Synthetic method of photosensitive composite hydrogel and application of photosensitive composite hydrogel in treatment of periodontitis

Abstract

The invention discloses a synthetic method of photosensitive composite hydrogel and application of the photosensitive composite hydrogel in treatment of periodontitis. The composition expression of the photosensitive composite hydrogel is MHA-B-coated Lipo, and the photosensitive composite hydrogel is prepared by the following steps: entrapping berberine hydrochloride by lipidosome with proton gradient, and then mixing with methacrylated hyaluronic acid, a photoinitiator and a cross-linking agent; according to the liposome with the proton gradient, a citric acid buffer solution is adopted for preparing blank liposome, and then alkali liquor is used for adjusting the pH value of an external phase to be alkalescent, so that the transmembrane gradient is formed. The liposome is used as a drug delivery carrier, berberine is entrapped in the liposome and is combined with the MHA hydrogel to form a composite sustained-release drug, and the composite sustained-release drug has the advantages of strong targeting property, good responsiveness, good biocompatibility and high biological safety; and the hydrogel has injectable and photosensitive cross-linking characteristics, is suitable for clinical minimally invasive operation, has the advantages of accurate immune regulation and control, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a synthesis method of a photosensitive composite hydrogel and application thereof in treating periodontitis, belonging to the technical field of biomedicine. Background Art

[0002] Periodontitis is a chronic inflammatory disease affecting approximately 48.6% of the adult population worldwide. It is one of the leading causes of tooth loss in adults and has a clear pathological and bidirectional regulatory relationship with systemic diseases such as diabetes and cardiovascular disease. Current clinical treatment relies on mechanical debridement combined with topical or systemic antibiotics. While this can control infection in the short term, it is difficult to reverse the immune metabolic imbalance and resulting tissue loss in the chronic inflammatory microenvironment. Long-term use of antibiotics also carries the risk of drug resistance.

[0003] In recent years, injectable photosensitive hydrogels have been widely studied for their excellent gelation and spatial adaptability in periodontal regeneration. Previous studies have demonstrated that hyaluronic acid (HA) modified with methacrylic anhydride (MA) can be used to construct photosensitive cross-linked MHA hydrogels, which rapidly form a gel under 365nm ultraviolet light, facilitating stable localized oral administration. However, HA itself has weak antimicrobial properties and lacks regulatory effects on the immune microenvironment, necessitating its combination with functional drugs to enhance therapeutic efficacy.

[0004] Berberine (B) is a natural isoquinoline alkaloid widely used in the treatment of digestive system diseases and exhibits excellent anti-inflammatory activity. Recent studies have found that it can effectively block the glycolysis-inflammation positive feedback loop in pro-inflammatory macrophages by inhibiting the activity of the key glycolytic enzyme PKM2, demonstrating its potential for immune metabolic reprogramming. However, its poor water solubility and low bioavailability make it difficult to stably release in the complex oral environment, limiting its clinical application.

[0005] Therefore, it is necessary to develop a berberine nano-drug delivery hydrogel with strong targeting, photosensitivity, and controlled sustained release to solve the above technical problems. Summary of the Invention

[0006] The purpose of the present invention is to construct a berberine nano-delivery hydrogel composite sustained-release drug (MHA-B@Lipo) with strong targeting, photosensitivity response and controllable sustained release, which is used for the metabolic regulation of pro-inflammatory macrophages and bone tissue repair in the periodontitis microenvironment, breaking through the problems of antibiotic dependence, unstable efficacy, insufficient immune regulation and so on in existing treatments, and has good application prospects and translational value.

[0007] In order to achieve the above-mentioned object, the present invention provides a photosensitive composite hydrogel, the composition expression of which is MHA-B@Lipo, wherein MHA is methacryloylated hyaluronic acid, B is berberine hydrochloride, and Lipo is liposome; the photosensitive composite hydrogel is prepared by encapsulating berberine hydrochloride in liposomes with a proton gradient and then mixing it with methacryloylated hyaluronic acid, a photoinitiator and a cross-linking agent; the liposomes with a proton gradient are prepared by using a citrate buffer to prepare blank liposomes, and then adjusting the pH of the external phase to a weak alkaline state with an alkaline solution to form a transmembrane gradient.

[0008] Preferably, the raw materials for preparing the photosensitive composite hydrogel include berberine hydrochloride, liposome and methacryloyl hyaluronic acid in a mass ratio of 1:10-20:0.2-5.

[0009] Preferably, the liposome comprises soybean lecithin, cholesterol and DSPE-PEG2000.

[0010] Preferably, the molar ratio of soybean lecithin:cholesterol:DSPE-PEG2000 is 20:5:1.

[0011] The present invention also provides a method for preparing the above-mentioned photosensitive composite hydrogel, comprising the following steps:

[0012] Step 1: Prepare blank liposomes using citrate buffer, then adjust the pH of the external phase to a weakly alkaline state with alkali solution to form a transmembrane gradient, thereby obtaining a liposome suspension with a proton gradient;

[0013] Step 2: dissolving berberine hydrochloride (BBR) in HEPES buffer, adding the liposome suspension obtained in step 1, and incubating under heating conditions to allow berberine hydrochloride to actively enter the liposome lumen with the help of a proton gradient; then adjusting the liposome particle size by extrusion through a polycarbonate membrane and centrifuging and purifying, collecting the supernatant to obtain a berberine liposome (Lipo-BBR) dispersion;

[0014] Step 3: Dissolve the MHA powder in the berberine liposome (Lipo-BBR) dispersion, add a photoinitiator and a cross-linking agent, stir and mix, and obtain a photosensitive composite hydrogel.

[0015] Preferably, the specific steps of preparing the blank liposomes in step 1 include: first preparing a liposome dry film by a thin film dispersion method, and then adding hot citrate buffer to hydrate the liposomes to form coarsely dispersed blank multilamellar liposomes.

[0016] Preferably, the pH value of the citrate buffer in step 1 is 3-5, more preferably 4.

[0017] Preferably, in step 1, the pH of the external phase is adjusted to 7-8, more preferably 7.8.

[0018] Preferably, the pH value of the HEPES buffer in step 2 is 7-8, more preferably 7.8.

[0019] Preferably, the photoinitiator in step 3 is Irgacure 2959, and the addition ratio of the photoinitiator is 0.05-0.2 wt% of the mass of MHA, more preferably 0.1 wt%; the crosslinking agent is N,N'-methylenebisacrylamide, and the addition ratio of the crosslinking agent is 0-1 / 2 of the mass of MHA, more preferably 1 / 3.

[0020] Preferably, in step 3, after stirring and mixing, a cross-linking reaction is initiated by light to prepare a gel.

[0021] Preferably, the preparation process of the MHA powder in step 3 comprises the following steps:

[0022] Step 1.1: dissolving hyaluronic acid in deionized water to obtain a hyaluronic acid aqueous solution;

[0023] Step 1.2: Add methacrylic anhydride dropwise to the hyaluronic acid aqueous solution under ice-water bath, adjust the pH of the reaction system to 8-9 with alkali solution, and stir the reaction under ice-water bath;

[0024] Step 1.3: After the reaction is completed, the MHA powder is separated and purified, and freeze-dried to obtain the MHA powder.

[0025] Preferably, the feeding ratio of the hyaluronic acid and methacrylic anhydride is 1 g: 7-8 mL.

[0026] Preferably, the separation and purification in step 1.3 comprises: adding ethanol after the reaction to precipitate the product, dissolving the precipitated product in water and performing dialysis purification.

[0027] The present invention also provides use of the photosensitive composite hydrogel in preparing products for treating periodontitis.

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

[0029] The present invention uses liposomes (Liposomal nanoparticles, Lipo) as drug delivery carriers, encapsulates berberine in the liposomes, and combines it with MHA hydrogel to form a composite sustained-release drug (MHA-B@Lipo), which has the following advantages: 1) Strong targeting: relying on HA-CD44 interaction to achieve targeted drug delivery to pro-inflammatory macrophages; 2) Good responsiveness: It can enhance B release in the periodontitis microenvironment (low pH, ROS); 3) Good biocompatibility: The construction material components are all natural or biomimetic biomaterials with high biosafety; 4) Easy operation: It has injectability and photosensitive cross-linking properties, and is suitable for clinical minimally invasive operations; 5) Precise immune regulation: It regulates macrophage phenotype through metabolic reprogramming and activates tissue self-repair mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the synthesis and application of MHA-B@Lipo hydrogel.

[0031] Figure 2 SEM scanning results of MHA-B@Lipo hydrogel.

[0032] Figure 3 Changes in storage modulus and loss modulus of hydrogel under UV irradiation.

[0033] Figure 4 In vitro testing of hydrogel biocompatibility.

[0034] Figure 5 Drug release under inflammatory environment (pH 5.5, 1 mM H2O2).

[0035] Figure 6 Confocal microscopy shows that the co-localization of MHA (green) and CD44 receptor (red) is significantly increased in LPS-activated mouse bone marrow-derived macrophages (mBMDM). DETAILED DESCRIPTION

[0036] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0037] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in the following examples, unless otherwise specified, were obtained from commercial sources.

[0038] Example

[0039] This study aims to construct a berberine-liposome-loaded photosensitive composite hydrogel (MHA-B@Lipo) for targeted immune regulation and bone regeneration in periodontitis. This material exhibits excellent injectability, biocompatibility, and UV cross-linking capabilities, enabling stable and sustained release of berberine in inflammatory environments.

[0040] 1. Preparation of berberine liposomes (Lipo-BBR)

[0041] Preparation of lipid film: Soybean lecithin: cholesterol: DSPE-PEG2000 = 20:5:1 (molar ratio), dissolved in chloroform / methanol (2:1, v / v), transferred to a round-bottom flask, and rotary evaporated at 40°C and -0.08 MPa to obtain a uniform lipid dry film; then, continue drying in a vacuum for ≥ 30 min to completely remove residual organic solvent.

[0042] Liposome hydration: Preheat 300 mmol / L, pH 4.0 citrate buffer to 55°C and add enough liquid to completely cover the film. Hydrate at 55°C for 30-50 minutes using a shaking water bath or spinner flask to form a coarsely dispersed blank multilamellar vesicle (MLV) suspension.

[0043] Proton gradient establishment: The obtained blank multilamellar liposome (MLV) suspension was placed at room temperature, and the external phase pH was slowly titrated to 7.8 using 1 mol / L NaOH solution while maintaining magnetic stirring to establish a proton gradient from intramembrane acidity (pH 4.0) to extramembrane alkalinity (pH 7.8), thereby obtaining a liposome suspension with a proton gradient.

[0044] Drug loading: Berberine hydrochloride (BBR) was dissolved in HEPES buffer at pH 7.8 (concentration of 1 mg / mL) and added to the liposome suspension with a proton gradient at a drug / lipid mass ratio of 1:10 to 1:20. The mixture was incubated at 60°C with gentle shaking for 10-15 minutes to allow BBR to actively enter the liposome lumen via the proton gradient.

[0045] Particle size control and purification: Liposomes were extruded through 0.5 μm, 0.2 μm, and 0.1 μm polycarbonate membranes 3-5 times per well to obtain berberine liposomes (Lipo-BBR) with a particle size of 100-200 nm and a PDI < 0.2. Free BBR was then removed by centrifugation at 8000 rpm and 4°C for 10 minutes, and the supernatant of the Lipo-BBR liposomes was collected for later use.

[0046] 2. Synthesis of Methacryl Hyaluronic Acid (MHA)

[0047] Dissolve 2.0 g of hyaluronic acid (HA) in 200 mL of deionized water and stir overnight at 4 °C in a dark environment until completely dissolved;

[0048] Slowly add 14.8 mL of methacrylic anhydride (MA, i.e., 7.4 mL / g HA) and maintain the temperature at 4°C.

[0049] Adjust the pH of the reaction system to 8.0-9.0 with NaOH solution (4 M for initial adjustment and 2 M for fine adjustment) and stir continuously at 4 °C for 24 h;

[0050] After the reaction is completed, an appropriate amount of ethanol is added to precipitate the modified product;

[0051] The precipitated product was transferred to a dialysis bag with a molecular weight cutoff of 8-14 kDa and dialyzed in deionized water for 7 days (the water was changed twice daily for the first 3 days and once daily for the next 4 days);

[0052] After dialysis, the solution was frozen and freeze-dried in a freeze dryer for 12-24 hours to obtain a white porous sponge-like methacryloyl hyaluronic acid (MHA) powder;

[0053] The MHA freeze-dried powder was sealed and stored at -20°C for later use.

[0054] 3. Construction of MHA-B@Lipo composite hydrogel

[0055] Take an appropriate amount of freeze-dried MHA powder and dissolve it in the supernatant of Lipo-BBR liposomes to prepare an MHA solution with a mass concentration of 2 wt%;

[0056] Add 0.1 wt% of the photoinitiator Irgacure 2959 and mix thoroughly. To improve the mechanical properties of the hydrogel, N,N'-methylenebisacrylamide can be added as an auxiliary crosslinker at a ratio of 1 / 3 of the MHA mass (optional).

[0057] Under dark conditions, stir at room temperature with low speed magnetic stirring (150-200 rpm) for 12-24 hours to fully dissolve MHA and mix it with Lipo-BBR to form a uniform injectable precursor solution;

[0058] The precursor liquid was injected into the mold or directly applied to the lesion area, and irradiated with 365nm ultraviolet light for 60 seconds (adjusted according to the specific application) to induce MHA cross-linking to form a three-dimensional hydrogel network structure, and finally the photosensitive composite hydrogel MHA-B@Lipo containing berberine liposomes was obtained.

[0059] 4. Results and Characterization

[0060] Particle size analysis: Using a particle size analyzer, the average particle size of the liposomes was about 100 nm, PDI < 0.2, and the particle size distribution was uniform;

[0061] Morphology observation: The prepared MHA-B@Lipo is a yellow translucent gel with good shear thinning rheology, which is easy to inject. The microstructure of the composite hydrogel was observed using a scanning electron microscope (SEM). Figure 2 As shown,

[0062] UV cross-linking effect: Dynamic shear rheometer was used to perform time rheological test on the hydrogel under UV irradiation, and the storage modulus and loss modulus of the hydrogel at different time points under UV irradiation were recorded. Figure 3 As shown, Figure 3 Results showed that the storage modulus (G') increased from 0.6 kPa to 22.8 kPa (MHA) and from 0.8 kPa to 29.8 kPa (MHA-B@Lipo) within 300 seconds. G' increased rapidly between 0 and 30 seconds and then stabilized. To minimize potential cytotoxicity, 30 seconds of UV irradiation was selected as the optimal gelation time for photocrosslinking and subsequent biological studies.

[0063] Biocompatibility test: Biocompatibility test results are as follows: Figure 4 As shown, Figure 4 The results showed that at an effective concentration of 5 μM, free drugs, liposome-encapsulated drugs, and composite hydrogels all exhibited good biocompatibility with mouse macrophages mBMDMs and MC3T3-E1 within 24 hours and 7 days.

[0064] Drug release performance under inflammatory environment: The release performance of MHA-B@Lipo under inflammatory environment (pH 5.5, 1mM H2O2) was tested in vitro. Figure 5 As shown, the results show that the material has good sustained-release ability, BBR release is accelerated under the in vitro simulated inflammatory environment (pH 6.0 / ROS), and has stimulus-responsive characteristics.

[0065] Targeting performance: Confocal microscopy showed that the co-localization of MHA (green) and CD44 receptor (red) increased significantly in LPS-activated mouse bone marrow-derived macrophages (mBMDM), as shown in Figure 2. Figure 6 shown.

[0066] In clinical practice, the MHA-B@Lipo composite hydrogel can be directly injected into periodontal lesions. It is then irradiated with a dental UV light device to crosslink and gel, forming a stable, controllable, sustained-release structure. In response to microenvironmental stimuli, the hydrogel releases berberine in the inflamed area, specifically regulating the metabolic state of local macrophages, alleviating inflammation and promoting bone regeneration. It is expected to become a breakthrough therapeutic material for alternative antibiotics.

[0067] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A photosensitive composite hydrogel, characterized in that: The composition expression of the photosensitive composite hydrogel is MHA-B@Lipo, wherein MHA is methacryloylated hyaluronic acid, B is berberine hydrochloride, and Lipo is liposome; the photosensitive composite hydrogel is prepared by encapsulating berberine hydrochloride in liposomes with a proton gradient and then mixing it with methacryloylated hyaluronic acid, a photoinitiator, and a cross-linking agent; the liposomes with a proton gradient are prepared by using a citric acid buffer to prepare blank liposomes, and then using an alkaline solution to adjust the pH of the external phase to a weak alkaline state to form a transmembrane gradient.

2. The photosensitive composite hydrogel according to claim 1, wherein The raw materials for preparing the photosensitive composite hydrogel include berberine hydrochloride, liposome and methacryloyl hyaluronic acid in a mass ratio of 1:10-20:0.2-5.

3. The photosensitive composite hydrogel according to claim 1, wherein The liposome comprises soybean lecithin: cholesterol: DSPE-PEG2000.

4. The photosensitive composite hydrogel according to claim 3, wherein The molar ratio of soybean lecithin:cholesterol:DSPE-PEG2000 is 20:5:

1.

5. The method for preparing the photosensitive composite hydrogel according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step 1: Prepare blank liposomes using citrate buffer, then adjust the pH of the external phase to a weakly alkaline state with alkali solution to form a transmembrane gradient, thereby obtaining a liposome suspension with a proton gradient; Step 2: dissolving berberine hydrochloride in HEPES buffer, adding the liposome suspension obtained in step 1, and incubating under heating conditions to allow berberine hydrochloride to actively enter the liposome lumen with the help of a proton gradient; then adjusting the liposome particle size by extrusion through a polycarbonate membrane and centrifuging and purifying, collecting the supernatant to obtain a berberine liposome dispersion; Step 3: dissolving the MHA powder in the berberine liposome dispersion, adding a photoinitiator and a cross-linking agent, stirring and mixing to obtain a photosensitive composite hydrogel.

6. The preparation method according to claim 5, wherein The specific preparation steps of the blank liposomes in step 1 include: firstly preparing a liposome dry film by a thin film dispersion method, and then adding hot citrate buffer to hydrate the liposomes to form coarsely dispersed blank multilamellar liposomes. And / or, the pH value of the HEPES buffer in step 2 is 7-8.

7. The preparation method according to claim 5, wherein The photoinitiator in step 3 is Irgacure 2959, and the addition ratio of the photoinitiator is 0.05-0.2 wt% of the mass of MHA; the crosslinking agent is N,N'-methylenebisacrylamide, and the addition ratio of the crosslinking agent is 0-1 / 2 of the mass of MHA.

8. The preparation method according to claim 5, wherein The preparation process of the MHA powder in step 3 comprises the following steps: Step 1.1: dissolving hyaluronic acid in deionized water to obtain a hyaluronic acid aqueous solution; Step 1.2: Add methacrylic anhydride dropwise to the hyaluronic acid aqueous solution under ice-water bath, adjust the pH of the reaction system to 8-9 with alkali solution, and stir the reaction under ice-water bath; Step 1.3: After the reaction is completed, the MHA powder is separated and purified, and freeze-dried to obtain the MHA powder.

9. The preparation method according to claim 8, wherein The feeding ratio of the hyaluronic acid and methacrylic anhydride is 1 g: 7-8 mL.

10. Use of the photosensitive composite hydrogel according to any one of claims 1 to 4 in the preparation of a product for treating periodontitis.

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