Rhein-chitosan composite nanoparticle-stabilized colon-targeted Pickering emulsion as well as preparation method and application thereof

Abstract

The invention discloses a colon-targeted Pickering emulsion stabilized by rhein-chitosan composite nanoparticles, a preparation method and an application of the colon-targeted Pickering emulsion. Rhein-chitosan is coupled and then crosslinked with sodium tripolyphosphate to form the rhein-chitosan composite nano-particles, and the nano-particles have better hydrophobicity and acid and alkali resistance stability; the nano-particle suspension and fish oil containing rhein are homogenized and emulsified at a high speed to obtain the Pickering emulsion entrapped with the rhein, and the Pickering emulsion has relatively high rhein drug loading capacity, better stability and good colon-specific release and slow release characteristics; and the traditional Chinese medicine composition has a dose-dependent relieving effect on dextran sodium sulfate induced ulcerative colitis mice, can inhibit inflammatory response and repair intestinal barrier functions, can effectively avoid intestinal nutrition loss caused by rhein, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a colon-targeted Pickering emulsion stabilized by rhein-chitosan composite nanoparticles, a preparation method and an application thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa. The primary pathophysiological characteristics of UC are twofold: a continuous inflammatory response in the colonic mucosa and an impairment of the colonic mucosal barrier function. While traditional therapeutic agents can alleviate symptoms, they still have numerous limitations. For example, traditional oral medications have low bioavailability due to the complex physiological environment of the gastrointestinal tract; long-term use can result in various adverse reactions and side effects; and the high cost and immunogenicity of biologics limit their widespread use.

[0003] Rhein (Rh), one of the characteristic anthraquinone components of rhubarb, demonstrates the therapeutic advantage of "multi-target synergy and multi-level intervention" in the treatment of colonic diseases. This synergistic network, formed through multiple levels including immune regulation, oxidative stress regulation, microbiome-host interactions, and intestinal barrier protection, underlies the multidimensional effects of Rh in the treatment of ulcerative colitis. However, Rh's poor water solubility results in low oral bioavailability, and its gastrointestinal irritation and diarrheal side effects reduce treatment compliance with Rh. These pharmacokinetic deficiencies, along with side effects, constitute a major obstacle to the clinical application of Rh.

[0004] Pickering emulsions (PEs), composed of a dispersed phase, a continuous phase, and solid nanoparticles, offer unique advantages in improving emulsion stability, delivering poorly soluble drugs, and providing sustained release. Despite this, the widespread application of PEs is limited by several factors, such as low drug loading, poor stability, and lack of targeted release. The properties of the nanoparticles are one of the primary factors influencing the properties of Pickering emulsions.

[0005] Chitosan nanoparticles (CS / TPPNPs) hold great promise for the preparation of poly(ethylene glycol) (PEs). However, the strong hydrophilicity and instability of CS / TPPNPs in alkaline environments weaken the overall stability of PEs, and their poor bioadhesion limits the construction of oral colon-targeted drug delivery systems. Therefore, structural modification of chitosan (CS) to improve the limitations of CS / TPPNPs is of great significance. The amino and hydroxyl groups of CS serve as active groups, providing abundant sites for chemical modification. By introducing functional groups or hydrophobic molecules, the physicochemical properties and biological functions of chitosan can be manipulated, such as improving hydrophilic and hydrophobic properties, enhancing acid and alkali stability, and enhancing mucosal adhesion. However, the purpose of currently commonly used modification reagents for chitosan modification is only to obtain a solid stabilizer with excellent properties, but it is unable to simultaneously improve drug loading and drug bioadhesion. Summary of the Invention

[0006] In response to the above-mentioned problems and deficiencies in the prior art, the present invention unexpectedly discovered that by introducing rhein into the chitosan backbone during the preparation of rhein Pickering emulsions, a novel derivative Rh-CS conjugate with amphiphilic characteristics was constructed. This, while retaining the biological functions of chitosan, optimizes chitosan's acid and alkali resistance, bioadhesion, and drug loading capacity. Furthermore, the Rh-CS conjugate was reacted with the crosslinker sodium tripolyphosphate (TPP) to synthesize novel nanoparticles, rhein-chitosan composite nanoparticles, for the preparation of Pickering emulsions and the construction of an oral colon-targeted drug delivery system. Specifically, the invention includes the following:

[0007] In a first aspect, the present invention provides a rhein-chitosan composite nanoparticle, wherein the preparation method of the rhein-chitosan composite nanoparticle comprises:

[0008] (1) coupling rhein and chitosan to prepare a rhein-chitosan conjugate;

[0009] (2) The rhein-chitosan conjugate was treated with sodium tripolyphosphate solution as a crosslinking agent to obtain rhein-chitosan composite nanoparticles.

[0010] Preferably, the step (1) comprises: dissolving rhein in DMSO, adding a catalyst, keeping the mixture in a dark ice bath for 30 minutes, and then adding the mixture dropwise to a chitosan solution; adjusting the pH of the solution to 5.5 using 1M NaOH, stirring the mixture for reaction, and obtaining a rhein-chitosan conjugate solution; and dialyzing and purifying the mixture to obtain a rhein-chitosan conjugate; wherein the catalyst is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or N-hydroxysuccinimide.

[0011] Preferably, the chitosan solution is prepared by suspending chitosan in ultrapure water, stirring for 30 minutes to fully swell the chitosan, and adjusting the pH value of the system to 2.5 using 1M HCl to completely dissolve the chitosan.

[0012] Preferably, the molar mass ratio of the chitosan amino groups to the rhein carboxyl groups is 5:2.

[0013] Preferably, the dialysis is performed for 72 hours, the dialysate is changed every 8 hours, the pH of the dialysate is 5.5, and the volume ratio of the dialysate to the sample solution is 100:1.

[0014] Preferably, the step (2) comprises: dissolving the rhein-chitosan conjugate in an acetic acid aqueous solution, adjusting the pH to 5.5 using a 1M NaOH aqueous solution; adding a sodium tripolyphosphate aqueous solution with a pH of 5.5 to the rhein-chitosan conjugate solution, and ultrasonicating for 30 minutes to generate rhein-chitosan composite nanoparticles.

[0015] Preferably, the concentration of the acetic acid solution is 1% (v / v).

[0016] Preferably, the mass ratio of the sodium tripolyphosphate to the rhein-chitosan conjugate is 0.25:1.

[0017] In a second aspect, the present invention provides use of the rhein-chitosan composite nanoparticles described in the first aspect in preparing a rhein Pickering emulsion.

[0018] In a third aspect, the present invention provides a rhein Pickering emulsion, wherein the preparation steps of the rhein Pickering emulsion are as follows: dissolving rhein in fish oil as the oil phase, using the rhein-chitosan composite nanoparticles described in the first aspect as a solid stabilizer, and high-speed homogenization and emulsification to obtain the rhein-loaded Pickering emulsion.

[0019] Preferably, the phase volume fraction is 0.5, and the concentration of rhein-chitosan composite nanoparticles is 0.5 mg / mL.

[0020] Preferably, the high-speed homogenization emulsification condition is 12000 rpm / 3 min.

[0021] In a fourth aspect, the present invention provides use of the rhein Pickering emulsion described in the third aspect in the preparation of a pharmaceutical preparation for treating ulcerative colitis.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] (1) The present application found that, when preparing rhein Pickering emulsion, the chitosan nanoparticles prepared by modifying chitosan in the existing way can improve the stability of chitosan nanoparticles, but cannot improve the drug loading capacity and drug bioadhesion; unexpectedly, the present application uses rhein to modify the structure of chitosan, which improves the amphiphilicity and acid and alkali stability of chitosan as expected, and further constructs a more stable colon-targeted Pickering emulsion; for the Pickering emulsion with rhein in the internal phase, the chemical modification of the solid particle stabilizer in the present application not only significantly improves the properties of chitosan itself, but also significantly improves the drug loading capacity of the functional ingredient rhein; moreover, the structural modification of chitosan in the present application unexpectedly improves the bioadhesion of chitosan, and the Pickering emulsion prepared thereby not only can respond to the colon environment to release the drug, but also has sustained-release performance.

[0024] (2) Compared with traditional rhein oral preparations, the oral colon-targeted and sustained-release Pickering emulsion constructed by the present application not only can realize colon targeting and sustained release to improve the bioavailability of rhein, but also can avoid the side effect of diarrhea caused by gastrointestinal irritation of rhein, and effectively avoid the loss of intestinal nutrients; it is shown that the drug delivery system constructed by the present application is expected to be used as a carrier for anti-ulcerative colitis drugs with intestinal irritation side effects in clinical application.

[0025] (3) The rhein Pickering emulsion prepared by the present application can inhibit inflammatory response by interfering with the TLR4 / NF-κB / MLCK pathway, repair intestinal barrier function by up-regulating tight junction protein ZO-1 and its related proteins claudin-3 and occludin, and has a dose-dependent alleviating effect on ulcerative colitis, which has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] The following drawings are used to provide further understanding of the present application and form a part of the specification, together with embodiments of the present application, to explain the present application, and do not constitute a limitation of the present application.

[0027] Figure 1 Synthesis of Rh-CS conjugate; (i) is stirring for 24 h under the condition of pH = 5.5 and room temperature; (ii) is dialysis and freeze-drying.

[0028] Figure 2 DSS-induced UC mouse model and drug treatment flowchart.

[0029] Figure 3Characterization of Rh-CS conjugate; wherein A is a Fourier transform infrared spectrum; B is a hydrogen nuclear magnetic resonance spectrum (CS and Rh-CS conjugates were prepared in 25% v / v CD3COOD in D2O, and Rh was prepared in DMSO-d6); C is an X-ray diffraction spectrum; D is a UV-visible spectrum; E is a scanning electron microscope image of CS; F is a scanning electron microscope image of the Rh-CS conjugate; GI is an X-ray photoelectron spectrum of CS; JL is an X-ray photoelectron spectrum of the Rh-CS conjugate.

[0030] Figure 4 Characterization of Rh-CS / TPPNPs; wherein, A is the turbidity of the nanoparticle suspension, and the insets are pictures of CS / TPPNPs (top) and Rh-CS / TPPNPs (bottom) samples; B is the hydrated particle size; C is the polydispersity coefficient; D is the particle size distribution of CS / TPPNPs, and the insets are the contact angle diagram and Tyndall effect diagram; E is the particle size distribution of Rh-CS / TPPNPs, and the insets are the contact angle diagram and Tyndall effect diagram; F is the transmission electron microscope image of Rh-CS NPs, and the inset is the particle size distribution diagram of the nanoparticles after 6 months of storage at room temperature; GI is the effect of pH on the hydrated particle size, polydispersity coefficient, and zeta potential of the nanoparticles; JL is the effect of NaCl on the hydrated particle size, polydispersity coefficient, and zeta potential of the nanoparticles.

[0031] Figure 5 Cell experiment results; A is cell viability; B is Calcein AM / PI staining image; C is cell uptake image; D is NO concentration level; EG is red blood cell hemolysis rate; ## p<0.01 compared with the BC group; * p<0.05, ** p<0.01 compared with the MC group; △△ p<0.01 compared with the 5-ASA group.

[0032] Figure 6 Physical and chemical characterization of CS / TPPPEs; A and E are microscopic images; B and F are droplet diameters; C and G are emulsion sample images; D and H are creaming indices; ** p<0.01 compared with Group; # p<0.05, ## p<0.01 compared with the c(NPs)=0.5 group.

[0033] Figure 7Physical and chemical characterization of Rh-CS / TPPPEs; A is the droplet diameter distribution of CS / TPPPEs, and the inset is a sample picture; B is the droplet diameter distribution of Rh-CS / TPPPEs, and the inset is a sample picture; C is the CLSM image of Rh-CS / TPPPEs; D is the apparent viscosity of Rh-CS / TPPPEs.

[0034] Figure 8 Physicochemical characterization of Rhein@Rh-CS / TPPPEs; A is the in vitro release curve of rhein; B is the in vivo distribution fluorescence image of the emulsion; CE is the in vivo pharmacokinetic curve of rhein; ** p<0.01 compared with rhein suspension; ## p<0.01 compared with Rhein@CS / TPPPEs.

[0035] Figure 9 Effects of Rhein@Rh-CS / TPPPEs on DSS-induced UC mice; A is mouse body weight; B is DAI score; C and D are colon lengths; E is H&E staining of colon tissue (200 μm); F is red blood cell count; G is white blood cell count; H is platelet count; I is hemoglobin content; ^ p<0.05, ^^ p<0.01 compared with BC group, * p<0.05, ** p<0.01 compared with the MC group.

[0036] Figure 10 Serum biochemical indicators related to liver and kidney toxicity in mice; A is aspartate aminotransferase (AST); B is alanine aminotransferase (ALT); C is alkaline phosphatase (ALP); D is blood urea nitrogen (BUN); E is creatinine (CREA); F is uric acid (UA); # p<0.05, ## p<0.01 compared with the BC group.

[0037] Figure 11 Effects of Rhein@Rh-CS / TPPPEs on the expression levels of inflammatory factors, related genes, and proteins; A is TNF-α; B is IL-6; C is IL-1β; D is NF-κB p65; E is TLR4; F is MLCK; G is immunohistochemical image (50 μm); H is claudin-3; I is occludin; J is ZO-1; ^ p<0.05, ^^ p<0.01 compared with the BC group; * p<0.05, ** p<0.01 compared with the MC group; #p<0.05, ## p<0.01 compared with the PC group.

[0038] Figure 12 Analysis of nutrient levels in mouse feces; A is the total polysaccharide content in feces; B is the total protein content in feces; C is the total fat content in feces; ^ p<0.05, ^^ p<0.01 compared with the BC group; # p<0.05, ## p<0.01 compared with the Rh group. DETAILED DESCRIPTION

[0039] The following specific examples and experimental examples further illustrate the present invention. Experimental methods for which specific conditions are not specified in the examples are generally carried out under conventional conditions or conditions recommended by the manufacturer; the general equipment, materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources. All experimental data were obtained by at least three replicates, and the results are expressed as mean ± standard deviation. If the data are normally distributed and the variances are homogeneous, the t-test is used for comparison between two groups, and one-way analysis of variance is used for statistical analysis between multiple groups. A p-value of less than 0.05 is considered statistically significant.

[0040] Example 1 Preparation and characterization of rhein-chitosan conjugate

[0041] (1) Chitosan was suspended in ultrapure water and stirred for 30 min to fully swell the chitosan. The pH value of the system was adjusted to 2.5 with 1 M HCl and magnetic stirring was carried out overnight until the chitosan was completely dissolved. Rhein was dissolved in DMSO, and catalysts EDC and NHS were added. The mixture was kept in an ice bath in the dark for 30 min. The rhein solution was then added dropwise to the chitosan solution at a uniform speed. The pH value of the system was adjusted to 5.5 with 1 M NaOH. The mixture was magnetically stirred at 500 rpm for 24 h under the conditions of light-proof, stable pH and room temperature to obtain a conjugate solution. The synthetic route is shown in the figure below. Figure 1 As shown; dialyzed in ultrapure water for 72 hours, with the dialysate changed every 8 hours; the purified conjugate sample solution was freeze-dried to obtain a cotton-like substance, namely the rhein-chitosan conjugate (Rh-CS conjugate).

[0042] (2) Fourier infrared spectrometer was used to obtain the infrared spectrum of Rh-CS conjugate; 400 MHz nuclear magnetic resonance spectrometer was used to obtain the nuclear magnetic resonance hydrogen spectrum of Rh-CS conjugate; X-ray diffractometer was used to obtain the X-ray diffraction spectrum of Rh-CS conjugate under the condition of 40 kV and 40 mA; ultraviolet visible scanning spectrophotometer was used to obtain the ultraviolet visible spectrum of Rh-CS conjugate; X-ray photoelectron spectrometer was used to analyze the X-ray photoelectron spectrum of Rh-CS conjugate; scanning electron microscope was used to observe the microstructure of Rh-CS conjugate; the reaction yield and coupling efficiency of Rh-CS conjugate were calculated according to the following formula (1) and (2).

[0043] Reaction yield (%) = (actual yield / theoretical yield) x 100 (1);

[0044] Coupling efficiency (%) = (emodin in conjugate / total emodin) x 100 (2).

[0045] The results are shown in Figure 3 The results of Fourier infrared spectrum, nuclear magnetic resonance hydrogen spectrum, X-ray diffraction spectrum, ultraviolet visible spectrum and X-ray photoelectron spectrum all prove the successful occurrence of amide reaction and verify the chemical structure of Rh-CS conjugate. The scanning electron microscope spectrum shows the microstructure of Rh-CS conjugate. According to the calculation, the reaction yield and coupling efficiency of Rh-CS conjugate are (84.361 ± 3.14) % and (23.884 ± 1.15) % respectively.

[0046] Example 2 Preparation and characterization of emodin-chitosan composite nanoparticles

[0047] (1) Rh-CS conjugate was dissolved in 1% (v / v) acetic acid aqueous solution (0.75 mg / mL), and 1M NaOH aqueous solution was used to adjust the pH to 5.5; crosslinking agent sodium tripolyphosphate solution was prepared in ultrapure water (0.75 mg / mL), and 1M HCl aqueous solution was used to adjust the pH to 5.5; crosslinking agent solution was added dropwise under continuous stirring, and ultrapure water was used to make up the volume, ultrasonic treatment for 30 min, to generate emodin-chitosan composite nanoparticles (0.5 mg / mL), which was Rh-CS / TPPNPs. Chitosan-sodium tripolyphosphate nanoparticles (CS / TPPNPs) were prepared by the same method as the control.

[0048] (2) The formation process of Rh-CS / TPPNPs was tracked by turbidity experiment. Turbidity was calculated according to formula (3), where A represents the absorbance of the solution at 600 nm. Malvern particle size analyzer was used to determine the hydration particle size (D H), polydispersity index (PDI), particle size distribution, and zeta potential. The morphology of Rh-CS / TPPNPs was observed by transmission electron microscopy. The hydrophobicity of Rh-CS / TPPNPs was evaluated by contact angle (θ). Furthermore, the effects of room temperature storage, different pH values, and different NaCl concentrations on the stability of Rh-CS / TPPNPs were investigated.

[0049] Turbidity = 1-10 -A (3).

[0050] Characterization results such as Figure 4 As shown: Turbidity test results prove the formation of nanoparticles ( Figure 4 As shown in A). When V TPP When the volume reaches 500 μL, the hydrated particle size of Rh-CS / TPP nanoparticles (D H ) reaches the minimum ( Figure 4 The polydispersity coefficient is between 0.1 and 0.2 ( Figure 4 (as shown in C in the figure), which may be because the tendency of nanoparticles to aggregate and the electrostatic repulsion between cationic amino groups have been balanced. At this time, the mass ratio of TPP:Rh-CS is 0.25:1, which is considered to be the optimal cross-linking ratio. The physicochemical characteristics of CS / TPPNPs and Rh-CS / TPPNPs at the optimal cross-linking ratio are shown in Figure 2. Figure 4 As shown in D and E, the transmission electron microscopy images of Rh-CS / TPPNPs are as follows Figure 4 As shown in Figure 5, the effect of pH on the hydrated particle size, polydispersity coefficient, and zeta potential of nanoparticles is shown in Figure 5. Figure 4 As shown in GI, the effect of NaCl on the hydrated particle size, polydispersity coefficient and zeta potential of nanoparticles is as follows Figure 4 As shown in Figure 1, the above results indicate that the Rh-CS / TPPNPs described in this application have long-term storage stability, acid and alkali stability, and high salt stability.

[0051] Example 3 Evaluation of in vitro biological activity of rhein-chitosan composite nanoparticles

[0052] The in vitro biological properties of rhein (Rh), CS / TPP NPs, and Rh-CS / TPP NPs were evaluated using colon adenocarcinoma cells (Caco-2) and inflammatory-related cells (RAW264.7). Cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and penicillin-streptomycin at 37°C in an atmosphere of 5% CO2.

[0053] (1) In vitro cell viability evaluation. RAW264.7 cells (5×10 4 cells / well) and Caco-2 cells (6×10 3Cells (100 cells / well) were plated and cultured for 24 h in 96-well plates. The cells were treated with different concentrations of Rh, CS / TPPNPs, and Rh-CS / TPPNPs for 24 h. Thiazolyl blue (5 mg / mL) was then added to the culture medium and incubated for 4 h. The absorbance was measured at 490 nm using a microplate reader. Cell viability was expressed according to formula (4).

[0054] Cell viability (%) = (OD 药物处理 -OD 空白 ) / (OD 对照 -OD 空白 )×100 (4).

[0055] (2) Evaluation of live and dead cells. Caco-2 cells or RAW264.7 cells were seeded in confocal culture dishes and treated with Rh (60 μM), CS / TPPNPs (0.4 mg / mL), and Rh-CS / TPPNPs (0.4 mg / mL) for 12 h. Calcein AM solution (Ex / Em = 494 / 517 nm) and PI solution (Ex / Em = 535 / 617 nm) were added and incubated at 37°C for 30 min. After washing with PBS, live cells (Calcein AM) and dead cells (PI) were observed using a laser confocal scanning microscope.

[0056] (3) In vitro cellular uptake assessment. FITC-CS / TPPNPs were prepared using FTIC probes instead of rhein. Briefly, FITC-CS conjugates are based on the reaction product between the isothiocyanate group of FITC and the primary amino group of chitosan. Caco-2 cells or RAW264.7 cells were cultured in confocal microscopy for 12 h, then incubated with FITC-CS / TPPNPs suspension (0.5 mg / mL) for 4 h, rinsed with PBS, fixed with 4% paraformaldehyde for 15 min, stained with DAPI for 15 min, and observed under a laser confocal scanning microscope.

[0057] (4) Evaluation of anti-inflammatory activity in vitro. RAW264.7 cells were plated in 12-well plates (2 × 10 5 Cells were inoculated in 400 μg / well (100 cells / well) for 24 hours and then subjected to different treatments. The blank control group received no treatment. The model control group was treated with 1 μg / mL lipopolysaccharide (LPS). The drug-treated groups were preincubated with 5-aminosalicylic acid (5-ASA), rhein, CS / TPPNPs, and Rh-CS / TPPNPs culture media for 4 hours, followed by treatment with 1 μg / mL LPS. After 24 hours, the actual level of nitric oxide (NO) in the cell culture was measured using the Griess kit.

[0058] (5) In vitro hemolysis evaluation. A 2% red blood cell suspension was mixed with equal volumes of Rh, CS / TPP NPs, and Rh-CS / TPP NPs at different concentrations and incubated at 37°C for 1 h. Ultrapure water and PBS were used as positive and negative controls, respectively. After centrifugation, the OD value of the supernatant at 540 nm was measured using a microplate reader. The hemolysis rate was calculated according to the following formula (5).

[0059] Hemolysis rate (%) = (OD 药物处理 -OD 阴性对照 ) / (OD 阳性对照 -OD 阴性对照 )×100(5).

[0060] The experimental results are as follows Figure 5 As shown, cytotoxicity assay ( Figure 5 As shown in A), live and dead cell staining experiment ( Figure 5 As shown in B), red blood cell hemolysis test ( Figure 5 EG) all proved that the Rh-CS / TPPNPs described in this application have good biocompatibility. Figure 5 (shown in C) proves that the Rh-CS / TPPNPs described in this application are easily taken up by cells, which helps them to exert their efficacy. In addition, Rh-CS / TPPNPs can significantly inhibit the release of NO induced by LPS in RAW264.7 cells ( Figure 5 D, p < 0.01), showing good anti-inflammatory potential.

[0061] Example 4 Preparation and Characterization of Pickering Emulsion

[0062] (1) Screening of the optimal oil-to-oil ratio and emulsifier concentration for Pickering emulsions

[0063] CS / TPPNPs were used as solid emulsifiers (concentrations were 0.25, 0.5, 1.0, and 1.5 mg / mL), and fish oil was used as dispersed phase (phase volume fraction CS / TPPP Pickering emulsions (CS / TPPPEs) were prepared using a D-160 hand-cranked homogenizer at 12,000 rpm / 3 min (0.3, 0.4, 0.5, and 0.6, respectively). Ultrapure water was used as the continuous phase. The droplet diameter and microstructure of the Pickering emulsions were investigated using a particle size analyzer and electron microscopy. After storage at room temperature for 48 h, the emulsification index (6) was calculated. The optimal formulation was screened based on the characterization results.

[0064] Creaming index (%) = (supernatant height / total emulsion height) × 100 (6).

[0065] The physical and chemical characterization results of CS / TPPPEs are as follows Figure 6 As shown. Figure 6 As shown in AD, when When the value is 0.5, the emulsion system prepared by the present invention is relatively uniform; after storage for 48 hours, the creaming index (CI) value is the smallest, so is the optimal volume ratio of the two phases; Figure 6 As shown in Figures E and H, when the CS / TPPNPs concentration is 0.5 mg / mL, the emulsion system is relatively uniform and the creaming index (CI) value is the smallest. Therefore, the optimal concentration of the solid emulsifier is 0.5 mg / mL.

[0066] (2) Using the best formula screened above, Rh-CS / TPPNPs were used instead of CS / TPPNPs as a solid emulsifier to prepare Rh-CS / TPPPickering emulsions (Rh-CS / TPPPEs). The characterization method was the same as above. In addition, 0.1% Nile red (Ex / Em=552 / 636nm) was used to label fish oil, and the morphology and type of Rh-CS / TPPPEs were observed using a laser confocal scanning microscope. The constant strain was set to 0.5%, the angular frequency test range was 0.1Hz-00Hz, and the apparent viscosity of Rh-CS / TPPPEs was evaluated at 25°C using a parallel plate rheometer.

[0067] The physicochemical characterization results of Rh-CS / TPP PEs are as follows Figure 7 As shown. Among them, Figure 7 A and B are the physicochemical characteristics of CS / TPP PEs and Rh-CS / TPPPEs, respectively. Figure 7 C in the middle is the microscopic image of Rh-CS / TPP PEs. Figure 7 D is the apparent viscosity result.

[0068] The above results show that Rh-CS / TPP PEs are oil-in-water emulsions; both CS / TPP PEs and Rh-CS / TPPPEs have gel-like rheology, but the Rh-CS / TPP PEs described in this application have a stronger gel structure, stronger deformation resistance, and better emulsion stability.

[0069] (3) The optimal formulation screened above was used, Rh-CS / TPPNPs were used as a solid emulsifier, and fish oil containing rhein was used as the oil phase to prepare Rhein-loaded Rh-CS / TPP Pickering emulsion (Rhein@Rh-CS / TPP PEs).

[0070] ① The in vitro release profile of rhein from Rhein@Rh-CS / TPPPEs was evaluated using the dialysis bag diffusion technique: First, 2 mL of sample was placed in a dialysis bag (MW = 3500) and then immersed in a sealed container containing 50 mL of simulated gastric fluid (SGF). The container was placed in a thermostatic shaker at 100 rpm and 37°C for 6 h. Subsequently, the dialysis bag was transferred to another sealed container containing 50 mL of simulated intestinal fluid (SIF) and allowed to stand for another 30 h. Analysis was performed using high-performance liquid chromatography (HPLC). The mobile phase consisted of methanol-0.1% formic acid (85:15, v / v), with a flow rate of 1.0 mL / min, a column temperature of 30°C, and a detection wavelength of 254 nm.

[0071] ② To evaluate the distribution of Rhein@Rh-CS / TPPPEs in the gastrointestinal tract after oral administration, a fluorescence and bioluminescence imaging system was used for research: DiR (DiIC18(7)) fluorescent probe was encapsulated in Rh-CS / TPPPEs (0.5 mg / kg) and administered orally to C57BL / 6J mice (SPF, male, 18 g ± 2 g). The biodistribution of the fluorescent signal in the mice was observed at fixed time points (ICG filter: Ex / Em = 749-790 nm / 810-860 nm). ③ The pharmacokinetic characteristics of Rhein@Rh-CS / TPPPEs were analyzed using HPLC. Rhein@Rh-CS / TPP PEs (25 mg / kg) were administered orally to SD rats (SPF, male, 200 g ± 20 g). Blood was collected at fixed time points. The plasma was treated with methanol and 1 M HCl, centrifuged, and the supernatant was extracted with ethyl acetate. The organic solvent was recovered and the residue was dried. The dried residue was then dissolved in methanol and analyzed by HPLC. Pharmacokinetic parameters, including maximum drug concentration (C max ), time to reach maximum drug concentration (T max ), the area under the curve from time 0 to ∞ (AUC 0-∞ ).

[0072] The physicochemical characterization results of Rhein@Rh-CS / TPP PEs are shown in Figure 2. Figure 8 As shown in Figure 2. Among them, the in vitro release curve of rhein is shown in Figure 2. Figure 8 As shown in Figure A, Rhein@Rh-CS / TPPpe exhibited the best stability and sustained release in simulated gastrointestinal fluid. Figure 8 As shown in Figure B, the Rh-CS conjugate has good bioadhesion and DIR@Rh-CS / TPPPEs has better stability in the stomach and intestine, which also suggests that it has colon-targeted release properties. Figure 8 C-E, the C of Rhein@Rh-CS / TPP PEs max values were lower than other groups, and the AUC 0-∞ values were 3.529 and 2.170 times of free rhein, respectively, indicating that it can significantly improve the oral bioavailability of rhein and has a certain sustained-release effect.

[0073] Example 5 Application of Rhein Pickering Emulsion in Ulcerative Colitis Mice

[0074] C57BL / 6J mice (SPF, male, 18 g ± 2 g) were from the Experimental Animal Center of Lanzhou University (animal quality certificate: SCXK (Gan) 2023-0003). All animal experiments were performed in accordance with the guidelines of the Lanzhou University Research Animal Subjects Committee. The mice were placed in a constant temperature environment, free to drink water and eat, and were adapted to the environment for one week before treatment.

[0075] The mice were randomly divided into 12 groups (Table 1). The 5% DSS-induced UC mouse model and drug treatment flowchart are shown in Figure 2 The mice were randomly divided into 12 groups. The blank control group (BC) mice were given purified water during the experiment; the rest of the groups were given 5% DSS solution to induce UC mouse model, and after 8 days, they were changed to purified water. From the 4th day, the other groups were given different drugs (Table 1, Figure 2 ), and the BC group was given normal saline.

[0076] Table 1 Animal grouping and drug intervention scheme

[0077]

[0078] Note: The dosage of the PE group (main treatment group) is calculated based on the total amount of rhein in the preparation. The dosage of the NP group is calculated based on the mass of Rh-CS / TPP NPs contained in the PE group. The dosage of the NC group (blank preparation group) is calculated based on the dosage volume converted from the dosage of the PE-M group. The dosage of the FO group is calculated based on the volume of fish oil contained in the PE-M group.

[0079] (1) Assessment of ulcerative colitis:

[0080] Daily disease activity index (DAI) assessment was based on the scoring criteria described in Table 2. After the experiment, blood was collected, and the contents of red blood cells (RBC), white blood cells (WBC), platelets (PLT), and hemoglobin (HGB) were analyzed. The mice were sacrificed by cervical dislocation, and the colon was isolated to measure its length. Then, the colon tissue was stained with hematoxylin-eosin (H&E) for histopathological analysis.

[0081] Table 2 Disease Activity Index (DAI) Scoring Table

[0082]

[0083] The results of the effects of Rhein@Rh-CS / TPP PEs on ulcerative colitis are as follows Figure 9 As shown. From the third day of modeling, mice began to have bloody stools and diarrhea ( Figure 9 As shown in B), weight loss ( Figure 9 Compared with the MC group, the PE group significantly improved weight loss (p < 0.01, Figure 9 (shown in A), changed the upward trend of DAI score (p<0.01, Figure 9 B), inhibited colon shortening (p < 0.01, Figure 9 The PE group could alleviate the typical symptoms of UC, reduce the infiltration of inflammatory cells and tissue damage, and the Rh group and PC group had similar effects ( Figure 9 As in the PC and Rh groups, the RBC and HGB concentrations in the PE group were increased ( Figure 9 The ratio of WBC and PLT decreased ( Figure 9 (Figure 3—figure supplement 1, p < 0.05). These results further confirm that Rhein@Rh-CS / TPP PEs have a favorable therapeutic effect on UC mice. Furthermore, high-dose Rh-CS / TPP NPs also exhibited anti-UC effects.

[0084] (2) Serum biochemical analysis:

[0085] After treatment, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA) and uric acid (UA) of mice were measured to evaluate liver and kidney safety.

[0086] The safety evaluation results of serum biochemistry are as follows Figure 10 Rhein@Rh-CS / TPP PEs had no hepatotoxicity or renal toxicity. In addition, Rhein@Rh-CS / TPPPEs and high-dose Rh-CS / TPPNPs could reverse DSS-induced liver and kidney damage in UC mice.

[0087] (3) Molecular biology experiments:

[0088] ① Enzyme-linked immunosorbent assay: The levels of major inflammatory cytokines (IL-1β, IL-6, and TNF-α) in colon tissue were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.

[0089] Quantitative real-time polymerase chain reaction: Total RNA was extracted from colon tissue for real-time quantitative polymerase chain reaction (qRT-PCR) analysis. The purity and concentration of total RNA were detected by ultra-low volume nucleic acid protein analyzer. RNA was reverse transcribed into cDNA template using M5Sprint qPCR RT kit. Then, cDNA was mixed with specific primers and qPCR SYBR Green Master Mix (Low Rox Plus), and then amplified on QuantStudioTM 5 Real-Time PCR instrument. The primer sequences of NF-κB p65, TLR4 and MLCK are shown in Table 3.

[0090] Table 3 Primer sequences for qRT-PCR detection

[0091]

[0092] Immunohistochemistry: The colon tissue sections were incubated with bovine serum albumin to minimize non-specific binding. Occludin, Zonula Occludens-1 (ZO-1), Claudin-3 antibodies were added respectively, and incubated at 4°C overnight. Then secondary antibody was added, followed by 3,3'-diaminobenzidine (DAB) staining. Finally, the sections were observed under a microscope for immunohistochemical (IHC) analysis.

[0093] The results of molecular biology are shown in Figure 11 The results of ELISA experiments are shown in A-C of Figure 11 The levels of TNF-a, IL-6 and IL-1β in the MC group were significantly higher than those in the BC group (p<0.01), indicating that the UC mouse model was successfully constructed; PC, Rh and PE groups could significantly inhibit the increase of these cytokines (p<0.05), proving that they could alleviate the inflammatory response of UC mice; the levels of TNF-a and IL-6 in the colon tissue of NP-H group mice were significantly reduced (p<0.05). The above results suggest that Rhein@Rh-CS / TPPPEs and high-dose Rh-CS / TPPNPs have anti-UC effects. The results of qRT-PCR experiments are shown in D-F of Figure 11 PC, Rh and PE groups could reverse the increase of NF-κB p65, TLR4 and MLCK mRNA levels in the colon tissue of UC mice (p<0.05), which might inhibit the activation of TLR4 / NF-κB / MLCK signaling pathway, regulate the overexpression of inflammation-related genes, and thus alleviate the inflammatory development and pathological process of UC; NP-H group significantly reduced the levels of TLR4 and NF-κB (p<0.05), suggesting that high-dose Rh-CS / TPPNPs might affect the TLR4 / NF-κB pathway. The results of IHC experiments are shown in Figure 11As shown in Figures GJ, the expressions of cludin-3, occludin, and ZO-1 were significantly upregulated in the PE-M and PE-H groups (p<0.05), indicating that these interventions may have the effect of repairing the "holes" in the intestinal epithelial barrier, thereby reducing the leakage of harmful substances into the intestine and alleviating the intestinal inflammatory response in UC mice.

[0094] (4) Evaluation of nutrients in mouse feces:

[0095] To verify whether Rhein@Rh-CS / TPPPEs can effectively prevent rhein-induced intestinal nutrient loss, we investigated the effects of different doses of rhein on fecal nutrient levels in UC mice. Mice in the BC, Rh, PE-H, PE-M, and PE-L groups were placed in metabolic cages to collect fresh feces. Fecal samples were dried, uniformly ground, and stored at -80°C. The fecal contents of total polysaccharides, total protein, and total fat were compared among the groups using the phenol-sulfuric acid method, the Coomassie brilliant blue method, and the acid hydrolysis method.

[0096] Evaluation results of nutrients in mouse feces Figure 12 Compared with the BC group, the total polysaccharide, total protein, and total fat contents in the Rh group were significantly increased (p < 0.05), indicating that rhein affected the intestinal absorption function and nutrient metabolism of mice, causing some nutrients to be excreted in feces. Compared with the Rh group, the PE group significantly reduced the total polysaccharide, total protein, and total fat contents in the mouse feces (p < 0.05), indicating that Rhein@Rh-CS / TPP PEs effectively reduced the intestinal irritation side effects caused by rhein, suggesting its wider application in the treatment of UC.

[0097] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A rhein-chitosan composite nanoparticle, characterized in that: The preparation method of the rhein-chitosan composite nanoparticles comprises: (1) coupling rhein and chitosan to prepare a rhein-chitosan conjugate; (2) The rhein-chitosan conjugate was treated with sodium tripolyphosphate solution as a crosslinking agent to obtain rhein-chitosan composite nanoparticles.

2. The rhein-chitosan composite nanoparticles according to claim 1, wherein The step (1) comprises: dissolving rhein in DMSO, adding a catalyst, keeping the mixture in an ice bath in the dark for 30 minutes, dripping the mixture into a chitosan solution, adjusting the pH to 5.5, stirring and reacting for 24 hours to obtain a rhein-chitosan conjugate solution; and dialysis and purification to obtain a rhein-chitosan conjugate; wherein the catalyst is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or N-hydroxysuccinimide.

3. The rhein-chitosan composite nanoparticles according to claim 2, wherein The chitosan solution is prepared by suspending chitosan in ultrapure water, stirring for 30 minutes to fully swell the chitosan, and adjusting the pH value to 2.5 to completely dissolve the chitosan.

4. The rhein-chitosan composite nanoparticles according to claim 3, wherein The molar mass ratio of the chitosan amino group to the rhein carboxyl group is 5:2; the dialysis is performed for 72 hours, the dialysate is changed every 8 hours, the pH of the dialysate is 5.5, and the volume ratio of the dialysate to the sample solution is 100:

1.

5. The rhein-chitosan composite nanoparticles according to claim 1, wherein The step (2) comprises: dissolving the rhein-chitosan conjugate in an acetic acid aqueous solution and adjusting the pH to 5.5; dripping a sodium tripolyphosphate aqueous solution with a pH of 5.5 into the rhein-chitosan conjugate solution, and ultrasonicating for 30 minutes to generate rhein-chitosan composite nanoparticles.

6. The rhein-chitosan composite nanoparticles according to claim 5, wherein The concentration of the acetic acid solution is 1% (v / v); the mass ratio of the sodium tripolyphosphate to the rhein-chitosan conjugate is 0.25:

1.

7. Use of the rhein-chitosan composite nanoparticles according to any one of claims 1 to 6 in the preparation of rhein Pickering emulsion.

8. A rhein Pickering emulsion, characterized in that: The preparation method of the rhein Pickering emulsion comprises: dissolving rhein in fish oil as the oil phase, using the rhein-chitosan composite nanoparticles according to any one of claims 1 to 6 as a solid stabilizer, and emulsifying to obtain the rhein-loaded Pickering emulsion.

9. The rhein Pickering emulsion according to claim 8, wherein The phase volume fraction is 0.5, the concentration of rhein-chitosan composite nanoparticles is 0.5 mg / mL; and the emulsification condition is 12000 rpm / 3 min.

10. Use of the rhein Pickering emulsion according to claim 8 or 9 in preparing a medicament for treating ulcerative colitis.

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