Structure-adjustable polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier and preparation method thereof

Abstract

The invention discloses a polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier with an adjustable structure and a preparation method of the polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier. The carrier is constructed by regulating calcium phosphate mineralization through polyphenol to form a flower-like structure and then adsorbing hyaluronic acid. The preparation method comprises the following steps: (1) preparing nanoflowers from polyphenol chelated calcium phosphate; (2) in-situ packaging of probiotics by the nanoflowers; and (3) coating the probiotic carrier with hyaluronic acid. The preparation method is simple in process and mild in condition, the prepared probiotic packaging body is excellent in biocompatibility, not only can improve gastrointestinal fluid environment tolerance, but also has the capabilities of active oxygen removal, inflammation targeting and intestinal colonization, and by synergistically reducing colitis, relieving tissue damage, repairing intestinal mucosal barrier and restoring intestinal flora balance, the intestinal tract intestinal colonization effect is improved. A new strategy is provided for the treatment of the inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of probiotic encapsulation, specifically relating to a structurally tunable polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier and its preparation method. Background Technology

[0002] Inflammatory bowel disease (IBD), a global epidemic, affects 6 to 8 million patients. Its chronic inflammatory environment disrupts intestinal homeostasis, leading to irreversible mucosal damage and increasing the risk of cancer by 2 to 6 times (Siew C Ng, et al. The Lancet Gastroenterology & Hepatology, 2017, 390, 2769). Although probiotic therapy has shown potential in restoring intestinal flora balance, oral delivery still faces three key obstacles: (1) insufficient bacterial survival due to gastric juice, digestive enzymes, and bile acids; (2) continuous intestinal peristalsis leading to decreased colonization and reduced probiotic utilization; and (3) high oxidative stress and low pH environment at the site of inflammation further inhibiting flora activity (AKhademhosseini, et al. Advanced Drug Delivery Reviews, 2020, 157, 37).

[0003] To address the aforementioned issues, various probiotic encapsulation strategies have been proposed in recent years. For example, Chinese patent CN117297099B utilizes metal ion-polyphenol coordination to form a protective layer on the surface of probiotics, while simultaneously introducing a sodium alginate coating, aiming to enhance the bacteria's tolerance to the gastrointestinal environment. However, as described in the literature (L. Gao, et al. Biomaterials, 2025, 321, 123323), loose encapsulation networks struggle to provide adequate protection in the complex intestinal environment. Another patent (CN113230280B) prepares multilayer encapsulated microcapsules of probiotics by electrostatically spraying sodium alginate, pectin, and soy protein isolate. Similarly, US patent US2024 / 0108047A1 mixes and dissolves probiotic powder with microcrystalline cellulose / starch, extrudes it into a core layer, and then coats it with atomized coating to obtain core-shell microcapsules. The aforementioned complex multi-layered encapsulation process may not only impair the activity of probiotics, but also make it difficult for the encapsulated material to open effectively at the site of action, or prevent the bacteria from being activated in time and thus expelled from the body. Furthermore, the complex pathological environment of IBD also requires encapsulation materials to be multifunctional to improve treatment efficiency.

[0004] Therefore, developing novel probiotic carriers that combine simplified and mild processes, tolerance to the gastrointestinal fluid environment, and the ability to scavenge reactive oxygen species, target inflammation, and colonize the intestines has become a core approach to solving the bottleneck in IBD treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a structurally tunable polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier and its preparation method, overcoming the shortcomings of existing technologies. In this study, we optimized the process flow, using polyphenols to regulate the mineralization of calcium phosphate to form a flower-like structure, followed by the adsorption of hyaluronic acid to form a high-efficiency probiotic carrier. Without affecting bacterial activity and improving bacterial encapsulation efficiency, the prepared probiotic encapsulated body exhibits good biocompatibility, high tolerability, and intestinal colonization ability. Simultaneously, it demonstrates synergistic therapeutic effects through inflammation targeting, scavenging reactive oxygen species, reducing the expression level of inflammatory factors, repairing the intestinal barrier, and restoring intestinal flora balance.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a method for preparing a structurally tunable polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier, characterized in that a flower-like mineralized structure formed by polyphenol chelation with calcium phosphate serves as the core for encapsulating probiotics, and hyaluronic acid is adsorbed on the outer surface of the high specific surface area mineralized structure to form a composite carrier. The probiotics are one or more selected from *Escherichia coli* Nissle 1917, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Lactobacillus paracasei*, *Lactobacillus acidophilus*, *Bifidobacterium*, and *Lactobacillus reuteri*. The polyphenols are one or more selected from tannic acid, gallic acid, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate.

[0008] Includes the following steps:

[0009] (1) Probiotic pretreatment: centrifuge, wash and resuspend in PBS buffer at pH 7.4;

[0010] (2) In-situ encapsulation: Polyphenol and CaCl2 solution are added to the bacterial suspension in sequence, and the mixture is reacted at 15-25℃ for 0.5-3h, and then centrifuged to remove free components;

[0011] (3) Hyaluronic acid coating: The product is resuspended in hyaluronic acid solution, incubated at 2-6℃ for 0.5-2h, and then centrifuged and washed;

[0012] (4) Freeze-drying: The product is freeze-dried at low temperature to obtain the multifunctional probiotic carrier.

[0013] In step (1), the concentration of the bacterial suspension is 1×10⁻⁶. 9 CFU / mL;

[0014] In step (2), the concentration of the polyphenol solution is 0.5–5 mg / mL, the concentration of the CaCl2 solution is 10–150 mM, and the solvent is ultrapure water.

[0015] In step (3), the hyaluronic acid has a molecular weight of 20-40 kDa and a solution concentration of 1-5 mg / mL.

[0016] In steps (1)-(3), the centrifugation temperature is 15-25℃, the rotation speed is 8000-12000rpm, and the time is 3-8min.

[0017] The beneficial effects of this invention include:

[0018] (1) The present invention is based on the polyphenol-mediated controlled mineralization of calcium phosphate to construct a flower-like structure carrier, and then achieves efficient encapsulation of probiotics under mild process conditions, and finally combines with hyaluronic acid to form a multifunctional composite system; the system maintains high probiotic activity while ensuring excellent biocompatibility.

[0019] (2) The polyphenol-calcium phosphate-hyaluronic acid probiotic carrier obtained maintains high structural stability in the complex gastrointestinal fluid environment, effectively protecting the survival of probiotics; its coating material can be degraded in the intestine, realizing precise release and timely activation of probiotics.

[0020] (3) The polyphenol component in the polyphenol-calcium phosphate-hyaluronic acid probiotic carrier endows it with reactive oxygen species scavenging ability, significantly alleviating oxidative stress; the hyaluronic acid component simultaneously provides inflammation targeting function and mucosal colonization ability, synergistically enhancing the bioavailability of probiotics. Validated using a dextran sulfate sodium (DSS)-induced colitis model, this carrier achieves synergistic treatment of IBD by reducing the expression level of inflammatory factors, alleviating tissue damage, repairing the intestinal mucosal barrier, and restoring the balance of intestinal flora. Attached Figure Description

[0021] Figure 1 This describes the microscopic morphology of encapsulated probiotics. Among them, Figure 1 (A) contains unencapsulated probiotics; Figure 1 (B) Probiotics are encapsulated in unstructured materials; Figure 1 (C) Probiotics are encapsulated in a flower-like structural material.

[0022] Figure 2 This is to encapsulate the activity of probiotics in complex environments. Among them, Figure 2 (A) represents the survival rate of probiotics after encapsulation. Figure 2 (B) represents the survival rate of encapsulated probiotics after 3 hours of treatment with simulated gastric juice; Figure 2 (C) represents the survival rate of encapsulated probiotics after 4 hours of treatment with simulated intestinal fluid; Figure 2 (D) represents the survival rate of probiotics after the entire process; Figure 2 (E) shows the bacterial morphology of encapsulated probiotics after treatment with simulated gastrointestinal fluid.

[0023] Figure 3To encapsulate the antioxidant effects of probiotics. Among them, Figure 3 (A) An image of intracellular reactive oxygen species (ROS) fluorescence in human clonal colon adenocarcinoma (Caco-2) cells treated with hydrogen peroxide (H2O2); Figure 3 (B) shows the semi-quantitative analysis results of the relative fluorescence intensity of ROS.

[0024] Figure 4 This is to encapsulate the ability of probiotics to adhere in the gut. Among them, Figure 4 (A) is a fluorescence in vivo imaging image of a mouse; Figure 4 (B) shows the semi-quantitative analysis results of relative fluorescence intensity.

[0025] Figure 5 The images show (A) of the mouse cecum and colon and (B) the quantitative statistical results of its length.

[0026] Figure 6 The expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β and IL-10 in mouse intestinal lysate.

[0027] Figure 7 This shows the presence of blood in the stool of mice. Detailed Implementation

[0028] All features disclosed in this specification, or steps in all methods or processes disclosed herein, may be combined in any way, except for mutually exclusive features and / or steps.

[0029] Any feature disclosed in this specification, unless otherwise stated, may be replaced by other equivalent or similar features. That is, unless otherwise stated, each feature is merely one example of a series of equivalent or similar features.

[0030] The preparation method of a structurally tunable polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier of the present invention will be described in detail below.

[0031] The product comprises a flower-like mineralized structure formed by polyphenol chelation of calcium phosphate as the core for encapsulating probiotics, and a composite carrier composed of hyaluronic acid (HA) adsorbed on the outer surface of the high specific surface area mineralized structure. The preferred probiotic is *Lactobacillus reuteri* (LR), and the preferred polyphenol is epigallocatechin gallate (EGCG).

[0032] In this invention, the selected EGCG solution concentration is 0-5 mg / mL, the selected CaCl2 solution concentration is 10-150 mM, the selected HA molecular weight is 20-40 kDa, and the solution concentration is 1-5 mg / mL.

[0033] In this invention, the solvents in both the CaCl2 and EGCG solutions are ultrapure water. After reacting at 15–25°C for 0.5–3 hours, the free components are removed by centrifugation. The centrifugation temperature is 15–25°C, the rotation speed is 8000–12000 rpm, and the time is 3–8 minutes.

[0034] The preparation method of the polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier of the present invention will be further described below with reference to specific embodiments and comparative examples.

[0035] Examples 1-7:

[0036] (1) Probiotic pretreatment: LR was cultured overnight at 37°C in MRS liquid medium. Then, it was centrifuged and washed three times with 1×PBS, followed by 1×10⁻⁶ probiotics. 9 The LR of CFU was resuspended in 1 mL of PBS solution with pH 7.4;

[0037] (2) In-situ encapsulation: Add 8 mL of PBS to the bacterial suspension, then add 500 μL of EGCG and 500 μL of CaCl2 solution (the concentrations of EGCG and CaCl2 solution are shown in Table 1). After reacting at 25 °C for 2 h, centrifuge and wash to remove excess material components.

[0038] (3) HA coating: The product was resuspended in 10 mL of HA solution, incubated at 4 °C for 1 h, and then centrifuged and washed to remove excess material components;

[0039] (4) Freeze-drying: The product is freeze-dried at low temperature to obtain the multifunctional probiotic carrier.

[0040] Comparative Example 1:

[0041] (1) Probiotic treatment: LR was cultured overnight at 37°C in MRS liquid medium. Then, it was centrifuged and washed three times with 1×PBS, followed by 1×10⁻⁶ probiotics. 9 The LR of CFU was resuspended in 1 mL of PBS solution with pH 7.4;

[0042] (2) Freeze-drying: The product is freeze-dried at low temperature to obtain probiotic freeze-dried powder.

[0043] Comparative Examples 2-3:

[0044] (1) Probiotic pretreatment: LR was cultured overnight at 37°C in MRS liquid medium. Then, it was centrifuged and washed three times with 1×PBS, followed by 1×10⁻⁶ probiotics. 9 The LR of CFU was resuspended in 1 mL of PBS solution with pH 7.4;

[0045] (2) In-situ encapsulation: Add 8 mL of PBS to the bacterial suspension, then add 500 μL of EGCG and 500 μL of CaCl2 solution (the concentrations of EGCG and CaCl2 solution are shown in Table 1). After reacting at 25 °C for 2 h, centrifuge and wash to remove excess material components.

[0046] (3) HA coating: The product was resuspended in 10 mL of HA solution, incubated at 4 °C for 1 h, and then centrifuged and washed to remove excess material components;

[0047] (4) Freeze-drying: The product is freeze-dried at low temperature to obtain a probiotic carrier.

[0048] Table 1. Formulations of Examples 1-7 and Comparative Examples 1-3, and viable bacterial counts after treatment with simulated gastrointestinal fluid.

[0049] sample EGCG (mg / mL) <![CDATA[CaCl2(mM)]]> viable bacterial count (CFUs) after treatment Example 1 1 10 <![CDATA[1.88×10 8 ]]> Example 2 1 30 <![CDATA[6.49×10 8 ]]> Example 3 1 70 <![CDATA[8.85×10 8 ]]> Example 4 1 100 <![CDATA[1.17×10 9 ]]> Example 5 0.5 100 <![CDATA[7.79×10 8 ]]> Example 6 2 100 <![CDATA[9.34×10 8 ]]> Example 7 5 100 <![CDATA[5.32×10 8 ]]> Comparative Example 1 0 0 <![CDATA[9.62×10 7 ]]> Comparative Example 2 5 10 <![CDATA[3.59×10 8 ]]> Comparative Example 3 0 100 <![CDATA[6.57×10 8 ]]>

[0050] As shown in Table 1, there are significant differences in the gastrointestinal tolerance of probiotics under different encapsulation parameters. When the EGCG concentration is fixed at 1 mg / mL, increasing the CaCl2 concentration enhances the stability of the encapsulation structure by promoting the formation of the phosphate mineralization layer, effectively improving the survival rate of probiotics in complex environments (as in Examples 1-4). When the CaCl2 concentration is constant at 100 mM, EGCG exhibits concentration-dependent regulation: medium concentrations of EGCG (0.5–2 mg / mL) guide the orderly assembly of calcium phosphate crystals through chelation, forming highly three-dimensional and appropriately sized nanoflower structures, significantly improving protective performance (Examples 4-6); however, high concentrations of EGCG (5 mg / mL) disrupt the crystal growth order due to coordination supersaturation, leading to structural defects and inhibition of bacterial activity (Example 7). In particular, the optimal synergistic combination (1 mg / mL EGCG + 100 mM CaCl2) formed a complete nanoflower structure, and its protection efficiency significantly surpassed that of the unencapsulated group (Comparative Example 1), the group without flower structure (Comparative Example 2), and the monocalcium phosphate system lacking EGCG (Comparative Example 3), fully verifying the synergistic mechanism of polyphenol-regulated crystal growth and calcium ion-enhanced mineralization.

[0051] The microstructure of representative encapsulated probiotics was observed using scanning electron microscopy. The results showed that the unencapsulated group (LR, Comparative Example 1) had a smooth bacterial surface. Figure 1 A); The surface of the non-florescent structure group (LR@Ca-E / HA, Comparative Example 2) shows a rough coating composed of a polyphenol-Ca network. Figure 1 B); while the structure-controlled nanoflower group (LR@CENFs / HA, Example 4) exhibits an overall encapsulation structure formed by the aggregation of mineralized nanoflowers, with the gaps between the petals filled by HA, forming a complete and dense encapsulation system. Figure 1 C).

[0052] Probiotic encapsulation survival rate testing showed that the survival rate of the LR@CENFs / HA group was approximately 95%, significantly higher than that of LR@Ca-E / HA (37.9%), confirming the high efficiency of the mineralization encapsulation process in ensuring bacterial activity. Figure 2 A). After treatment with simulated gastrointestinal fluid (SGF / SIF) ( Figure 2 In groups B and C, the survival rates of the encapsulated groups (LR@Ca-E / HA and LR@CENFs / HA) were higher than those of the unencapsulated LR group. The environmental tolerance of the LR@CENFs / HA group was 4.3 times and 16.2 times that of the LR@Ca-E / HA group, respectively. Figure 2 D). Further morphological changes were observed using scanning electron microscopy. It was found that the unencapsulated LR group showed massive cell disintegration and death after SGF / SIF treatment, the LR@Ca-E / HA group showed local structural damage, while the LR@CENFs / HA group maintained an intact cell morphology after the orderly degradation of the nanoflower structure, confirming that it has the dual functions of structural protection and responsive targeted release.

[0053] To verify the antioxidant capacity of the probiotic carrier, a Caco-2 cell oxidative stress model induced by H2O2 was used to semi-quantitatively analyze the ROS scavenging ability of the encapsulation system. For example... Figure 3 As shown in Figure A, compared to the LR group, both the LR@Ca-E / HA and LR@CENFs / HA groups significantly reduced intracellular ROS levels (increased green fluorescence), with the LR@CENFs / HA group showing the most significant reduction. Further semi-quantitative analysis confirmed ( Figure 3 B) The fluorescence intensity of the LR@CENFs / HA group was only 1 / 4 that of the LR group (p<0.001). These results indicate that the CENFs / HA carrier can efficiently scavenge ROS, endowing the system with excellent antioxidant function.

[0054] Fluorescent labeling combined with in vivo imaging was used to monitor the adhesion of probiotics in the intestine. Four hours after oral administration, the LR@CENFs / HA group showed significant fluorescence in the colonic region of mice. Figure 4 A) Its intensity was 2.9 times that of the LR group (p<0.01) and 1.8 times that of the LR@Ca-E / HA group (p<0.05), respectively. Figure 4 B) This indicates that the LR@CENFs / HA group achieves long-term retention and enhanced colonization of probiotics in the intestinal microenvironment through mineralization structure stability and HA-mediated inflammatory targeting effects.

[0055] In a DSS-induced mouse colitis model, the colon length in the LR@CENFs / HA treatment group recovered to near-healthy levels compared to the model group (DSS). Figure 5 A), which is about 1.5 times better than the DSS group ( Figure 5B); Enzyme-linked immunosorbent assay (ELISA) further confirmed that the LR@CENFs / HA group could effectively inhibit the expression of pro-inflammatory factors (IL-6, TNF-α, IL-1β) while increasing the level of IL-10 anti-inflammatory factor, and its overall efficacy was significantly better than that of the LR and LR@Ca-E / HA groups. Figure 6 Finally, by observing the fecal condition of the mice, LR@CENFs / HA essentially eliminated the symptoms of bloody stool and diarrhea by day 9, and its symptom relief speed and extent both surpassed those of other treatment groups. Figure 7 The study comprehensively verified that the encapsulation system alleviates IBD through a synergistic effect of "structural protection and functional regulation".

[0056] In summary, this invention proposes a structurally tunable polyphenol-calcium phosphate-hyaluronic acid (HHA) multifunctional probiotic delivery carrier and its preparation method. By precisely controlling the EGCG / CaCl2 ratio to construct a flower-like mineralized carrier core, and leveraging its high specific surface area and structural stability to efficiently load HA, a polyphenol-calcium phosphate-HA ternary composite carrier is formed. Its flower-like mineralized structure provides a physical barrier against gastrointestinal fluid erosion, the HA coating layer endows it with inflammation-targeting and mucosal colonization capabilities, and the polyphenol component scavenges ROS to alleviate oxidative stress, significantly improving the synergistic therapeutic effect of IBD.

[0057] Although the present invention has been described above in conjunction with exemplary embodiments, those skilled in the art should understand that various modifications and variations to the above embodiments without departing from the spirit and scope of the claims also fall within the protection scope of this patent.

Claims

1. A structurally tunable polyphenol-calcium phosphate-hyaluronic acid multifunctional probiotic delivery carrier, characterized in that, A flower-like mineralized structure formed by polyphenol chelation of calcium phosphate serves as the core for encapsulating probiotics, and hyaluronic acid is adsorbed on the outer surface of the high specific surface area mineralized structure to form a composite encapsulation.

2. The multifunctional probiotic delivery carrier as described in claim 1, characterized in that, The probiotics mentioned are one or more of Escherichia coli Nissle 1917, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus acidophilus, Bifidobacterium, and Lactobacillus reuteri.

3. The multifunctional probiotic delivery carrier as described in claim 1, characterized in that, The polyphenols mentioned are one or more of tannic acid, gallocatechin, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate.

4. The method for preparing the multifunctional probiotic carrier according to any one of claims 1-3, characterized in that... Includes the following steps: (1) Probiotic pretreatment: centrifuge, wash and resuspend in PBS buffer at pH 7.4; (2) In-situ encapsulation: Polyphenol and CaCl2 solution are added to the bacterial suspension in sequence, and the mixture is reacted at 15-25℃ for 0.5-3h, and then centrifuged to remove free components; (3) Hyaluronic acid coating: The product is resuspended in hyaluronic acid solution, incubated at 2-6℃ for 0.5-2h, and then centrifuged and washed; (4) Freeze-drying: The product is freeze-dried at low temperature to obtain the multifunctional probiotic carrier.

5. The preparation method according to claim 4, characterized in that, In step (1), the concentration of the bacterial suspension is 1×10⁻⁶. 9 CFU / mL.

6. The preparation method according to claim 4, characterized in that, In step (2), the concentration of the polyphenol solution is 0.5-5 mg / mL, the concentration of the CaCl2 solution is 10-150 mM, and the solvent is ultrapure water.

7. The preparation method according to claim 4, characterized in that, In step (3), the hyaluronic acid has a molecular weight of 20-40 kDa and a solution concentration of 1-5 mg / mL.

8. The preparation method according to claim 4, characterized in that, In steps (1)-(3), the centrifugation temperature is 15-25℃, the rotation speed is 8000-12000rpm, and the time is 3-8min.

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