Preparation method of microgel nanoparticles with colon-targeted retention and flora regulation-anti-inflammatory synergistic effect

Abstract

The invention discloses a preparation method of microgel nanoparticles with colon targeted retention and flora regulation-anti-inflammatory synergistic effect, and belongs to the technical field of functional food targeted delivery, and the preparation method comprises the following steps: (1) dissolving lactoferrin and curcumin in deionized water and absolute ethyl alcohol respectively, and stirring uniformly; obtaining a lactoferrin solution and a curcumin solution for later use; (2) mixing the lactoferrin solution and the curcumin solution, magnetically stirring, heating to obtain a mixed solution of a lactoferrin / curcumin compound, and centrifuging to remove redundant curcumin; (3) respectively dissolving the citrus pectin and CaCl2 in deionized water to prepare a citrus pectin solution and a CaCl2 solution; and (4) mixing the citrus pectin solution with the mixed solution obtained in the step (2), dropwise adding the CaCl2 solution obtained in the step (3), magnetically stirring, and centrifuging to obtain the microgel nanoparticles. The problems that curcumin is unstable and low in oral bioavailability are solved, and the effects of regulating intestinal flora and relieving inflammation are achieved.

Description

Technical Field

[0001] This invention belongs to the field of functional food targeted delivery technology, specifically relating to a method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammatory properties. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory bowel disease of unknown etiology, characterized by destructive and relapsing tissue disease, including local immune dysregulation, gut microbiota dysbiosis, and impaired intestinal barrier. With rapid economic development, the incidence of IBD is rising sharply. Due to its recurrent and difficult-to-cure nature, it seriously endangers public health. According to data from the Optum Research Database (2007–2016), IBD patients incur a direct or indirect cost of $15,000 annually. Currently, IBD treatment typically relies on anti-inflammatory drugs, immunosuppressants, and biologics, but these all have adverse effects such as gut microbiota imbalance, increased drug resistance, headaches, nausea, osteoporosis, indigestion, mood disorders, and cardiovascular problems, and a high relapse rate. Therefore, in-depth exploration of the targets of action and new treatment strategies for IBD is of great practical significance for the scientific prevention and treatment of IBD.

[0003] Numerous studies have confirmed that polyphenols in food and plant-based foods can effectively prevent and improve colonic damage and immune system disorders caused by IBD. They can be used as adjunctive therapy for IBD and for dietary intervention, and are abundant, safe, and effective. Curcumin, an active polyphenol extracted from the rhizome of the turmeric plant, possesses various pharmacological activities, including anti-tumor, anti-inflammatory, hypoglycemic, and antioxidant effects. Multiple animal models and human clinical trials have shown that curcumin can produce good preventive and therapeutic effects against IBD through multiple pathways and targets, with high patient acceptance, good efficacy, relative safety, and low cost. Furthermore, curcumin preferentially accumulates in the intestines, colon, and liver, providing a theoretical basis for its positive role in IBD prevention and treatment. Despite its good pharmacological effects and safety, curcumin's poor water solubility, chemical instability, high intestinal metabolic rate, and rapid excretion limit its oral bioavailability, severely restricting its application in the prevention and treatment of IBD.

[0004] Employing specialized microencapsulation technology to stabilize curcumin while improving its solubility and biocompatibility is one of the most effective methods for solving the aforementioned challenges. Composite aggregation is a self-aggregation phenomenon based on electrostatic interactions between two or more charged macromolecules (e.g., polysaccharide-protein, polysaccharide-polysaccharide, protein-protein). Composite aggregation offers mild preparation conditions, high yield, and high efficiency, and the prepared microcapsules exhibit excellent performance in terms of environmental tolerance and controlled release. It can achieve the encapsulation, transport, and controlled release of sensitive or active substances, and has become a research hotspot in the field of microencapsulation technology both domestically and internationally. For example, Chinese patent CN115414340A discloses a method for preparing curcumin nanomedicine, its application, and a method for constructing a three-dimensional tumor model in conjunction with it; Chinese patent CN115252815A discloses a curcumin complex and its preparation method; and Chinese patent CN110897161B discloses soybean polypeptide-based nanoparticles with high curcumin loading and their pH-driven preparation method and application. This demonstrates that micro-nano-scale complexes formed by biomolecular complex condensation reactions can achieve the effects of stable encapsulation and controlled release of curcumin.

[0005] Designing core-shell nanocarriers is one of the most effective strategies for reducing drug dissolution rates in nanocarriers, offering the advantages of high drug loading efficiency and controlled drug release. For example, Oshi et al. developed chitosan / alginate-loaded curcumin crystal core-shell nanoparticles (CAP1AG4CH5@CUNCs, 421 ± 14 nm) for oral administration, aiming to achieve colon-targeted delivery to alleviate IBD. Their study found that these nanoparticles were mainly distributed in the colon and had a high release rate, effectively reducing inflammation-related symptoms in a mouse colitis model, indicating that they can serve as a highly efficient colon-targeted oral delivery system for the prevention and treatment of IBD. Recently, Wu et al. designed an oral colon-targeted adhesive core-shell nanocarrier using folic acid-zein as the core and pectin as the shell, significantly improving the bioavailability of glycyrrhizic acid and its anti-inflammatory effects at specific sites in the intestine. These results confirm that core-shell nanocarriers can be precisely delivered to the colon, effectively alleviating IBD symptoms by inhibiting the inflammatory response through curcumin. However, the aforementioned nanoparticles failed to address the issues of curcumin's high intestinal metabolic rate and rapid excretion in the body, resulting in low oral bioavailability. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing microgel nanoparticles with synergistic effects of colon-targeted retention, gut microbiota regulation, and anti-inflammation. Specifically, a curcumin-loaded microgel is prepared by using a double-shell raw material consisting of colon-targeting citrus pectin (CP) and absorption-promoting lactoferrin (LF) through a complex coagulation reaction. The efficacy of the curcumin-loaded microgel in alleviating IBD in mice is also investigated. This invention uses LF and CP as research materials to prepare curcumin-loaded microgels through a complex coagulation reaction, which is expected to solve the problems of curcumin instability and low oral bioavailability. Furthermore, the prepared microgel is applied to IBD mice to achieve the effects of regulating gut microbiota and alleviating inflammation.

[0007] A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammation includes the following steps: (1) Dissolve lactoferrin and curcumin in deionized water and anhydrous ethanol respectively to obtain lactoferrin solution and curcumin solution for later use; (2) Mix the lactoferrin solution and curcumin solution of step (1) in equal volumes, stir magnetically and then heat to obtain a mixture of lactoferrin / curcumin complex, and centrifuge to remove excess curcumin. (3) Citrus pectin and CaCl2 were dissolved in deionized water to prepare citrus pectin solution and CaCl2 solution, respectively; (4) Mix the citrus pectin solution from step (3) with the mixture from step (2) at a ratio of 1:3, stir magnetically, then add the CaCl2 solution from step (3), stir magnetically, and centrifuge to obtain microgel nanoparticles.

[0008] Furthermore, in step (1), lactoferrin is dissolved in deionized water and curcumin is dissolved in anhydrous ethanol, and both are then magnetically stirred at room temperature for 1 h.

[0009] Furthermore, the mass concentration of the lactoferrin solution in step (1) is 3.0-5.5 mg / mL; and the mass concentration of the curcumin solution is 0.5-3.0 mg / mL.

[0010] Furthermore, the magnetic stirring described in step (2) is performed at room temperature for 30 min.

[0011] Furthermore, the heat treatment described in step (2) is performed at 90°C for 30 min.

[0012] Furthermore, in step (3), after dissolving citrus pectin and CaCl2 in deionized water, both were magnetically stirred at room temperature for 2 h.

[0013] Furthermore, the mass concentration of the citrus pectin solution in step (3) is 1.0-3.5 mg / mL; and the mass concentration of the CaCl2 solution is 55.5 mg / mL.

[0014] Furthermore, the citrus pectin solution from step (3) and the mixture from step (2) are mixed at a volume ratio of 1:3 in step (4).

[0015] Furthermore, the magnetic stirring described in step (4) is performed at room temperature for 30 min.

[0016] The beneficial effects of this invention are: (1) The present invention uses LF and CP as the matrix for composite coagulation reaction. The materials used are not only biocompatible and safe and non-toxic, but also have good biological activity. The microgel nanoparticles prepared solve the problem of Cur instability and optimize the encapsulation rate of Cur.

[0017] (2) The preparation process of this invention is simple, low in cost, easy to industrialize, green, environmentally friendly and pollution-free, providing technical support for the research and development of Cur.

[0018] (3) In this invention, the prepared microgel is dispersed in water to form a homogeneous system, which is then administered orally to IBD model mice. The results show that this strategy not only significantly improves the stability and sensory quality of curcumin and increases its retention in the colon, but also effectively alleviates the symptoms of IBD. Attached Figure Description

[0019] Figure 1 These are images showing how the loading rate of Cur in LF / CP-Cur MN changes with different component concentration gradients in each of the single-factor analyses optimized in Examples 1-3.

[0020] Figure 2 These are images from Example 3 showing the dispersion state of Cur, LF / CP, and LF / CP-Cur MN in aqueous solution and their surface morphology characteristics under a scanning electron microscope.

[0021] Figure 3 This is an image showing the crystal structure of LF / CP-Cur MN characterized by X-ray diffraction (XRD) analysis in Example 3.

[0022] Figure 4 These are images showing the systematic characterization of the chemical structure of LF / CP-Cur MN by FT-IR in Example 3.

[0023] Figure 5 These are images reflecting the thermal stability of Cur, LF / CP, and LF / CP-Cur MN nanoparticles in Example 3.

[0024] Figure 6 This is a graph showing the effect of LF / CP-Cur MN on alleviating clinical symptoms in IBD mice in an application example.

[0025] Figure 7 This is a pathological section image showing the effect of LF / CP-Cur MN on the inflammatory response in IBD mice in an application example.

[0026] Figure 8 This is a diagram showing the inhibition of inflammatory cytokines in the serum of IBD mice by LF / CP-Cur MN in an application example.

[0027] Figure 9 This is a graph showing the effect of LF / CP-Cur MN on intestinal mucus in IBD mice in an application example.

[0028] Figure 10 This is a graph showing the residual amount of Cur and LF / CP-Cur MN in the colon of IBD mice in an application example.

[0029] Figure 11 This is a diagram showing the effects of Cur, LF / CP--Cur MN on the gut microbiota of IBD mice in an application example. Detailed Implementation

[0030] The specific embodiments of the present invention will be further described in detail with reference to the accompanying drawings, examples, and comparative examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0031] Example 1: A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammation, comprising the following steps: (1) Lactoferrin (LF) and curcumin (Cur) were dissolved in deionized water and anhydrous ethanol, respectively, and magnetically stirred at room temperature for 1 h to obtain lactoferrin (LF) solution and curcumin (Cur) solution for later use; the mass concentration of lactoferrin (LF) solution was 4.0 mg / mL; the mass concentration of curcumin (Cur) solution was 1.5 mg / mL; (2) Mix the lactoferrin (LF) solution and curcumin (Cur) solution of step (1) in equal volumes, stir magnetically at room temperature for 30 min, and then heat at 90°C to obtain a mixture of lactoferrin / curcumin (LF / Cur); (3) Citrus pectin (CP) and CaCl2 were dissolved in deionized water and magnetically stirred at room temperature for 2 h to obtain citrus pectin (CP) solution and CaCl2 solution; the mass concentration of the citrus pectin (CP) solution was 3.0 mg / mL; the mass concentration of the CaCl2 solution was 55.5 mg / mL. (4) Mix the citrus pectin (CP) solution from step (3) with the mixture from step (2) at a volume ratio of 1:3, then add the CaCl2 solution from step (3) dropwise, and stir magnetically for 30 minutes at room temperature to obtain core-shell lactoferrin pectin calcium-loaded curcumin microgel nanoparticles (LF / CP-Cur MN).

[0032] In this Example 1, the experiment on the concentration of citrus pectin (CP) solution also included tests at different concentrations. Specifically, all other methods and amounts remained unchanged, only the mass concentration of the citrus pectin (CP) solution was varied, with tests conducted at 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, and 3.5 mg / mL. The final changes in curcumin (Cur) loading are shown in the attached figure. Figure 1 As shown in Figure C, the optimal mass concentration of citrus pectin (CP) solution is 3.0 mg / mL.

[0033] Example 2: The main process steps of Example 2 are the same as those of Example 1, except that the mass concentration of CP solution is 1.0 mg / mL and the mass concentration of Cur is 1.5 mg / mL.

[0034] In Example 2, the experiment on Cur solution concentration also included tests at different concentrations. Specifically, all other methods and dosages remained unchanged, only the mass concentration of the Cur solution was varied. Tests were conducted at 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, and 3.0 mg / mL. The final changes in Cur loading are shown in the attached figure. Figure 1 As shown in Figure A, the optimal mass concentration of the Cur solution is 1.5 mg / mL.

[0035] Example 3: The main process steps of Example 3 are the same as those of Example 1, except that the mass concentration of CP solution is 1.0 mg / mL and the mass concentration of LF solution is 3.0 mg / mL.

[0036] In Example 3, the experiment on LF solution concentration also included tests at different concentrations. Specifically, all other methods and dosages remained unchanged, only the mass concentration of the LF solution was varied. Tests were conducted at 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, and 5.5 mg / mL. The final changes in Cur loading are shown in the attached figure. Figure 1 As shown in Figure B, the optimal mass concentration of the LF solution is 3.0 mg / mL.

[0037] Example 4: The main process steps of Example 4 are the same as those of Example 1, except that the mass concentration of Cur solution is 1.5 mg / mL, the mass concentration of CP solution is 1.0 mg / mL, and the mass concentration of LF solution is 3.0 mg / mL. The Cur encapsulation efficiency of the prepared LF / CP-Cur MN is as high as 30.75%. The particle size of the LF / CP-Cur MN solution was measured to be ~208 nm and the potential was ~-32.1 mV using a Malvern particle size analyzer (Zetasizer Nano ZS90, Malvern Panalytical, Worcestershire, England).

[0038] Example 5: Evaluation of the physicochemical properties of LF / CP-Cur MN: (1) Surface morphology of LF / CP-Cur MN: As attached Figure 2 As shown in Figure A, Cur is insoluble in water at room temperature. After mixing the core-shell LF / CP and LF / CP-Cur MN at room temperature for 3 hours, both formed a uniformly dispersed solution in water. The solution of LF / CP-Cur MN turned yellow due to Cur, which indicates that the core-shell LF / CP effectively encapsulates Cur, masking the hydrophobic regions of Cur and improving its water solubility.

[0039] Small amounts of dried Cur, LF / CP, and LF / CP-Cur MN samples were fixed onto the sample stage and sputter-coated with gold for 1 min. The surface morphology of the LF / CP-Cur MN samples from Example 3 was analyzed using a COXEM EM-30plus scanning electron microscope under high vacuum conditions. Figure 2 As shown in Figure B), the Cur solid is granular with a high degree of fragmentation, while the LF / CP surface is flocculent, smooth, loose, and porous. Furthermore, the LF / CP-Cur MN surface exhibits a flocculent and porous morphology, indicating that CP successfully encapsulates LF and Cur.

[0040] (2) Crystal structure analysis of LF / CP-Cur MN: The crystal structure of LF / CP-Cur MN in Example 3 was analyzed using a Dandong Haoyuan DX 2800 X-ray diffractometer. The crystallinity and amorphous properties of the Cur, LF / CP, and LF / CP-Cur MN samples were analyzed using a Cu Kα radiation source (λ = 0.154 nm). XRD patterns were recorded at 40 kV and 40 mA within the 2θ range of 5°–50° using copper Kα radiation (λ = 0.1541 nm) at a scan rate of 4°min. -1 Measurement time 0.2, step width angle 0.02, tube voltage 30, tube current 20.

[0041] Appendix Figure 3 XRD patterns of Cur, LF / CP, and LF / CP-Cur MN are shown. Cur exhibits multiple characteristic peaks (7.9°, 8.8°, 14.5°, 15.9°, 17.2°, and 28.1°) in the 2θ range from 5° to 50°, which are attributed to the crystalline nature of Cur. In contrast, no characteristic peaks were observed in the spectrum of LF / CP-Cur MN. This observation indicates that Cur transforms from a crystalline state to an amorphous state after encapsulation, further suggesting that Cur is dispersed at the molecular level in LF / CP, and the amorphous state is beneficial for human absorption and utilization. Furthermore, core-shell LF / CP also did not show any crystalline peaks.

[0042] (3) Structural analysis of LF / CP-Cur MN: The Nexus 670 Fourier transform infrared spectrometer (PerkinElmer, USA) was used in the wavenumber range of 500–4000 cm⁻¹. -1 Infrared spectra of LF, CP, Cur, LF / CP, and the LF / CP-Cur MN sample from Example 3 were obtained from potassium bromide microspheres and compared with the infrared spectrum of air. The "chemical fingerprint" of LF / CP-Cur MN was analyzed (see appendix). Figure 4 (As shown). In the Cur spectrum, approximately 3505 cm⁻¹ -1 1025 cm -1 and 1260 cm -1 The three characteristic absorption peaks at [location] correspond to the stretching vibration of the phenolic hydroxyl group (-OH), the COC vibration, and the ketone group of Cur, respectively. Furthermore, the CP spectrum shows an absorption peak at approximately 10¹⁵ cm⁻¹. -1 This can be attributed to the stretching and bending vibrations of the CO bonds within the pyranose ring of the carbohydrate, 1740 cm. -1 The absorption peak at 1606 cm⁻¹ corresponds to the stretching vibrations of the methyl ester group (COOCH₃) and the undissociated carboxylic acid group (COOH), while the absorption peak at 1606 cm⁻¹ corresponds to the stretching vibrations of the methyl ester group (COOCH₃) and the undissociated carboxylic acid group (COOH). -1 The nearby peaks belong to the carboxylate anion (COO). - The asymmetric stretching vibration of amide I is observed. The LF spectrum reveals the characteristic amide I band (1636 cm⁻¹). -1 (C=O stretching vibration) and amide II band (1517 cm) -1 (NH bending vibration and CN stretching vibration). The LF / CP complex spectrum retains the characteristic peaks of both components, but at 1606 cm⁻¹... -1 The disappearance of the peak position indicates an interaction between the carboxylate group of CP and the amino or calcium ion of LF. Notably, the composite nanoparticle spectrum shows an interaction at 1744 cm⁻¹. -1 1636 cm-1 1514 cm -1 1260 cm -1 and 1025 cm -1 The characteristic vibrational peaks corresponding to CP, LF, and LF / CP are clearly preserved nearby, confirming the composite properties of the material. The LF / CP-Cur MN spectrum at 3505 cm⁻¹... -1 The characteristic peak of curcumin phenol hydroxyl group at 1740 cm⁻¹ -1 The disappearance of the asymmetric stretching peak of the CP carboxylate group indicates that curcumin has been successfully encapsulated within nanoparticles.

[0043] (4) Thermal stability analysis: Two-dimensional scanning calorimetry (DSC) thermograms of Cur, LF / CP, and LF / CP-Cur MN from Example 3 were measured using a DSC822e thermal analyzer (Mettler Toledo Co., Switzerland). Approximately 3.0 mg of sample was placed in an aluminum pot and then sealed with an aluminum lid. Thermal analysis was then performed from 25 to 300 °C under a dry nitrogen atmosphere at a flow rate of 20 mL / min and a heating rate of 10 °C / min. Using the recorded DSC curve of the sealed empty pot as a reference, the results showed that the thermal analysis curve of Cur exhibited a strong endothermic peak at approximately 190 °C (e.g., ...). Figure 5 As shown in the figure, this is due to the melting of Cur crystals, further indicating the high crystallinity of Cur. However, few characteristic peaks of Cur were observed in the thermal analysis of LF / CP-Cur MN nanoparticles, indicating the loss of most of the Cur crystalline structure, as observed in the XRD analysis, and further demonstrating that Cur was successfully encapsulated in LF / CP nanoparticles, forming a high-energy amorphous morphology. Furthermore, LF / CP and LF / CP-Cur MN nanoparticles exhibit a broad endothermic peak at approximately 80 °C. These data confirm the amorphous nature of Cur in LF / CP-Cur MN nanoparticles.

[0044] Application example: Testing the anti-inflammatory effect of LF / CP-Cur MN prepared in Example 3 on IBD mice: (1) Construction and treatment of IBD mouse model: Mice were placed in an institutional animal care facility with a 12:12 light-dark cycle. After one week of acclimatization, 32 C57BL / 6 mice were randomly divided into four groups: a blank control group (CON), a 2% DSS treatment group (DSS), a 2% DSS+Cur treatment group (DSS+Cur), and a 2% DSS+LF / CP-Cur MN treatment group (DSS+LF / CP-Cur MN). Mice in the IBD model group were fed 2% DSS water. Mice in the Cur and LF / CP-Cur MN groups were fed 2% DSS water and received Cur and LF / CP-Cur MN feeding interventions at a dose of 10 mg / (kg·Bw) per day, respectively. Administration continued for 11 days, during which time all animals were allowed free access to standard laboratory food and designated water.

[0045] (2) Clinical symptoms For 11 consecutive days, the mice were weighed at regular intervals each day, and the consistency of their stool and the presence of bloody stool were carefully observed. The DAI score was determined by summing the scores for weight, diarrhea, and bloody stool according to the DAI scoring system in Table 1.

[0046] Table 1. Scoring System for Disease Activity Index ; DSS-induced IBD mice showed a decreasing body weight trend (see appendix) Figure 6 (See Figure A in the table). Furthermore, diarrhea, even bloody stools, appeared from day 5 onwards, showing a statistically significant difference compared to the CON group (p<0.001), indicating the successful establishment of the IBD mouse model. The body weight of both the LF / CP-Cur MN-treated and Cur-treated IBD mice showed a gradual upward trend on day 8, indicating comparable intervention effects. In addition, after day 7, the DAI score of the IBD mice continued to increase, while the Cur and LF / CP-Cur MN-treated groups showed a decrease. Moreover, the DAI score of the DSS+LF / CP-Cur MN group decreased earlier than that of the DSS+Cur group (see Appendix). Figure 6 (Figure B in the text).

[0047] The colon length of IBD mice (5.67±0.28 cm) was significantly shorter than that of the normal control group (8.27±0.19 cm), and the difference between the groups was statistically significant (p<0.001). (See appendix) Figure 6 (See Figures C and D). The colon length of IBD mice treated with LF / CP-Cur MN was 15.52% longer than that of IBD mice, but not significantly different from that of IBD mice treated with Cur. This indicates that the anti-inflammatory effect of core-shell LF / CP-encapsulated Cur is comparable to that of Cur.

[0048] (3) Preparation and observation of mouse colon pathological sections Mouse colons were fixed by immersion in 4% paraformaldehyde solution for 24 hours and then embedded in paraffin. Paraffin sections (3 μm thick) were dewaxed by washing twice with xylene and then with different concentrations of ethanol (100, 95, 90, 80, 70, and 50%), and stained with HE or AB-PAS according to the methods described in the kit. After dehydration by washing with a gradient of ethanol (50, 70, 80, 90, 95, and 100%) and xylene, sections were mounted with a permanent mounting agent (neutral resin). Images were obtained at ×20 magnification using a Nikon Eclipse Ti microscope (Melville, NY).

[0049] The submucosa of normal mice is much more structurally intact than that of IBD model mice (see appendix). Figure 7 The lamina propria and crypts of IBD mice showed moderate edema and extensive mononuclear cell infiltration. The submucosa of IBD mice showed moderate mononuclear cell infiltration and severe edema. Due to severe colitis, numerous erosions were present on the intestinal epithelial surface of IBD model mice. However, only mild mononuclear infiltration and mild edema were observed in the submucosa of mice treated with LF / CP-Cur MN nanoparticles and Cur, respectively, indicating a reduction in inflammatory infiltration and crypt damage induced by DSS. DSS-induced mice exhibited clinical signs of IBD, including weight loss, elevated DAI scores, significantly shortened colon length, and extensive inflammatory cell infiltration. However, treatment with LF / CP-Cur MN nanoparticles slowed the rate of weight loss and DAI score reduction in IBD mice, and the colon length was not statistically significant compared to normal mice, with no extensive inflammatory cell infiltration, consistent with the effects of Cur-treated mice. All data indicate that the medicinal properties of the Cur-loaded LF / CP-Cur MN nanoparticles were not lost and that they had a mitigating effect on the clinical symptoms of IBD mice.

[0050] (4) Inflammatory cytokines: The levels of pro-inflammatory cytokines (TNF-α) in the serum of IBD mice were determined by enzyme-linked immunosorbent assay (ELISA) in the LF / CP-CurMN sample of Example 3. α IL-6 and IL-1 β Inhibitory effect of ) (Appendix) Figure 8 Fresh mouse blood was incubated at 37°C for 30 min, then centrifuged (12000 rpm) for 10 min to obtain serum, which was then stored at -20°C. Serum IL-1 levels were measured using an ELISA kit. β TNF- α The levels of IL-6 and the inhibitory effects on cellular inflammatory factors are shown in the attached figure. Figure 8 From the appendix Figure 8 It can be seen that TNF-α in the serum of IBD mice α IL-6 and IL-1 β Compared to normal mice, TNF-α levels were elevated in the serum of mice in the LF / CP-Cur MN group and the Cur group. α IL-6 and IL-1 β The levels were lower than those in IBD mice, and the inhibitory effect of the LF / CP-Cur MN group on pro-inflammatory cytokines was similar to that in the Cur group.

[0051] IBD mice have a large number of inflammatory cytokines, including IL-1, in the intestinal inflammatory sites. β Pro-inflammatory cytokines such as TNF-α and IL-6 are important secretory factors in the inflammatory response. Cur intervention reduced serum TNF-α levels in IBD mice. α IL-6 and IL-1 β The levels of serum pro-inflammatory factor TNF-α in IBD treated with LF / CP-Cur MN decreased by 48.94%, 76.32%, and 44.04%, respectively. α IL-6 and IL-1 β The levels decreased by 57.19%, 84.95%, and 46.58%, respectively, indicating that the Cur-loaded LF / CP-Cur MN had a strong effect, and the inflammatory activity of mice treated with LF / CP-Cur MN was significantly reduced.

[0052] (5) Distribution of intestinal mucus Mucus-filled goblet cells are distributed in the lamina propria of the colon, and mucus covers the surface of the colonic epithelial cells, including an internally attached mucus layer and a loose mucus layer (such as...). Figure 9 The internally adhered mucus layer prevents bacteria and toxins from leaving intestinal cells, while the loose mucus provides habitat and nutrients for symbiotic bacteria. Confocal microscopy revealed that normal mice have mucus-filled goblet cells in their lamina propria and mucus on the surface of the colonic epithelium, while IBD mice have almost no mucus-filled goblet cells in their lamina propria and no mucus in the intestinal lumen. In mice administered LF / CP-Cur MN nanoparticles and IBD mice treated with Cur, mucus-filled goblet cells and mucus on the surface of the colonic epithelium were observed in their lamina propria. These results indicate that Cur-loaded LF / CP-Cur MN nanoparticles effectively inhibit the destruction of goblet cells in the intestines of IBD mice and promote mucus secretion, strengthening the first physical barrier of the intestinal mucus barrier.

[0053] (6) Curl residue in the colon: The mouse colon was blotted dry with filter paper, weighed, and chopped. It was then placed in a centrifuge tube, and 500 μL of 50% ethanol was added. The mixture was shaken for 1 h, and then centrifuged at 4000 rpm for 20 min. The supernatant was collected. 5 mg of curcumin was dissolved in 50% ethanol and diluted to volume in a 100 mL volumetric flask to obtain a 0.05 mg / mL curcumin solution. 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mL of this solution were transferred to eight 20 mL centrifuge tubes, and 50% ethanol was added to a total volume of 10 mL. The absorbance was measured at 432 nm, a standard curve was plotted, and the curcumin content in the mouse colon was calculated.

[0054] The residual amount of curcumin in the colon was detected. The results showed that in the colonic tissue of DSS-induced IBD model mice, the residual amount of curcumin detected in the orally administered free curcumin group was 0.03281 ± 0.00254 mg / g, which was significantly lower than that in the LF / CP-Cur MN group (0.0451 ± 0.00127 mg / g) (see attached). Figure 10 The LF / CP-Cur MN loading technology can significantly improve the colonic delivery efficiency of Cur. The mechanism of action may be that the nanoparticle encapsulation effectively reduces the degradation of Cur by gastric acid, and LF / CP assists in enhancing the penetration ability of LF / CP-Cur MN in the colonic mucus layer, indicating that LF / CP-Cur MN improves the bioavailability of Cur.

[0055] (7) Changes in gut microbiota: Precisely weighed fecal samples (0.25–0.50 g or 200 μL) were mixed with buffer SA (500 μL), SC (100 μL), and grinding beads (0.25 g). After vortexing and low-temperature grinding, the mixture was lysed at 70°C for 15 min, and the supernatant was collected by centrifugation. Subsequently, specific amplification was performed on different microbial groups: bacteria (16S V3+V4, V4+V5, V4 region), endophytes (16S V3+V4), fungi (ITS1, ITS2), archaea (16S V3+V4), and eukaryotes (18S V4, V7). PCR products were detected by 2% agarose gel electrophoresis, purified using an ampoule XT bead recovery kit, and finally sequenced at both ends (2 × 300 bp) on the MiSeq platform.

[0056] At the door level (attached) Figure 11 Figure A in the figure) and genus level (attached) Figure 11The analysis results (Figure B) showed that the difference in microbial community between the DSS+LF / CP-Cur MN group and the CON group was the smallest, indicating that LF / CP-Cur MN maintained the microbial community in IBD mice. Specifically, at the phylum level, LF / CP-Cur MN could reduce pathogenic microbiota (such as Proteobacteria) in IBD mice. Proteobacteria The relative abundance of ) was significantly increased, while the abundance of beneficial Firmicutes phylum ( Firmicutes ) and Bacteroidetes ( Bacteroidota The relative abundance of ) was analyzed. At the genus level, LF / CP-Cur MN inhibited the growth of multiple pathogenic bacteria (including Enterobacteriaceae). Enterorhadus ), Enterobacteriaceae ( Enterobacter ) and Parasartella spp. ( Parasutterella The growth of bacteria that produce short-chain fatty acids (such as lactobacilli) is promoted, while simultaneously promoting the growth of bacteria that produce short-chain fatty acids (such as lactobacilli). Lactobacillus (such as) intestinal probiotics and Bacteroides ( Bacteroides This embodiment illustrates that core-shell lactoferrin pectin calcium-loaded curcumin microgel nanoparticles effectively improve the stability of curcumin, prolong the residual amount of curcumin in the colon, and have a dual effect of regulating intestinal flora and anti-inflammation.

[0057] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammatory properties, characterized in that, Includes the following steps: (1) Dissolve lactoferrin and curcumin in deionized water and anhydrous ethanol respectively to obtain lactoferrin solution and curcumin solution for later use; (2) Mix the lactoferrin solution and curcumin solution of step (1) in equal volumes, stir magnetically and then heat to obtain a mixture of lactoferrin / curcumin complex, and centrifuge to remove excess curcumin. (3) Citrus pectin and CaCl2 were dissolved in deionized water to prepare citrus pectin solution and CaCl2 solution, respectively; (4) Mix the citrus pectin solution from step (3) with the mixture from step (2) at a ratio of 1:3, stir magnetically, then add the CaCl2 solution from step (3), stir magnetically, and centrifuge to obtain microgel nanoparticles.

2. The method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammation according to claim 1, characterized in that, In step (1), lactoferrin was dissolved in deionized water and curcumin was dissolved in anhydrous ethanol, and both were magnetically stirred at room temperature for 1 h.

3. A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammatory according to claim 1, characterized in that, The mass concentration of the lactoferrin solution in step (1) is 3.0-5.5 mg / mL; the mass concentration of the curcumin solution is 0.5-3.0 mg / mL.

4. A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammation according to claim 1, characterized in that, The magnetic stirring described in step (2) involves stirring at room temperature for 30 minutes.

5. A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammation according to claim 1, characterized in that, The heating treatment described in step (2) is performed at 90°C for 30 minutes.

6. A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammation according to claim 1, characterized in that, In step (3), after dissolving citrus pectin and CaCl2 in deionized water, both were magnetically stirred at room temperature for 2 h.

7. A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammation according to claim 1, characterized in that, The mass concentration of the citrus pectin solution in step (3) is 1.0-3.5 mg / mL; the mass concentration of the CaCl2 solution is 55.5 mg / mL.

8. The method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammatory according to claim 1, characterized in that, The citrus pectin solution from step (3) and the mixture from step (2) are mixed at a volume ratio of 1:

3.

9. A method for preparing microgel nanoparticles with colon-targeted retention and synergistic effects of gut microbiota regulation and anti-inflammation according to claim 1, characterized in that, The magnetic stirring described in step (4) involves stirring at room temperature for 30 minutes.

Images