A probiotic-containing hydrogel and a preparation method and application thereof

Abstract

The present application relates to the technical field of biomedical materials and drug delivery system, and particularly relates to a water gel containing probiotics and a preparation method and application thereof in the treatment of oral ulcer. The water gel is prepared by taking sodium alginate as a base material, taking chitosan as an auxiliary structural material, constructing a three-dimensional porous network structure through ionic crosslinking, and embedding probiotics in the three-dimensional porous network structure, so as to obtain the water gel containing probiotics. The water gel has a stable three-dimensional porous structure, good probiotic release performance, and obvious inhibitory effect on Staphylococcus aureus and Candida albicans. The water gel can be used as an oral local drug delivery system, and has a good application prospect in the treatment of recurrent aphthous ulcer and other oral microecological related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and drug delivery systems, specifically to a probiotic-containing hydrogel, its preparation method, and its application in the treatment of oral ulcers. Background Technology

[0002] Recurrent aphthous ulcers are a common chronic inflammatory disease of the oral mucosa, with an incidence rate of approximately 20%–25% in the general population. This disease is characterized by recurrent attacks, significant pain, and a long course, severely impacting patients' quality of life. Currently, commonly used clinical treatments include topical corticosteroids, antibiotics, and analgesics. However, existing treatment methods have the following problems: 1. The drug has a short residence time in the oral cavity, making it difficult to maintain an effective concentration; 2. Long-term use of hormones may lead to mucosal atrophy or secondary infection; 3. Antibiotic abuse may disrupt the oral microecological balance.

[0003] Recent studies have shown that oral microecological imbalance is one of the important pathogenic factors of recurrent aphthous ulcers. Therefore, regulating the oral microecological environment through probiotic supplementation has become a new treatment strategy. However, free probiotics are easily affected by factors such as saliva rinsing, enzymatic degradation, and oxidative stress in the oral environment, resulting in a low survival rate and difficulty in long-term colonization at the lesion site. Therefore, it is necessary to develop a delivery system that can protect probiotics and achieve sustained release. Summary of the Invention

[0004] The purpose of this invention is to provide a probiotic-containing hydrogel, its preparation method, and its application in the treatment of oral ulcers.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a probiotic-containing hydrogel involves using sodium alginate as the matrix material and chitosan as the auxiliary structural material. A three-dimensional porous network structure is constructed by ion crosslinking, and probiotics are encapsulated therein to obtain the probiotic-containing hydrogel.

[0006] To elaborate further, 1) Prepare a sodium alginate solution; 2) Prepare a chitosan solution, and then vacuum dry it to obtain chitosan lyophilized powder; 3) Add the probiotic suspension to the sodium alginate solution prepared above and mix well. After mixing, add chitosan lyophilized powder and then perform ionic cross-linking under the action of a cross-linking agent to form a hydrogel.

[0007] The sodium alginate solution is prepared by dissolving sodium alginate in sterile water to obtain a 2wt% sodium alginate solution; the chitosan lyophilized powder is prepared by dissolving chitosan in an aqueous acetic acid solution to obtain a chitosan solution with a concentration of 5-15 mg / mL, and then vacuum drying to obtain chitosan lyophilized powder.

[0008] The probiotic suspension was prepared by culturing probiotics in Lactobacillus delbrueckii medium (MRS) to most growth phases, then collecting the bacterial suspension by centrifugation, and finally resuspending it in sterile water to obtain a concentration of 10. 8 ~10 9 CFU / mL bacterial suspension.

[0009] The probiotic is *Lactobacillus reuteri*.

[0010] The crosslinking agent is a calcium chloride solution.

[0011] A probiotic-containing hydrogel is prepared by the method described above, and a hydrogel with a three-dimensional porous network structure is prepared by the method described above.

[0012] An application of the probiotic-containing hydrogel, specifically its use in the preparation of a drug for treating oral ulcers. Compared with the prior art, the present invention has the following advantages: This invention relates to a hydrogel using sodium alginate as the matrix material and chitosan as the auxiliary structural material. Through ionic cross-linking, the two materials can form a stable interpenetrating three-dimensional porous network structure through electrostatic interactions, encapsulating the probiotic *Lactobacillus reuteri* within it. The hydrogel exhibits good biocompatibility, bioadhesion, and mechanical properties, enabling effective protection and sustained release of the probiotics. The structure and physicochemical properties of the hydrogel were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and rheological testing. Its functional characteristics were evaluated through in vitro release and antibacterial experiments. The results show that the hydrogel possesses a stable three-dimensional porous structure, excellent probiotic release performance, and significant inhibitory effects on oral pathogens, particularly *Staphylococcus aureus* and *Candida albicans*, and can regulate the oral microecological environment. Therefore, this hydrogel can be used as a local oral drug delivery system, showing promising application prospects in the treatment of recurrent aphthous ulcers and other oral microecological-related diseases. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the adhesion properties of the probiotic-loaded sodium alginate / chitosan hydrogel provided in an embodiment of the present invention; wherein, A is... L. reuteri -Inverted image of SA / CS1-5 hydrogel, B is... L. reuteri -SA / CS1-5 hydrogel mold diagram, C is... L. reuteri -SA / CS1-5 hydrogel lyophilized powder image, D is... L. reuteri -SA / CS1-5 hydrogel adhesion diagram; a: L. reuteri -SA / CS1; b: L. reuteri -SA / CS2; c: L. reuteri -SA / CS3; d: L. reuteri -SA / CS4; e: L. reuteri -SA / CS5.

[0014] Figure 2 Scanning electron microscope (SEM) images and Fourier transform infrared (FTIR) spectra of the hydrogel provided in this embodiment of the invention; wherein, A is... L. reuteri SEM image of SA / CS1 hydrogel, B is... L. reuteri SEM image of SA / CS2 hydrogel, C represents... L. reuteri -SEM image of SA / CS3 hydrogel, D is L. reuteri -SEM image of SA / CS4 hydrogel, E is L. reuteri -SEM image of SA / CS5 hydrogel, F is L. reuteri FTIR image of SA / CS1-5 hydrogel.

[0015] Figure 3 The hydrogel rheological property test results provided in this embodiment of the invention; wherein, A is L. reuteri - Plot of storage modulus (G') and loss modulus (G'') of SA / CS1-5 hydrogel as a function of angular frequency; B is L. reuteri -Graph showing the variation of storage modulus (G') and loss modulus (G'') of SA / CS1-5 hydrogel with strain.

[0016] Figure 4 The hydrogel swelling performance test results provided in this embodiment of the invention; wherein, A is... L. reuteri -Graph showing the swelling changes of SA / CS1-5 hydrogel in PBS buffer, B represents L. reuteri -Graph showing the swelling changes of SA / CS1-5 hydrogel in artificial saliva.

[0017] Figure 5 The hydrogel degradation performance test results provided in this embodiment of the invention; wherein, A is... L. reuteri -Graph showing the degradation of SA / CS1-5 hydrogel in PBS buffer. B represents... L. reuteri -Graph showing the degradation of SA / CS1-5 hydrogel in artificial saliva.

[0018] Figure 6 The results of the probiotic release performance test in the hydrogel provided in this embodiment of the invention; wherein, A is L. reuteri -SA / CS1-5 Hydrogel in vitro release colony count, B is L. reuteri -SA / CS1-5 hydrogel releases colony count after 30 days of storage.

[0019] Figure 7 The antibacterial performance test results in the hydrogel provided in this embodiment of the invention; wherein, A is... L. reuteri -Inhibition rate of SA / CS1-5 hydrogel against Staphylococcus aureus. B represents... L. reuteri -Inhibition rate of SA / CS1-5 hydrogel against Candida albicans. C represents... L.reuteri - Colony count after co-culturing SA / CS1-5 hydrogel with Staphylococcus aureus / Candida albicans. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0021] This invention relates to a sodium alginate / chitosan hydrogel loaded with probiotics, prepared using an ionic crosslinking method. First, chitosan is dissolved in acetic acid solution to prepare a chitosan solution. Then, sodium alginate is dissolved in sterile water to form a sodium alginate solution. A probiotic suspension is added to the sodium alginate solution and mixed thoroughly. Next, chitosan is added to form a mixed solution, and an ionic crosslinking reaction is carried out using calcium chloride solution to form the hydrogel. The resulting hydrogel forms a three-dimensional porous network structure, enabling effective encapsulation and sustained release of probiotics. This hydrogel exhibits good swelling and degradation properties in an artificial saliva environment and can form a stable bioadhesive structure on the oral mucosa surface.

[0022] All of the following examples were obtained commercially; chitosan was purchased from Shanghai Maclean Biotechnology Co., Ltd., with a molecular weight of medium viscosity, 200-400 mPas; sodium alginate was purchased from Shanghai Maclean Biotechnology Co., Ltd., with a molecular weight of viscosity, 200±20 mPas.

[0023] Lactobacillus reuteri is a type of Lactobacillus reuteri ( Lactobacillus reuteri PB-LR09, L. reuteri Purchased from Shaanxi Junhe Biotechnology Co., Ltd. Staphylococcus aureus ( Canidia Albicans CMCC(B)26003) and Candida albicans ( Canidia Albicans CMCC(F)98001 was purchased from Shanghai Luwei Technology Co., Ltd. Lactobacillus delbrueckii medium (MRS), tryptic soy peptone medium (TSB), Sabouraud dextrose agar (SDA), and agar were all purchased from Beijing Luqiao Technology Co., Ltd.

[0024] Example 1: Preparation of hydrogel 1) Dissolve chitosan (1% by mass) in a 1 wt% aqueous acetic acid solution to prepare a 1% chitosan solution. Then, stir magnetically until the powder is completely dissolved. After autoclaving, freeze dry in a vacuum freeze dryer to obtain chitosan freeze-dried powder.

[0025] 2) Dissolve sodium alginate in sterile water to prepare a 2wt% sodium alginate solution.

[0026] 3) After culturing *Lactobacillus reuteri* to the logarithmic growth phase, the bacterial cells were collected by centrifugation, washed three times with sterile water, and then diluted with sterile water to a concentration of 10. 8 ~10 9 CFU / mL bacterial suspension (the concentration prepared in this example is 10) 8 (CFU / mL).

[0027] The process of culturing *Lactobacillus reuteri* to the logarithmic growth phase involves inoculating the strain at a 2% inoculum into *Lactobacillus delbrueckii* medium (MRS) and culturing it at 37 ℃ with shaking at 150 r / min until the logarithmic growth phase is reached, ready for use.

[0028] 4) Take 2 mL of bacterial suspension and add it to 2 mL of sodium alginate solution and mix well. Then add chitosan lyophilized powder to make the final chitosan concentrations in the system as follows: 5 mg / mL (a); 7.5 mg / mL (b); 10 mg / mL (c); 12.5 mg / mL (d); 15 mg / mL (e).

[0029] 5) After mixing thoroughly, the mixed solution is added dropwise to a 2% calcium chloride solution for ionic cross-linking, forming five hydrogels of different concentrations. L. reuteri -SA / CS1— L. reuteri -SA / CS5).

[0030] The surface morphology and surface functional groups of the hydrogels obtained at different concentrations were determined by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). See [link to relevant documentation]. Figure 1 ,Depend on Figure 1 The five types of samples that have been successfully prepared can be observed using the inversion method. L. reuteri -SA / CS1-5 hydrogel. When the test tube is tilted, observe that no liquid drips down the wall; this indicates gel formation, as shown in Figure 1A. Separately... L. reuteri -SA / CS1-5 hydrogels can be placed in different molds to form hydrogels of different shapes, and then freeze-dried into lyophilized powders, as shown in Figures 1B and 1C. Figure 1D shows... L. reuteri -SA / CS1-5 hydrogel adheres to the pigskin and can resist water erosion.

[0031] Structural characterization of the hydrogels (SA / CS1—SA / CS5) obtained above at different concentrations: The microstructure of the hydrogel was observed using scanning electron microscopy, and the results showed that the hydrogel has a typical three-dimensional porous network structure (see [link]). Figure 2 When the CS concentration is 12.5 mg / mL, the pores inside the hydrogel become smaller and more irregular, and this phenomenon becomes more pronounced when the CS concentration increases to 15 mg / mL.

[0032] Depend on Figure 2 As can be seen from the Fourier transform infrared spectroscopy analysis, at 3421 cm⁻¹... -1 Peaks for hydroxyl and amino vibrations; 1420 cm⁻¹ -1 This is the peak for carboxyl vibration; 1024 cm⁻¹ -1 The peak at 1635 cm⁻¹ indicates a stable hydrogen bond structure between sodium alginate and chitosan, representing a vibrational peak in the polysaccharide skeleton. -1 and 1543 cm -1 The absorption intensity is enhanced at 3421 cm⁻¹, and at 3421 cm⁻¹. -1 The O–H / –NH2 stretching band at that location shifted slightly towards a lower wavenumber, confirming that... L. reuteri It was successfully incorporated into the SA / CS composite hydrogel.

[0033] Meanwhile, the properties of the hydrogel obtained above were further measured, specifically as follows: The rheological properties were characterized using a rotational rheometer (MCR 302e, Antonpah, Austria) at 37 °C. First, strain scans (0.01–1000%) were performed at a fixed angular frequency of 5 rad / s to determine the linear viscoelastic region (LVR). Subsequently, frequency scans were performed within the LVR at a constant strain of 1%, covering an angular frequency range of 0.01–100 rad / s. The changes in storage modulus (G′) and loss modulus (G″) with angular frequency were recorded. Figure 3 Conclusion: In the angular frequency range of 0.1 to 100 rad / s, the storage modulus G' was consistently greater than the loss modulus G'', and this state remained remarkably stable. This phenomenon reveals the excellent rigidity and toughness of the hydrogel, exhibiting remarkably stable viscoelastic solid properties. G' remained essentially stable up to 300% strain, indicating that the hydrogel can maintain its three-dimensional network structure. When the strain reached 300%, G' and G'' intersected, and subsequently G' rapidly decreased, indicating the collapse of the hydrogel network. This hydrogel possesses excellent resistance to deformation, and 300% strain is the critical point for structural instability.

[0034] Will L. reuteri -SA / CS1-5 hydrogel (8 mm in diameter, 2 mm in height) was freeze-dried. Samples were placed at 37°C and agitated (90 r / min) in PBS buffer (pH 7.2) and artificial saliva (pH 6.8) for 24 hours. Samples were collected at predetermined time intervals (0, 2, 4, 6, 8, 10, 12, and 24 h). Figure 4 ).in conclusion: L. reuteri The SA / CS1-5 series hydrogels maintained structural integrity and exhibited stable swelling behavior in both PBS buffer and artificial saliva, reaching swelling equilibrium in approximately 6 hours. The swelling ratio of the hydrogels in PBS buffer was significantly higher than that in artificial saliva, indicating that an acidic environment inhibits their swelling ability. The swelling ratio of the hydrogels decreased with increasing chitosan concentration.

[0035] Will L.reuteri -SA / CS1-5 hydrogels (15 mm in diameter, 2 mm in height) were freeze-dried to constant weight. The samples were then immersed in 20 mL of PBS buffer (pH 7.2) and artificial saliva (pH 6.8) and incubated with shaking at 37 °C and 120 r / min. At predetermined time points (0, 1, 3, 5, 10, 15, 20, and 30 days), the samples were removed, gently rinsed with deionized water to remove residual salts, and freeze-dried to constant weight. Figure 5 Conclusion: In the initial stage (within 1 day), swelling was the dominant phenomenon, followed by degradation, which gradually became the dominant process, especially during the period from 5 to 15 days. The degradation rate was rapid, manifested as the collapse and decomposition of the three-dimensional network structure, leading to gel weight loss. After 30 days, all hydrogels retained 46.6%–78.6% of their initial weight in both media, indicating that they have excellent water retention capacity and structural stability at normal temperatures, effectively delaying water loss and exhibiting good long-term structural stability.

[0036] Example 2 Probiotic Release Experiment The hydrogel prepared in the above example was placed in MRS medium and cultured with shaking at 37 °C.

[0037] Probiotic release levels were measured at 4 h, 8 h, 12 h, and 24 h (see [reference]). Figure 6 ).

[0038] Depend on Figure 6 The results showed that the hydrogel could continuously release probiotics, with the hydrogel having the best release effect at a chitosan concentration of 10 mg / mL; specifically, L. reuteriThe SA / CS3 hydrogel significantly outperformed other formulations in both probiotic release performance and long-term survival protection. In the initial release phase (4 h and 8 h), there was no significant difference in release levels among all groups. p >0.05); after 12 h, the release levels in the CS3 and CS4 groups were significantly higher than those in the CS1 group ( p <0.05); by 24 h, the CS3 group showed the best release performance ( p <0.05, achieving an optimal balance between mesh permeability and structural stability. After storage at 4 ℃ for 30 days, the CS3 group showed the highest probiotic survival rate (6.9×10). 6 CFU·mL -1 , p <0.05), thanks to the protective microenvironment provided by its suitable SA / CS network, which can reduce external pressure, maintain hydration, facilitate material exchange, and prevent excessive cell diffusion.

[0039] Example 3 Antibacterial performance test The hydrogels prepared in the above examples were co-cultured with Staphylococcus aureus and Candida albicans, respectively. Specifically, Staphylococcus aureus ( S. aureus ) and Candida albicans ( C. albicans The cells were cultured in TSB and SDA media (37°C, 150 r / min) respectively until they entered the exponential growth phase. They were then collected by centrifugation and cultured in fresh TSB medium (for...). S. aureus ) or SDA medium (for C. albicans Resuspend. Then, add 5 mL S. aureus (10) 8 - 10 9 CFU mL - 1 )or C. albicans suspension (10 5 - 10 6 CFU mL - 1 )and L. reuteri SA / CS1-5 hydrogels (10 mm in diameter, 2 mm in height) were mixed separately. The mixtures were incubated at 37°C with shaking at 150 r / min for 24 h. After co-incubation, the mixtures were plated onto the corresponding agar plates for analysis. S. aureus or C. albicans The number of bacterial colonies.

[0040] Depend on Figure 7 The results showed that the hydrogel inhibited Staphylococcus aureus by approximately 45% and Candida albicans by approximately 70%, as detailed in the attached images. L. reuteriBoth SA / CS1–5 hydrogels exhibited broad-spectrum antibacterial activity against oral pathogens (Staphylococcus aureus and Candida albicans), and the antibacterial effect was chitosan concentration-dependent. High concentration group ( L. reuteri -SA / CS3–5 showed significantly better antibacterial effects than the low concentration group ( L. reuteri -SA / CS1–2), which has an inhibition rate of 35.6%–49.1% against Staphylococcus aureus and 65.4%–72.7% against Candida albicans. It can be seen that the hydrogel obtained by the present invention can significantly inhibit the proliferation of two representative oral pathogens (Staphylococcus aureus and Candida albicans), thereby reshaping the healthy microenvironment.

[0041] The above results indicate that, L. reuteri -SA / CS3 hydrogel is an optimal formulation with potential application value in the treatment of recurrent aphthous ulcers (RAU). This hydrogel is not a potent bactericide, but rather an excellent microecological regulator. Its effects are sufficient to inhibit the excessive proliferation of opportunistic pathogens, creating conditions conducive to ulcer healing.

Claims

1. A method for preparing a probiotic-containing hydrogel, characterized in that: Using sodium alginate as the matrix material and chitosan as the auxiliary structural material, a three-dimensional porous network structure is constructed by ion crosslinking, and probiotics are encapsulated in it to obtain a probiotic-containing hydrogel.

2. The method for preparing probiotic-containing hydrogel according to claim 1, characterized in that: 1) Prepare a sodium alginate solution; 2) Prepare a chitosan solution, and then vacuum dry it to obtain chitosan lyophilized powder; 3) Add the probiotic suspension to the sodium alginate solution prepared above and mix well. After mixing, add chitosan lyophilized powder and then perform ionic cross-linking under the action of a cross-linking agent to form a hydrogel.

3. The method for preparing probiotic-containing hydrogel according to claim 2, characterized in that: The sodium alginate solution is prepared by dissolving sodium alginate in sterile water to obtain a 2wt% sodium alginate solution; the chitosan lyophilized powder is prepared by dissolving chitosan in an aqueous acetic acid solution to obtain a chitosan solution with a concentration of 5-15 mg / mL, and then vacuum drying to obtain chitosan lyophilized powder.

4. The method for preparing probiotic-containing hydrogel according to claim 2, characterized in that: The probiotic suspension was prepared by culturing probiotics in Lactobacillus delbrueckii medium (MRS) to most growth phases, then collecting the bacterial suspension by centrifugation, and finally resuspending it in sterile water to obtain a concentration of 10. 8 ~10 9 CFU / mL bacterial suspension.

5. The method for preparing probiotic-containing hydrogel according to claim 4, characterized in that: The probiotic is *Lactobacillus reuteri*.

6. A method for preparing a probiotic-containing hydrogel according to claim 1, characterized in that: A hydrogel with a three-dimensional porous network was prepared according to the method described in claim 1.

7. An application of the probiotic-containing hydrogel according to claim 6, characterized in that: The application of the probiotic-containing hydrogel in the preparation of drugs for treating oral ulcers.

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