanol, mixed and sonicated and stirred at room temperature to obtain a baicalin and calcium carbonate nanoparticle complex; the baicalin and calcium carbonate nanoparticle complex, phospholipid material and cholesterol were dissolved in ethanol, mixed evenly and added to deionized water while stirring, then stirred at room temperature and sonicated in a water bath to obtain a suspension of the phospholipid membrane-loaded baicalin and calcium carbonate nanoparticle complex.

[0022] (2) Weigh 4-hydroxyphenylboronic acid (PBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt (EDC) and N-hydroxysuccinimide (NHS) and dissolve them in DMSO. Stir to activate, and then mix thoroughly with methacrylic anhydride gelatin aqueous solution (GelMA) to obtain 4-hydroxyphenylboronic acid modified methacrylic anhydride gelatin (GM-B).

[0023] (3) 4-hydroxyphenylboronic acid-modified methacrylic anhydride gelatin (GM-B) and photosensitizer were dissolved in a phospholipid membrane-coated complex suspension of baicalin and calcium carbonate nanoparticles, and then thoroughly mixed with PVA solution to obtain co-loaded baicalin and calcium carbonate nanoparticles. 2+ ROS-responsive in-situ photosensitive gel.

[0024] Preferably, in step (1), in order to load baicalin into calcium carbonate nanoparticles, the mass ratio of baicalin to calcium carbonate nanoparticles is 1:1-3:1; the mass ratio of phospholipid material to cholesterol is 1:1-3:1.

[0025] Preferably, in step (2), to successfully graft 4-hydroxyphenylboronic acid (PBA) onto methacrylic anhydride gelatin to release the drug under ROS conditions, the mass ratio of the methacrylic anhydride gelatin to 4-hydroxyphenylboronic acid (PBA) is 1:1 to 1:3.

[0026] Preferably, in step (3), in order to successfully synthesize the hydrogel and shorten the gelation time of the hydrogel, the mass ratio of the 4-hydroxyphenylboronic acid modified methacrylic anhydride gelatin (GM-B) and PVA is 1:1 to 1:3.

[0027] The present invention also provides the above-mentioned co-loaded baicalin and Ca 2+ The application of ROS-responsive in-situ photosensitive gel in the preparation of drugs for treating periodontitis. The drug is applied to the periodontal pocket via in-situ light curing. Upon ROS stimulation at the periodontal lesion site, the gel undergoes boric acid bond breakage, gradually degrading and releasing baicalin and calcium. 2+ The two work synergistically to enhance the cytotoxic clearance of neutrophils by M2 macrophages, restore the host immune balance, alleviate excessive inflammatory response, and achieve synergistic treatment of periodontitis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The photosensitive gel provided by this invention enables precise delivery of drugs to the lesion site, reduces the distribution of drugs in normal tissues, improves drug bioavailability, and reduces toxic side effects;

[0030] The photosensitive gel provided by this invention possesses ROS-responsive drug release characteristics, degrading and releasing drugs only at the site of periodontitis lesions (high concentration of ROS), thereby achieving the release of baicalin and calcium. 2+ Synchronous and controlled release avoids fluctuations in blood drug concentration caused by rapid drug release, thereby improving treatment efficacy;

[0031] The photosensitive gel provided by this invention has strong adhesion. The photosensitive gel can be cured in situ under light and adheres tightly to the surface of the periodontal pocket mucosa, effectively resisting saliva erosion, prolonging the drug retention time, and ensuring that the drug can play a full role at the lesion site.

[0032] The photosensitive gel provided by this invention contains baicalin and Ca 2+ They have a synergistic effect; when used together, they can synergistically enhance the cytotoxic clearance of neutrophils by M2 macrophages, restore the host's immune balance, alleviate excessive inflammatory response, and repair alveolar bone in the treatment of periodontitis. This can significantly improve the treatment effect of periodontitis and provide a new formulation for the treatment of periodontitis. Attached Figure Description

[0033] Figure 1 The appearance of the solution of the phospholipid membrane-coated complex of baicalin and CaNP (L-BA-CaNP) of the present invention;

[0034] Figure 2 The particle size characterization results of the phospholipid membrane-encapsulated complex of baicalin and CaNP (L-BA-CaNP) and CaNP and BA-CaNP are shown below.

[0035] Figure 3 The results of the potential characterization of the complex of baicalin and CaNP encapsulated in the phospholipid membrane of the present invention (L-BA-CaNP) and CaNP and BA-CaNP.

[0036] Figure 4 The transmission electron microscopy (TEM) characterization results of the phospholipid membrane-encapsulated complex of baicalin and CaNP (L-BA-CaNP) and CaNP of the present invention;

[0037] Figure 5 This is the EDS spectrum of the complex (L-BA-CaNP) of phospholipid membrane-encapsulated baicalin and CaNP of the present invention.

[0038] Figure 6 This invention co-loads baicalin and Ca 2+UV-response phase transition diagram of ROS-responsive in-situ photosensitive gel (GM-B / PVA@L-BA-CaNP);

[0039] Figure 7 This invention co-loads baicalin and Ca 2+ Scanning electron microscopy (SEM) characterization results of ROS-responsive in-situ photosensitive gel (GM-B / PVA@L-BA-CaNP);

[0040] Figure 8 This invention co-loads baicalin and Ca 2+ Characterization results of the swelling behavior of ROS-responsive in-situ photosensitive gel (GM-B / PVA@L-BA-CaNP);

[0041] Figure 9 This invention co-loads baicalin and Ca 2+ Characterization results of the release behavior of ROS-responsive in-situ photosensitive gel (GM-B / PVA@L-BA-CaNP);

[0042] Figure 10 This invention co-loads baicalin and Ca 2+ H&E staining of rats with ROS-responsive in situ photosensitive gel (GM-B / PVA@L-BA-CaNP) treated with periodontitis;

[0043] Figure 11 This invention co-loads baicalin and Ca 2+ Inflammation-related immunohistochemical staining images and quantitative analysis results of tissue sections of rats with ROS-responsive in situ photosensitive gel (GM-B / PVA@L-BA-CaNP) treated with periodontitis;

[0044] Figure 12 This invention co-loads baicalin and Ca 2+ Micro-CT images and quantitative analysis results of alveolar bone in rats with ROS-responsive in situ photosensitive gel (GM-B / PVA@L-BA-CaNP) treated with periodontitis. Detailed Implementation

[0045] The present invention will be further illustrated below through specific embodiments. The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments.

[0046] All raw materials used in the embodiments of this invention are commercially available.

[0047] Example 1: Preparation and characterization of a phospholipid membrane-coated complex of baicalin and calcium carbonate nanoparticles (L-BA-CaNP)

[0048] (1) Preparation of calcium carbonate nanoparticles (CaNP)

[0049] 2046 parts of the surfactant dodecyl bromide were dissolved in 5 mL of the co-surfactant n-butanol, followed by the addition of 27 mL of the oil phase n-hexane. The mixture was stirred in a 50°C water bath for 30 minutes to form an emulsion. Then, 0.1 mL of a 2 mol / L calcium chloride aqueous solution and 0.1 mL of a 2 mol / L sodium carbonate aqueous solution were added to the oil phase, and the mixture was stirred at room temperature for 12 hours. The product was collected by centrifugation, washed with n-butanol and ethanol respectively, and dried to obtain CaNP.

[0050] (2) Preparation of L-BA-CaNP

[0051] Weigh out 5 parts of baicalin and 5 parts of CaNP, dissolve them separately in 5 ml of ethanol, mix and sonicate, stir at room temperature for 24 h, wash with pure water, and dry to obtain BA-CaNP.

[0052] Ten parts of the prepared BA-CaNP were dispersed in ethanol and mixed with 7 parts of DOTAP. The mixture was sonicated for 20 min, and the precipitate was collected by centrifugation. The precipitate was dispersed in an ethanol solution of 22 parts of DPPC, 3 parts of lysoPC, and 19 parts of cholesterol. The organic phase was slowly added dropwise to 10 ml of deionized water in a 50 °C water bath using a syringe while stirring. The mixture was then stirred at room temperature for 4 h. The mixture was sonicated in a water bath for 5 min to obtain a homogeneous suspension. Finally, the suspension was extruded through a 0.45 µm filter to obtain the phospholipid membrane-coated baicalin and calcium carbonate nanoparticle complex (L-BA-CaNP).

[0053] (3) Particle size, potential, TEM image, and EDS energy spectrum

[0054] The particle size, PDI, and potential of L-BA-CaNP were determined using a Malvern particle size analyzer. Figure 2 and Figure 3 As shown, the particle size of CaNP was 105±5.59 nm, and the potential was -14.87±1.10 mV. Compared with CaNP, the particle size of BA-CaNP increased to 120.60±1.25 nm, possibly due to the physical adsorption of BA and CaNP. Phospholipid film coating further increased the particle size of L-BA-CaNP to 165.60±2.73 nm. After being coated with the cationic phospholipid material on the surface, the potential of L-BA-CaNP reversed to 42.43±0.49 mV, indicating that the phospholipid film was successfully coated on the surface of L-BA-CaNP.

[0055] The morphology and structure of CaNP and L-BA-CaNP were examined using transmission electron microscopy (TEM), such as... Figure 4 As shown, CaNP is spherical, and L-BA-CaNP forms an encapsulation structure for BA-CaNP. Figure 5As shown, the main elements of L-BA-CaNP include Ca, O, N, and P. The presence of Ca and O further confirms the basic composition of CaNP, while the detection of N and P indicates the successful introduction of phospholipid materials.

[0056] Example 2: Co-loading of baicalin and Ca 2+ Formulation screening of ROS-responsive in situ photosensitized gel (GM-B / PVA@L-BA-CaNP)

[0057] (1) Preparation of L-BA-CaNP

[0058] Five parts of baicalin and five parts of CaNP prepared in Example 1 were weighed and dissolved in 5 ml of ethanol respectively. After mixing and sonicating, the mixture was stirred at room temperature for 24 h, washed with pure water, and dried to obtain BA-CaNP.

[0059] Ten parts of the prepared BA-CaNP were dispersed in ethanol and mixed with 7 parts of DOTAP. The mixture was sonicated for 20 min, and the precipitate was collected by centrifugation. The precipitate was dispersed in an ethanol solution of 22 parts of DPPC, 3 parts of lysoPC, and 19 parts of cholesterol. The organic phase was slowly added dropwise to 10 ml of deionized water in a 50 °C water bath using a syringe while stirring. The mixture was then stirred at room temperature for 4 h. The mixture was sonicated in a water bath for 5 min to obtain a homogeneous suspension. Finally, the suspension was extruded through a 0.45 µm filter to obtain the L-BA-CaNP suspension.

[0060] (2) Preparation of 4-carboxyphenylboronic acid modified methacrylamide gelatin (GM-B)

[0061] Weigh 500 parts of GelMA and add 25 ml of pure water, stirring at 37°C until completely dissolved. Then weigh 500 parts of PBA, 47.5 parts of EDC, and 11.5 parts of NHS, dissolve in 15 ml of DMSO, and activate on a magnetic stirrer for 2 hours. After activation, slowly add the PBA solution dropwise to the GelMA solution. After the addition is complete, heat the reaction in a 45°C water bath for 24 hours. After the reaction is complete, transfer the reaction solution to a MW (14000) dialysis bag and dialyze at 30°C for 3 days. After dialysis, freeze-dry to obtain a white, fluffy solid, which is 4-carboxyphenylboronic acid modified methacrylamide gelatin (GM-B).

[0062] (3) Preparation of GM-B / PVA@L-BA-CaNP

[0063] Weigh 15 parts of GM-B and 0.02 parts of LAP and dissolve them in 200 µl of L-BA-CaNP suspension. Mix with PVA solution and stir repeatedly for 3 min to obtain co-loaded baicalin and Ca... 2+The ROS-responsive photosensitive gel (GM-B / PVA@L-BA-CaNP) has a mass ratio of GM-B to PVA of 1:1 to 1:3.

[0064] Using the gelation time of GM-B / PVA@L-BA-CaNP under UV irradiation as the evaluation index, the optimal formulation of GM-B / PVA@L-BA-CaNP with different mass ratios of GelMA and PVA per unit volume was selected using the vial inversion method. Specifically, nine 1ml portions of GM-B / PVA@L-BA-CaNP were measured and placed in separate vials. The vials were then irradiated with UV light, and the rapid gelation of the samples was observed. The experiment was repeated three times. The results are shown in Table 1. The fastest gelation speed was observed when the mass ratio of GM-B to PVA was 1:1, allowing for rapid gelation and retention at the site of periodontitis, meeting the requirements for periodontal drug administration.

[0065] Table 1. Gel time for different formulations

[0066] Prescription number GM-B (mg) PVA (mg) gelation time 1 15 15 2 s 2 15 30 3 s 3 15 45 6 s

[0067] Example 3: Co-loading of baicalin and Ca 2+ Preparation and characterization of ROS-responsive photogel (GM-B / PVA@L-BA-CaNP)

[0068] (1) Preparation of L-BA-CaNP

[0069] Five parts of baicalin and five parts of CaNP prepared in Example 1 were weighed and dissolved in 5 ml of ethanol respectively. After mixing and sonicating, the mixture was stirred at room temperature for 24 h, washed with pure water, and dried to obtain BA-CaNP.

[0070] Ten parts of the prepared BA-CaNP were dispersed in ethanol and mixed with 7 parts of DOTAP. The mixture was sonicated for 20 min, and the precipitate was collected by centrifugation. The precipitate was dispersed in an ethanol solution of 22 parts of DPPC, 3 parts of lysoPC, and 19 parts of cholesterol. The organic phase was slowly added dropwise to 10 ml of deionized water in a 50 °C water bath using a syringe while stirring. The mixture was then stirred at room temperature for 4 h. The mixture was sonicated in a water bath for 5 min to obtain a homogeneous suspension. Finally, the suspension was extruded through a 0.45 µm filter to obtain the L-BA-CaNP suspension.

[0071] (2) Preparation of 4-carboxyphenylboronic acid modified methacrylamide gelatin (GM-B)

[0072] Weigh 500 parts of GelMA and add 25 ml of pure water. Stir at 37°C until completely dissolved. Then weigh 500 parts of PBA, 47.5 parts of EDC, and 11.5 parts of NHS and dissolve them in 15 ml of DMSO. Activate the solution on a magnetic stirrer for 2 hours. After activation, slowly add the PBA solution dropwise to the GelMA solution. After the addition is complete, heat the reaction mixture in a 45°C water bath for 24 hours. After the reaction is complete, transfer the reaction solution to a MW (14000) dialysis bag and dialyze at 30°C for 3 days. After dialysis, freeze-dry the solution to obtain a white, fluffy solid, which is GM-B.

[0073] (3) Preparation of GM-B / PVA@L-BA-CaNP

[0074] Dissolve 15 parts of PVA in an aqueous solution at 90℃ to obtain a PVA solution; weigh 15 parts of GM-B and 0.02 parts of LAP and dissolve them in 200µl of L-BA-CaNP suspension, then mix with the PVA solution and stir repeatedly for 3 min to obtain co-loaded baicalin and Ca... 2+ The reactive oxygen species responsive photosensitive gel (GM-B / PVA@L-BA-CaNP).

[0075] (4) Scanning electron microscopy characterization of GM-B / PVA@L-BA-CaNP

[0076] After freeze-drying GM-B / PVA@L-BA-CaNP, it was sputter-coated with gold to prepare a scanning electron microscope (SEM) sample. The gel morphology was observed and photographed under an electron microscope, and the results are as follows: Figure 6 As shown, the gel has a porous network structure, which is advantageous for load-based drug delivery systems.

[0077] (5) Temperature response phase transition results of GM-B / PVA@L-BA-CaNP

[0078] like Figure 7 As shown, it can be observed from a macroscopic perspective that GM-B / PVA@L-BA-CaNP transforms into a gel state upon UV irradiation, indicating that it has temperature-responsive characteristics.

[0079] (6) Determination of the swelling rate of GM-B / PVA@L-BA-CaNP

[0080] Dissolve 15 parts of PVA in an aqueous solution at 90℃ to obtain a PVA solution; weigh 15 parts of GM-B and 0.02 parts of LAP and dissolve them in 200µl of pure water, then mix them with the PVA solution and stir repeatedly for 3 minutes to obtain GM-B / PVA.

[0081] like Figure 8As shown, both GM-B / PVA and GM-B / PVA@L-BA-CaNP rapidly absorbed water and swelled within 12 hours, with a significant increase in swelling rate. After 12 hours, the swelling rate gradually slowed down, eventually reaching swelling equilibrium within 24 hours. The swelling behavior of GM-B / PVA@L-BA-CaNP was similar to that of GM-B / PVA, but due to the introduction of L-BA-CaNP, the swelling rate of GM-B / PVA@L-BA-CaNP was slightly lower. The hydrogel's ability to absorb water and swell to form a stable gel state within a short time indicates a sustained and slow release of the drug, thereby improving local drug concentration and therapeutic efficacy. This demonstrates that GM-B / PVA@L-BA-CaNP possesses excellent swelling properties and can provide sustained and effective drug release in the treatment of periodontitis.

[0082] (7) Determination of the release rate of GM-B / PVA@L-BA-CaNP

[0083] Dissolve 15 parts of PVA in an aqueous solution at 90℃ to obtain a PVA solution; weigh 15 parts of GM-B and 0.02 parts of LAP and dissolve them in 200µl of pure water, then mix them with the PVA solution and stir repeatedly for 3 minutes to obtain GM-B / PVA.

[0084] like Figure 9 As shown, the degradation rates of GM-B / PVA and GM-B / PVA@L-BA-CaNP were relatively slow in the PBS environment, and they were not completely degraded even after 7 days. However, in the PBS environment containing H2O2, the degradation rates of both were significantly accelerated, and they were almost completely degraded by the fifth day. This indicates that the loading of L-BA-CaNP does not significantly change the overall structure of the hydrogel, and that GM-B / PVA@L-BA-CaNP can respond rapidly and degrade quickly in a high ROS environment, making it suitable for the treatment of periodontitis.

[0085] Example 4: Co-loading of baicalin and Ca 2+ Preparation of ROS-responsive photogel (GM-B / PVA@L-BA-CaNP)

[0086] (1) Preparation of L-BA-CaNP

[0087] Weigh 10 parts of baicalin and 5 parts of CaNP prepared in Example 1, dissolve them in ethanol respectively, mix and sonicate, stir at room temperature for 24 hours, wash with pure water, and dry to obtain BA-CaNP;

[0088] 15 parts of the prepared BA-CaNP were dispersed in ethanol and mixed with 10 parts of DOTAP. The mixture was sonicated for 20 min, and the precipitate was collected by centrifugation. The precipitate was dispersed in an ethanol solution of 20 parts DSPC, 5 parts LPE, and 35 parts cholesterol. The organic phase was slowly added dropwise to 10 ml of deionized water in a 50 °C water bath using a syringe while stirring. The mixture was then stirred at room temperature for 4 h. The mixture was sonicated in a water bath for 5 min to obtain a homogeneous suspension. Finally, the suspension was extruded through a 0.45 µm filter to obtain the L-BA-CaNP suspension.

[0089] (2) Preparation of 4-carboxyphenylboronic acid modified methacrylamide gelatin (GM-B)

[0090] Weigh 500 parts of GelMA and add 25 ml of pure water. Stir at 37°C until completely dissolved. Then weigh 1000 parts of PBA, 60 parts of EDC, and 20 parts of NHS and dissolve them in 25 ml of DMSO. Activate the solution on a magnetic stirrer for 2 hours. After activation, slowly add the PBA solution dropwise to the GelMA solution. After the addition is complete, heat the reaction mixture in a 45°C water bath for 24 hours. After the reaction is complete, transfer the reaction solution to a MW (14000) dialysis bag and dialyze at 30°C for 3 days. After dialysis, freeze-dry the solution to obtain a white, fluffy solid, which is GM-B.

[0091] (3) Preparation of GM-B / PVA@L-BA-CaNP

[0092] Dissolve 10 parts of PVA in an aqueous solution at 90℃ to obtain a PVA solution; weigh 10 parts of GM-B and 0.02 parts of TPO and dissolve them in 200µl of L-BA-CaNP suspension, then mix with the PVA solution and stir repeatedly for 3 min to obtain co-loaded baicalin and Ca 2+ The reactive oxygen species responsive photosensitive gel (GM-B / PVA@L-BA-CaNP).

[0093] Example 5: Co-loading of baicalin and Ca 2+ Preparation of ROS-responsive photogel (GM-B / PVA@L-BA-CaNP)

[0094] (1) Preparation of L-BA-CaNP

[0095] Weigh 15 parts of baicalin and 5 parts of CaNP prepared in Example 1, dissolve them in ethanol respectively, mix and sonicate, stir at room temperature for 24 hours, wash with pure water, and dry to obtain BA-CaNP.

[0096] 20 parts of the prepared BA-CaNP were dispersed in ethanol and mixed with 15 parts of DOTAP. The mixture was sonicated for 20 min, and the precipitate was collected by centrifugation. The precipitate was dispersed in an ethanol solution of 30 parts of DMPC, 10 parts of lysoPC, and 40 parts of cholesterol. The organic phase was slowly added dropwise to 10 ml of deionized water in a 50 °C water bath using a syringe while stirring. The mixture was then stirred at room temperature for 4 h. The mixture was sonicated in a water bath for 5 min to obtain a homogeneous suspension. Finally, the suspension was extruded through a 0.45 µm filter to obtain the L-BA-CaNP suspension.

[0097] (2) Preparation of 4-carboxyphenylboronic acid modified methacrylamide gelatin (GM-B)

[0098] Weigh 500 parts of GelMA and add 25 ml of pure water. Stir at 37°C until completely dissolved. Then weigh 500 parts of PBA, 47.5 parts of EDC, and 11.5 parts of NHS and dissolve them in 15 ml of DMSO. Activate the solution on a magnetic stirrer for 2 hours. After activation, slowly add the PBA solution dropwise to the GelMA solution. After the addition is complete, heat the reaction mixture in a 45°C water bath for 24 hours. After the reaction is complete, transfer the reaction solution to a MW (14000) dialysis bag and dialyze at 30°C for 3 days. After dialysis, freeze-dry the solution to obtain a white, fluffy solid, which is GM-B.

[0099] (3) Preparation of GM-B / PVA@L-BA-CaNP

[0100] Dissolve 25 parts of PVA in an aqueous solution at 90℃ to obtain a PVA solution; weigh 25 parts of GM-B and 0.02 parts of LAP and dissolve them in 200µl of L-BA-CaNP suspension, then mix with the PVA solution and stir repeatedly for 3 min to obtain co-loaded baicalin and Ca... 2+ The reactive oxygen species responsive photosensitive gel (GM-B / PVA@L-BA-CaNP).

[0101] Example 6: Co-loading of baicalin and Ca 2+ Evaluation of the efficacy of ROS-responsive photosensitive hydrocoagulation in treating periodontitis in rats

[0102] One hundred healthy male SPF-grade rats were acclimatized for two weeks. Except for the control group, all other groups were anesthetized intraperitoneally with 3% sodium pentobarbital (30 mg / kg). The rats were placed supine on the experimental table, their heads and limbs fixed, and their mandibles were pulled to fully expose the left maxillary first molar. A 0.2 mm ligature wire was passed through the gap between the first and second molars and tied at the neck of the first molar, with a knot tied on the palatal side. Excess ligature wire was trimmed, and the knot was buried under the gum. Four weeks later, the periodontal condition of the rats was observed. Rats that successfully established the model were randomly divided into the following groups: Control group, Model group, Periodicline group, GM-B / PVA@L-BA-CaNP group, GM-B / PVA@BA-CaNP group, GM-B / PVA@L-CaNP group, GM-B / PVA@L-BA group, and GM / PVA@L-BA-CaNP group. Every 3 days, 20 µL of the corresponding preparation was injected submucosioperiosteally into the buccal and palatal central alveolar ridge crest of the maxillary first molar, followed by UV irradiation for about 2 seconds. The normal group and the model group were injected with the same volume of physiological saline.

[0103] The preparation method of GM-B / PVA@L-BA-CaNP is the same as in Example 3;

[0104] Preparation method of GM-B / PVA@BA-CaNP: The difference from Example 3 is that phospholipid materials and cholesterol are not added in step (1), and the rest is the same as in Example 3. In short: BA and CaNP are dissolved in anhydrous ethanol and mixed thoroughly to obtain a baicalin and CaNP nanoparticle complex (BA-CaNP); activated PBA, EDC, and NHS are added dropwise to GelMA aqueous solution, reacted for 24 h, and then lyophilized by dialysis to obtain GM-B; GM-B is dissolved in BA-CaNP, then mixed thoroughly with PVA solution, and then photosensitizer is added and stirred evenly to obtain GM-B / PVA@BA-CaNP.

[0105] Preparation method of GM-B / PVA@L-CaNP: The difference from Example 3 is that baicalin (BA) is not added, otherwise it is the same as Example 3. In short: CaNP, phospholipid material and cholesterol are dissolved in anhydrous ethanol and mixed thoroughly. The organic phase is slowly added dropwise to 10 ml of deionized water at 50°C with a syringe while stirring. After stirring at room temperature for 4 h, phospholipid membrane-coated CaNP nanoparticles (L-CaNP) are obtained. Activated PBA, EDC and NHS are added dropwise to GelMA aqueous solution. After reacting for 24 h, GM-B is obtained by dialysis and lyophilization. GM-B is dissolved in L-CaNP, then mixed thoroughly with PVA solution, and then photosensitizer is added and stirred thoroughly to obtain GM-B / PVA@L-CaNP.

[0106] Preparation method of GM-B / PVA@L-BA: The difference from Example 3 is that calcium carbonate nanoparticles (CaNP) are not added, otherwise the same as in Example 3. In short: BA, phospholipid material and cholesterol are dissolved in anhydrous ethanol and mixed thoroughly. The organic phase is slowly added dropwise to 10 ml of deionized water at 50°C with a syringe while stirring. After stirring at room temperature for 4 h, phospholipid membrane-coated baicalin (L-BA) is obtained. Activated PBA, EDC and NHS are added dropwise to GelMA aqueous solution. After reacting for 24 h, GM-B is obtained after dialysis and lyophilization. GM-B is dissolved in L-BA, then mixed thoroughly with PVA solution, and then photosensitizer is added and stirred thoroughly to obtain GM-B / PVA@L-BA.

[0107] Preparation method of GM / PVA@L-BA-CaNP: The difference from Example 3 is that GelMA is not modified with PBA, otherwise it is the same as Example 3. In short: BA and CaNP are dissolved in anhydrous ethanol and mixed thoroughly to obtain a baicalin and CaNP nanoparticle complex (BA-CaNP); the prepared BA-CaNP, phospholipid material and cholesterol are dissolved in anhydrous ethanol and mixed thoroughly. The organic phase is slowly added dropwise to 10 ml of deionized water in a 50°C water bath using a syringe while stirring. After stirring at room temperature for 4 h, phospholipid membrane-coated baicalin and CaNP nanoparticles (L-BA-CaNP) are obtained; GelMA is dissolved in L-BA-CaNP, then mixed thoroughly with PVA solution, and then photosensitizer is added and stirred thoroughly to obtain GM / PVA@L-BA-CaNP.

[0108] Four weeks after drug administration, SD rats were euthanized, the left maxilla was separated, blood stains were washed with physiological saline, and tissue near the first molar was collected, rinsed, and stored at -80℃. Excess soft tissue was removed with fine scissors, and the specimen was fixed in 4% paraformaldehyde for 48 hours. Standard procedures such as decalcification, dehydration, embedding, and sectioning were performed. The histological changes of periodontal tissues after 4 weeks were observed using the hematoxylin and eosin (H&E) staining method.

[0109] H&E staining results are as follows Figure 10As shown, the periodontal tissue structure in the Control group was clear, with intact and tightly arranged periodontal fibers, no alveolar bone resorption, and almost no inflammatory cell infiltration. In the Model group, the interdental papillae disappeared, the junctional epithelium migrated towards the root, the periodontal fiber structure was disordered, periodontal pockets were formed, and a large number of inflammatory cells were visible. Reduced inflammatory cell infiltration was observed in the GM-B / PVA@L-BA-CaNP, GM-B / PVA@BA-CaNP, GM-B / PVA@L-CaNP, GM-B / PVA@L-BA, and GM / PVA@L-BA-CaNP groups. Among these, the GM-B / PVA@L-BA-CaNP group showed a clear epithelial structure, and its anti-inflammatory effect was superior to the other groups.

[0110] Immunohistochemical staining was performed on each group using IL-1β and IL-10 as inflammatory markers, and the results are as follows: Figure 11 As shown in the figure, compared with the Model, the expression of pro-inflammatory cytokine IL-1β was decreased and the expression of anti-inflammatory cytokine IL-10 was increased in the GM-B / PVA@L-BA-CaNP group, GM-B / PVA@L-CaNP group, GM-B / PVA@L-BA group, and GM / PVA@L-BA-CaNP group. Specifically, the expression of pro-inflammatory cytokine IL-1β was significantly decreased and the expression of anti-inflammatory cytokine IL-10 was significantly increased in the GM-B / PVA@L-BA-CaNP group. This is because BA in the GM-B / PVA@L-BA-CaNP group reduces the secretion of inflammatory factors by regulating the conversion of M1 macrophages to M2 macrophages. Furthermore, Ca in the GM-B / PVA@L-BA-CaNP group... 2+ It is expected to exert an adjunctive anti-inflammatory effect by promoting periodontal bone remodeling and inhibiting the infiltration of inflammatory cells into deeper bone tissue. Therefore, in BA and Ca 2+ The combined effect of these substances further enhances the anti-inflammatory ability of GM-B / PVA@L-BA-CaNP.

[0111] Three-dimensional reconstruction of rat alveolar bone was performed using Micro-CT technology, and the results are as follows: Figure 12 As shown in the diagram, the control group had smaller gaps between teeth and intact alveolar bone, while the model group had more pronounced gaps and significant alveolar bone loss. The GM-B / PVA@L-BA-CaNP group showed significantly reduced gaps and marked alveolar bone repair, demonstrating superior treatment efficacy compared to other groups. This is partly due to the presence of Ca in GM-B / PVA@L-BA-CaNP. 2+ It can promote osteoblast protein expression and promote osteogenic differentiation of periodontal ligament stem cells; on the other hand, BA in GM-B / PVA@L-BA-CaNP is expected to inhibit the differentiation of M1 macrophages into osteoclast precursors by inhibiting the secretion of inflammatory factors, and help promote the regeneration of periodontal bone tissue.

Claims

1. A method co-loaded with baicalin and Ca 2+ The ROS-responsive in-situ photosensitive gel is characterized by, This includes a complex of phospholipid membrane-encapsulated baicalin and calcium carbonate nanoparticles, as well as a ROS-responsive gel matrix. In the phospholipid membrane-coated complex of baicalin and calcium carbonate nanoparticles, the components by weight are: baicalin 1-10 parts, calcium carbonate nanoparticles 1-20 parts, phospholipid material 30-50 parts, and cholesterol 10-30 parts. The ROS-responsive gel matrix comprises the following components by weight: 10-30 parts of 4-hydroxyphenylboronic acid-modified methacrylic anhydride gelatin, 10-30 parts of PVA, and 0.01-0.05 parts of photosensitizer.

2. The co-loaded baicalin and Ca according to claim 1 2+ The ROS-responsive in-situ photosensitive gel is characterized by, The phospholipid membrane-coated composite of baicalin and calcium carbonate nanoparticles has an average particle size of 160-170 nm and a potential of 40-45 mV.

3. The co-loaded baicalin and Ca according to claim 1 2+ The ROS-responsive in-situ photosensitive gel is characterized by, The phospholipid material is selected from one or more of dipalmitoylcholine DPPC, distearylcholine DSPC, dimyristoylphosphatidylcholine DMPC, soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin phosphatidic acid, phosphatidylglycerol, dipalmitoylphosphatidylserine DPPS, dioleoylphosphatidylserine DOPS, lysophosphatidylethanolamine LPE, lysophosphatidylcholine LysoPC, dimyristoylphosphatidylethanolamine DMPE, dioleoylphosphatidylglycerol DOPG, DOP-DEDA, DOTMA, Dlin-MC3-DMA, DODMA, or DOTAP, preferably one or more of dipalmitoylcholine DPPC, lysophosphatidylcholine LysoPC, or DOTAP.

4. The co-loaded baicalin and Ca according to claim 1 2+ The ROS-responsive in-situ photosensitive gel is characterized by, The photosensitizer is one or more of 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO), lithium phenyl 2,4,6-trimethylbenzoylphosphonate (LAP), 2-isopropylthioxanthone (ITX), and 2-hydroxy-2-methyl-1-phenyl-1-propanone.

5. The co-loaded baicalin and Ca according to any one of claims 1-4 2+ A method for preparing ROS-responsive in-situ photosensitizing gel, characterized in that, Includes the following steps: (1) Baicalin and calcium carbonate nanoparticles were dissolved in ethanol, mixed and sonicated and stirred at room temperature to obtain a baicalin and calcium carbonate nanoparticle complex; the baicalin and calcium carbonate nanoparticle complex, phospholipid material and cholesterol were dissolved in ethanol, mixed evenly and added to deionized water while stirring, then stirred at room temperature and sonicated in a water bath to obtain a suspension of the phospholipid membrane-loaded baicalin and calcium carbonate nanoparticle complex. (2) Weigh 4-hydroxyphenylboronic acid PBA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide salt EDC and N-hydroxysuccinimide NHS and dissolve them in DMSO. Stir to activate, and then mix thoroughly with the aqueous solution of methacrylic anhydride gelatin to obtain 4-hydroxyphenylboronic acid modified methacrylic anhydride gelatin. (3) 4-hydroxyphenylboronic acid-modified methacrylic anhydride gelatin and photosensitizer were dissolved in a phospholipid membrane-coated complex suspension of baicalin and calcium carbonate nanoparticles, and then thoroughly mixed with PVA solution to obtain co-loaded baicalin and calcium carbonate nanoparticles. 2+ ROS-responsive in-situ photosensitive gel.

6. The preparation method according to claim 5, characterized in that, In step (1), the mass ratio of baicalin to calcium carbonate nanoparticles is 1:1-3:1; the mass ratio of phospholipid material to cholesterol is 1:1-3:

1.

7. The preparation method according to claim 5, characterized in that, In step (2), the mass ratio of the methacrylic anhydride gelatin and 4-hydroxyphenylboronic acid PBA is 1:1 to 1:

3.

8. The preparation method according to claim 5, characterized in that, In step (3), the mass ratio of the 4-hydroxyphenylboronic acid-modified methacrylic anhydride gelatin and PVA is 1:1 to 1:

3.

9. The co-loaded baicalin and Ca according to any one of claims 1-4 2+ Application of ROS-responsive in-situ photosensitive gel in the preparation of drugs for treating periodontitis.

Images