Injectable temperature-sensitive hydrogel for oral mucosa protection and preparation method thereof

Abstract

The invention discloses injectable temperature-sensitive hydrogel for oral mucosa protection and a preparation method of the injectable temperature-sensitive hydrogel, and relates to the field of biological materials.The preparation method comprises the following steps that poloxamer F127 and gelatin are added into deionized water and stirred until the poloxamer F127 and the gelatin are completely dissolved, and transparent sol is formed; slowly dropwise adding an APBA solution into the transparent sol at 0-4 DEG C, and stirring to form a reversible borate bond network and construct a dynamic cross-linking system; adding AA2G into the dynamic cross-linking system, stirring and mixing in a dark place, carrying out ultrasonic dispersion to completely dissolve and disperse the AA2G into the dynamic cross-linking system, finally adding QCS into the reaction system, stirring and mixing for 30 minutes at 100 rpm in a dark place, and carrying out ultrasonic dispersion; and heating to 37 DEG C, adjusting the pH value to be neutral, carrying out sterile filtration, and subpackaging to obtain the temperature-sensitive hydrogel.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, and particularly to oral mucosal protection materials, specifically an injectable thermosensitive hydrogel for oral mucosal protection and its preparation method. Background Technology

[0002] Oral wounds (such as intractable oral ulcers, post-periodontal surgery wounds, and delayed healing after tooth extraction) heal particularly slowly and complexly due to their unique location (moist, mobile, and subject to saliva rinsing). Compared to healthy patients, the wound sites of diabetic patients are more susceptible to bacterial infection, inflammatory imbalance, impaired angiogenesis, and increased oxidative stress due to the overlapping pathological microenvironment of diabetes. These pathological processes severely hinder normal mucosal healing; therefore, wound healing disorders in diabetic patients are a serious clinical problem that urgently needs to be addressed.

[0003] Ideal oral wound dressings for diabetes not only need to meet the requirements of conventional oral dressings (such as ease of application, good retention, and comfort), but also must be able to proactively address the unique pathological conditions of diabetic wounds in order to overcome the treatment challenges of traditional dressings in complex microenvironments.

[0004] While existing technologies such as oral dressings and thermosensitive hydrogels have been used to promote wound healing, they both have significant shortcomings and are unable to address the multiple pathological problems of diabetic oral wounds. First, traditional ointments, pastes, patches, or ordinary sprays are difficult to retain stably in the moist, highly mobile oral environment and are easily washed away by saliva, resulting in poor retention and difficulty in adhesion. More importantly, due to the pathological characteristics of diabetic wounds, such as persistent inflammation, susceptibility to infection, and impaired healing-related signaling pathways, existing dressings lack targeted and effective intervention components or designs, and the passive protection they provide is insufficient to overcome the healing obstacles caused by diabetes.

[0005] II. Existing thermosensitive hydrogels, as carriers for in-situ molding, have shown potential in the field of wound dressings and are a potential option for oral and diabetic wound care. However, existing thermosensitive hydrogel systems for oral wounds often have the following key drawbacks when targeting the specific scenario of diabetic oral wounds: First, existing systems are not specifically designed for the diabetic microenvironment and lack the responsiveness and regulatory capacity to address the diabetic microenvironment (hyperglycemia, high oxidative stress, chronic inflammation, and susceptibility to infection). They also lack the ability to load or intelligently release targeted drugs such as potent anti-inflammatory agents, broad-spectrum antibacterial agents / antibiofilm agents, antioxidants, and pro-angiogenic factors, making it difficult to synergistically address multiple pathological factors.

[0006] Secondly, because diabetic patients have impaired tissue repair capabilities and immune status, the biocompatibility requirements for materials are higher. Some existing gel materials or their degradation products may trigger stronger irritation or hinder healing in a high-sugar environment, and their biocompatibility and safety have often not been fully verified.

[0007] Third, existing thermosensitive hydrogels focus on physical barriers and have limited functions (such as only basic antibacterial function). They fail to integrate multiple synergistic effects such as physical protection, controlled drug release, and active regulation of the microenvironment (such as regulating macrophage polarization, scavenging reactive oxygen species, and promoting angiogenesis), making it difficult to systematically address the complex biological processes of diabetic wound healing.

[0008] In summary, existing technologies are insufficient to meet the clinical needs of oral wound care for diabetic patients. There is an urgent need to develop an injectable, temperature-sensitive hydrogel that, through material design and synergistic action of active ingredients, integrates physical isolation, controlled drug release, and active microenvironment regulation. This hydrogel should intelligently release anti-inflammatory, antibacterial, antioxidant, and angiogenic drugs targeting the diabetic microenvironment; maintain on-demand release and high biocompatibility even in high-glucose environments; synergistically exert physical protection and microenvironment regulation effects; and improve sprayability, injectability, gelation speed, and stability. Such dressings have significant clinical importance and practical value in enhancing the effectiveness of oral wound care for diabetic patients. Summary of the Invention

[0009] This invention addresses the pressing issue that existing thermosensitive hydrogels struggle to simultaneously achieve injectability, oral retention, and multifunctional synergy in the oral environment of diabetic patients. It provides an injectable thermosensitive hydrogel for oral mucosal protection and its preparation method, effectively resolving the clinical dilemma of oral wound care for diabetic patients.

[0010] This invention is achieved using the following techniques: This invention provides an injectable thermosensitive hydrogel (F127 / Gel-QCS / APB@AA2G, FGQA) for oral mucosal protection, comprising the following components: Poloxamer F127 serves as the physical cross-linking backbone; Gelatin provides reactive amino (-NH2) groups, which enhance mucosal adhesion and promote cell migration. APBA (aldehyde phenylboronic acid) forms a dynamic reversible borate bond with the AA2G vicinal diol group, which acts as a blood glucose response switch for on-demand drug release upon bond cleavage during hyperglycemia, and also serves as a dynamic crosslinking agent. AA2G (ascorbic acid-2-glucosidase) has a strong antioxidant and healing-promoting effect. It competitively binds to GLUT1 on the cell membrane with glucose molecules, and after entering the cell, it activates related signaling pathways and corrects the microenvironment disorder caused by high glucose. QCS (chitosan quaternary ammonium salt) carries a strong positive charge, which can enhance the structure of the hydrogel and has antibacterial and hemostatic functions.

[0011] Deionized water is used as a solvent to ensure the activity of the components is maintained during the low-temperature preparation process.

[0012] Furthermore, based on deionized water, The mass-to-volume ratio of poloxamer F127 to deionized water is 15-25%. The mass-to-volume ratio of gelatin to deionized water is 3-8%. The mass-to-volume ratio of APBA to deionized water is 0.5-2%. The mass-to-volume ratio of AA2G to deionized water is 1-5%. The mass-to-volume ratio of QCS to deionized water is 0.5-3%.

[0013] Furthermore, the prepared injectable thermosensitive hydrogel has a viscosity of ≤50 mPa·s at room temperature, a gelation time of ≤10s at 37℃, and resists saliva rinsing for ≥6 hours.

[0014] The three-tiered cascade synergistic anti-glycation and healing-promoting mechanism of this invention: 1. APBA-based dynamic bond exchange competing with GLUT1 (glucose transporter 1)

[0015] This mechanism aims to reduce excessive glucose input to cells at the wound site, alleviating high glucose stress at its source. Glucose responsiveness: The functional cross-linking agent in the gel network—aldeoxyphenylboronic acid (APBA)—can form reversible borate ester bonds with the vicinal diol structure in the antioxidant AA2G. When the local glucose concentration at the wound site increases, glucose (also containing the vicinal diol structure) competitively binds to APBA, leading to partial exchange of borate ester bonds and the intelligent release of AA2G. The released AA2G, with a molecular structure similar to glucose, can competitively bind to GLUT1 on the cell membrane (expressed at higher glucose levels), occupying its transport channel and thus reducing excessive glucose uptake by the cell. According to molecular docking simulations and surface plasmon resonance results, the AA2G binding domain has a high affinity (binding energy ≤ -7.3 kcal / mol), effectively inhibiting excessive glucose from entering the cell through GLUT1. The 2-NBDG fluorescence tracing experiment verified that HGF cells treated with the gel of this invention showed a significant decrease in glucose uptake, with an inhibition rate of up to 81.4%. By reducing the intracellular glucose load, the source pressure of subsequent advanced glycation end products (AGEs) formation and oxidative stress was directly alleviated.

[0016] 2. End-of-life scavenging and repair – the antioxidant properties of AA2G and its synergistic effect against glycation.

[0017] This mechanism aims to eliminate existing harmful substances and promote repair, blocking the high-glycemic damage chain downstream. It effectively scavenges reactive oxygen species (ROS): AA2G, a stable vitamin C derivative, can be enzymatically released into active vitamin C (VC) at the wound site. In vitro and in vivo experiments have confirmed that this component can directly neutralize excess ROS, effectively reducing oxidative stress-induced cell damage. In the high-glycemic environment of diabetes, ROS is a key factor driving the rapid formation of advanced aging processes (AGEs). In this invention, AA2G, through its powerful ROS scavenging, can block this major oxidative pathway of AGE formation. Simultaneously, vitamin C, as an essential cofactor for proline hydroxylase, is crucial for collagen synthesis and maturation. Therefore, this mechanism not only indirectly combats glycation damage through antioxidation but also directly promotes extracellular matrix reconstruction, improving healing quality from multiple levels.

[0018] 3. Endogenous metabolic regulation and regeneration repair activation – FGF1-Hippo signaling axis

[0019] The most crucial finding of this invention is that the aforementioned microenvironment remodeling can directly activate the endogenous repair program of key wound cells (fibroblasts). Analysis of fibroblasts in a diabetic wound model treated with the hydrogel of this invention using RNA sequencing (RNA-seq) and Western blotting (WB) revealed a significant upregulation of fibroblast growth factor 1 (FGF1) expression, correcting the abnormal activation of the hippo signaling pathway in diabetic patients. This upregulation of FGF1 is not an isolated event; it is an important intracellular metabolic regulator with insulin-like effects, which can further synergistically enhance the hypoglycemic effect of the first-line mechanism within the cell, forming an internal-external glucose regulatory network.

[0020] This invention also provides a method for preparing an injectable thermosensitive hydrogel for oral mucosal protection, comprising the following steps: a) Pre-melting of temperature-sensitive skeleton substrate Add poloxamer F127 and gelatin to deionized water at 4°C, maintain the temperature at 4°C and stir at 300 rpm for 2 hours to prevent gelatin denaturation, until completely dissolved to form a transparent sol (GF127).

[0021] b, Dynamic covalent crosslinking At 0~4℃, APBA solution (aldehyde phenylboronic acid solution) was slowly added dropwise to the transparent sol obtained in step a, while maintaining the pH at 7.4 and stirring at 200 rpm for 2 hours to form a reversible Schiff base network.

[0022] c, Antioxidant integration AA2G was added to the dynamic crosslinking system constructed in step b, stirred and mixed under light-protected conditions, and ultrasonically dispersed to completely dissolve and disperse AA2G in the dynamic crosslinking system. This step ultimately yielded a dual dynamic covalent network based on Schiff base and borate ester.

[0023] The reaction mechanism is as follows: APBA possesses both -CHO and -B(OH)2, and can undergo two different types of covalent reactions with GF127 obtained in step a and AA2G added in step c, namely: ① Schiff base reaction: GF127-NH2 + APBA-CHO → GF127-N=CH-APBA +H2O ②Borate ester bonding: AA2G-C(OH)-C(OH)-+ APBA-B(OH)2 → AA2G-COB-APBA d. Integration of antibacterial and hemostatic functions QCS was added to the dynamic cross-linking system constructed in step c. Under light-protected conditions, the mixture was stirred at 100 rpm for 30 min and ultrasonically dispersed to completely dissolve and disperse the QCS in the dynamic cross-linking system. This strengthened the structure of the reaction system and endowed the gel with antibacterial and hemostatic functions, making it suitable for diabetic wounds prone to infection.

[0024] QCS is mainly integrated in two ways: first, the residual primary amine groups can participate in the covalent cross-linking of Schiff bases; second, the physical adsorption of their positive charges.

[0025] e, Gelization regulation The product obtained in step d was heated to 37°C to simulate oral temperature, allowing observation of gelation behavior. The gelation time was ≤10s. The pH was adjusted to 7.0~7.8 using 0.1 M NaOH or HCl to neutralize the acidity of APBA and prevent hydrolysis of AA2G. After aseptic filtration, the product was dispensed using a 0.22 μm filter membrane to obtain a temperature-sensitive hydrogel (F127 / Gel-QCS / APB@AA2G, FGQA), which can be stored at room temperature.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention discloses an injectable thermosensitive hydrogel for oral mucosal protection and its preparation method, which has excellent injectability and barrier stability: room temperature viscosity ≤50 mPa·s, gelation within 10 seconds at 37℃, resistance to saliva rinsing for ≥6 hours; it remains stable at room temperature and can be stored at room temperature.

[0027] 2. More importantly, this invention reveals for the first time and experimentally confirms that the gel can significantly upregulate the expression of FGF1 in wound fibroblasts, thereby activating endogenous insulin-like metabolic regulation and strong pro-proliferation and pro-angiogenic signals, which is a key mechanism for achieving high-quality regeneration of diabetic refractory wounds.

[0028] 3. The hydrogel disclosed in this invention achieves stable adhesion and self-healing in the moist, easily infected, and difficult-to-heal oral environment of diabetic patients through multiple dynamic networks. It synergistically exerts antibacterial, hemostatic, and antioxidant effects, precisely reduces local tissue sugar uptake, corrects local microenvironment disorders, stimulates the expression of endogenous growth factors, and promotes the repair of local tissue functions. It provides a multi-functional intelligent responsive, on-demand release drug treatment strategy for diabetic oral mucosa. Attached Figure Description

[0029] Figure 1 This diagram illustrates the molecular structures of poloxamer F127, gelatin, and GF127 of the present invention.

[0030] Figure 2 This diagram illustrates the injectable temperature-sensitive gel sol (room temperature) to gel (37°C) conversion of the present invention.

[0031] Figure 3 The example demonstrates the injectability of the thermosensitive gel obtained, showing rapid gelation upon heating to a 37-degree platform.

[0032] Figure 4 The rheological curves of the thermosensitive gel obtained in the examples are shown as a function of temperature.

[0033] Figure 5 The thermosensitive gel obtained in the example shows a porous structure under a scanning electron microscope.

[0034] Figure 6 This diagram illustrates the superposition model of AA2G and glucose in the GLUT1 transporter binding domain obtained through molecular docking simulation.

[0035] Figure 7 This diagram shows a fluorescence microscope illustration and statistical chart of 2-NBDG fluorescence tracer for glycogen uptake in fibroblasts.

[0036] Figure 8 This diagram illustrates the wound healing process in animal models used in the examples and comparative examples.

[0037] Figure 9 The results of HE staining in animal models of the examples and comparative examples are shown.

[0038] Figure 10 The MASSON staining results of the animal models in the examples and comparative examples are shown.

[0039] Figure 11 CCK8 experiments show the statistical graph of HGF cell survival rates after 24 and 48 hours of treatment with thermosensitive gels at different drug loading concentrations.

[0040] Figure 12 The changes in serum liver and kidney function indicators (ALT, AST, UREA, CREA) in diabetic mice of the examples and comparative examples are shown.

[0041] Figure 13 The images show fluorescence microscopy images of thermosensitive gel extracts with different drug loading concentrations used to assess the cytotoxicity of HGF cells using the Calcein-AM / PI double staining method. Detailed Implementation

[0042] Specific embodiments of the present invention will be described below. Example 1

[0043] The present invention discloses a method for preparing an injectable thermosensitive hydrogel for oral mucosa protection, comprising the following steps: a) Pre-melting of temperature-sensitive skeleton substrate like Figure 1 Poloxamer F127 and gelatin were added to deionized water at 4°C, and the mixture was stirred at 300 rpm for 2 hours until completely dissolved. Magnetic stirring was used in this embodiment to form a transparent sol (GF127).

[0044] b, Dynamic covalent crosslinking APBA solution was slowly added dropwise to the transparent sol prepared in step a at 0~4℃, while maintaining the pH at 7.4. The mixture was stirred at 200 rpm for 2 h to form a reversible Schiff base network and construct a dynamic cross-linking system.

[0045] c, Antioxidant integration AA2G was added to the dynamic crosslinking system constructed in step b, stirred and mixed under light-protected conditions, and ultrasonically dispersed to completely dissolve and disperse AA2G in the dynamic crosslinking system, ultimately forming a reversible network based on dual dynamic covalent bonds (Schiff base-boronic acid ester).

[0046] d. Integration of antibacterial and hemostatic functions QCS was added to the dynamic cross-linking system constructed in step c. Under light-protected conditions, the mixture was stirred at 100 rpm for 30 min and ultrasonically dispersed to completely dissolve and disperse the QCS in the dynamic cross-linking system, thereby strengthening the structure of the reaction system and endowing the gel with antibacterial and hemostatic functions.

[0047] e, Gelization regulation The product obtained in step d was heated to 37°C, and its gelation behavior was observed. The gelation time was found to be ≤10s. Figure 2The pH was adjusted to 7.0–7.8 using 0.1 M NaOH or HCl, and the mixture was aseptically filtered and dispensed using a 0.22 μm filter membrane to prepare a temperature-sensitive hydrogel (F127 / Gel-QCS / APB@AA2G, FGQA), which was stored at room temperature.

[0048] In the above steps, based on deionized water, the mass-volume ratio of poloxamer F127 to deionized water is 18%, the mass-volume ratio of gelatin to deionized water is 5%, the mass-volume ratio of APBA to deionized water is 1.5%, the mass-volume ratio of AA2G to deionized water is 2.5%, and the mass-volume ratio of QCS to deionized water is 3%.

[0049] The properties of the prepared thermosensitive hydrogel were validated. The injectability of the hydrogel was observed, such as... Figure 3 The hydrogel was injected onto a 37°C heating platform, and its gelation properties and gelation time were observed; the gelation time was 8 ± 2 s. The gelation temperature was measured at 32.3°C using a rotational rheometer. Figure 4 In vitro simulated saliva flushing experiments (flow rate 1 mL / min) showed that the gel effectively resisted flushing for ≥6 hours. After low-temperature freeze-drying, scanning electron microscopy revealed that the thermosensitive gel structure was loose and porous, as shown in... Figure 5 . Figure 6 Molecular docking simulations showed that AA2G has a high affinity for the GLUT1 binding domain (binding energy ≤ -7.3 kcal / mol), effectively inhibiting the excessive entry of external glucose into the cell via GLUT1. Glucose uptake in fibroblasts was detected using 2-NBDG fluorescence tracing. The fluorescence intensity of the gel-treated group (HGT) decreased by 81.4% compared to the high glucose control group (HG), validating at the cellular level that AA2G can competitively bind to GLUT1. Figure 7 As shown. Comparative Example

[0050] PBS spray was used as Comparative Example 1, and commercially available triamcinolone acetonide oral paste was used as Comparative Example 2. The efficacy of the two comparative examples and the gel prepared in the example was compared in a diabetic animal model.

[0051] animal models

[0052] The animal model used was a type 2 diabetic mouse induced by a high-fat diet and streptozotocin. Random blood glucose levels were ≥11.2 mmol / L, n=15 mice / group. A 2 mm diameter oral mucosal wound was created in the oral cavity of each diabetic mouse. The mice were randomly divided into three groups and treated with Comparative Example 1 (PBS injection), Comparative Example 2 (triamcinolone acetonide oral paste application), and Example 3 (thermosensitive gel injection, 20 μL / time). All treatments were administered once daily. The wound area was recorded on days 0, 3, 7, and 14 post-modeling.

[0053] The test results are as follows Figure 8 After 14 days of treatment, the wound closure rate in the example reached 95.7%, which was significantly better than that in the control group.

[0054] Histological analysis

[0055] After euthanizing the animal, tissue samples were taken from the wound for histological analysis.

[0056] H&E staining showed that the epithelial layer in the example group was completely regenerated with minimal inflammatory cell infiltration; Masson staining further showed that the collagen in the example group was densely and orderly arranged, such as... Figure 9 , 10 This demonstrates that, compared to the comparative example, the hydrogel provided by the present invention significantly accelerates the healing process of oral mucosal wounds in diabetic patients.

[0057] RNA sequencing

[0058] To further explore its healing-promoting mechanism, we performed RNA sequencing analysis on the treated fibroblasts. The results showed a significant increase (10.32-fold) in the expression of the key regeneration factor FGF1, which was verified by Western blotting (WB). This indicates that the hydrogel of this invention not only improves the local microenvironment but also directly upregulates endogenous tissue regeneration processes, including promoting cell proliferation and migration, driving angiogenesis, improving wound healing disorders in diabetic patients, and effectively combating the delayed healing caused by diabetes.

[0059] Security verification

[0060] To ensure the safety of clinical application, a safety evaluation of this invention was conducted.

[0061] 1. Cytotoxicity: like Figure 11 HGF cell viability was detected using the CCK-8 assay: >100% (100 μg / mL gel extract); indicating that the gel extract was non-cytotoxic. The Calcein-AM / PI double staining method was used to assess the cytotoxicity of the temperature-sensitive gel extract to HGF cells; the results showed that the viable cell rate in the 100 μg / mL gel extract group was ≥98%. Figure 13 .

[0062] 2. Systemic toxicity: like Figure 12 After 14 days of treatment, the liver and kidney function indicators (ALT, AST, UREA, CREA) of diabetic rats were not significantly different from those of the control group (p>0.05).

[0063] The combined results of in vitro and in vivo experiments demonstrate that the hydrogel of the present invention exhibits excellent biocompatibility and safety at effective therapeutic doses.

[0064] The scope of protection claimed by this invention is not limited to the specific embodiments described above. Moreover, for those skilled in the art, this invention can have various modifications and alterations. Any modifications, improvements, and equivalent substitutions made within the concept and principles of this invention should be included within the scope of protection of this invention.

Claims

1. An injectable thermosensitive hydrogel for oral mucosal protection, characterized in that, It includes the following components: poloxamer F127, gelatin, APBA, AA2G, QCS, and deionized water.

2. The injectable thermosensitive hydrogel for oral mucosa protection according to claim 1, characterized in that, The mass-to-volume ratio of poloxamer F127 to deionized water is 15-25%. The mass-to-volume ratio of the gelatin to deionized water is 3-8%. The mass-to-volume ratio of APBA to deionized water is 0.5-2%. The mass-to-volume ratio of AA2G to deionized water is 1-5%. The mass-to-volume ratio of QCS to deionized water is 0.5-3%.

3. The injectable thermosensitive hydrogel for oral mucosa protection according to claim 1, characterized in that, The injectable thermosensitive hydrogel has a viscosity of ≤50 mPa·s at room temperature and a gelation time of ≤10 s at 37°C.

4. A method for preparing an injectable thermosensitive hydrogel for oral mucosa protection according to any one of claims 1 to 3, characterized in that, Includes the following steps: a) Pre-melting of temperature-sensitive skeleton substrate Add poloxamer F127 and gelatin to deionized water at 4°C, maintain the temperature at 4°C and stir for 2 hours until completely dissolved to form a transparent sol. b, Dynamic covalent crosslinking At 0~4℃, APBA solution was slowly added dropwise to the transparent sol obtained in step a and stirred to form a reversible Schiff base network. c, Antioxidant integration AA2G was added to the dynamic crosslinking system constructed in step b, stirred and mixed under light-protected conditions, and ultrasonically dispersed to completely dissolve and disperse AA2G in the dynamic crosslinking system, thus obtaining a dual dynamic covalent network based on Schiff base and borate ester. d. Integration of antibacterial and hemostatic functions Add QCS to the product obtained in step c and stir and mix well under light-protected conditions; e, Gelization regulation The product obtained in step d was heated to 37°C, and the pH was adjusted to 7.0~7.8 using 0.1 M NaOH or HCl. After sterile filtration, it was dispensed to obtain a temperature-sensitive hydrogel.

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