Oral targeted nano delivery system containing geniposide and preparation method and application thereof

Abstract

The application discloses an oral targeted nano delivery system containing gardenoside as well as a preparation method and application of the oral targeted nano delivery system, and particularly relates to the technical field of medicines.The method comprises the following steps: synthesizing MSNs by a Stober method; after removing a template, further modifying amino groups on the surface of the MSNs to obtain MSN@NH2; adding HA activated by NHS / EDC into a suspension of the MSN@NH2 to obtain MSN-HA through reaction; preparing Chi / Alg@MSN-HA NPs by using the MSN-HA, a chitosan solution and sodium alginate; and loading gardenoside into the Chi / Alg@MSN-HA NPs according to a proportion to prepare gardenoside-loaded Chi / Alg-GE@MSN-HA NPs.The nano targeted delivery system is constructed, a drug administration site is changed, local positioning release of the gardenoside in the colon is realized, the anti-UC (ulcerative colitis) efficacy is improved, and a solution to the safety problem of the gardenoside is provided.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to an oral targeted nanodelivery system containing geniposide, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease characterized by insidious pathogenesis, diarrhea, hematochezia, and intestinal damage. In traditional Chinese medicine (TCM) diagnosis, UC is predominantly classified as damp-heat accumulation in the intestines. Gardenia (Gardenia jasminoides Ellis), a plant in the Rubiaceae family, is a traditional Chinese medicine. According to the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), gardenia is bitter and cold in nature, entering the heart, lung, and triple burner meridians. It has the effects of purging fire and relieving irritability, clearing heat and promoting diuresis, cooling blood and detoxifying, and is used for damp-heat jaundice, carbuncles, etc. Geniposide (GE) is the main active ingredient in gardenia that exerts its anti-inflammatory and other pharmacological effects. Many scholars have conducted basic research on the pharmacodynamic activity and toxicology of geniposide, demonstrating its potential application value in UC. However, rapid degradation after oral administration leads to a decrease in the amount of drug reaching the colon, thus affecting its efficacy, and increasing the dosage may lead to hepatotoxicity. Wang Enli et al.'s research comparing the toxicity of geniposide administered via different routes indicated that gavage showed greater hepatotoxicity than injection, possibly related to its breakdown into genipin in the gastrointestinal tract. Furthermore, oral administration of geniposide for treating colitis often results in higher doses, significantly increasing its hepatotoxicity. Feng Xiaoyi et al.'s research also indicated that high doses of geniposide can lead to kidney damage. Currently, geniposide is primarily administered via enema for colitis treatment, but this is inconvenient. Therefore, finding an oral geniposide treatment method for colitis without compromising its efficacy remains a pressing problem.

[0003] Chinese patent CN 107865822 A discloses a method for preparing and applying a hydrogel drug sustained-release carrier material incorporating nanoporous silica. This patent also mentions using gelatin (GE), sodium alginate (SA), and nanoporous silica (MSN) as raw materials, first using Ca... 2+ A GE-SA semi-interpenetrating composite hydrogel containing MSN was prepared by physical cross-linking. The hydrogel was then modified with a polyelectrolyte membrane using chitosan CS to obtain a stable GE-SA-CS(MSN) composite hydrogel. The sustained-release effect of this composite hydrogel material on drugs was investigated using BSA, MH, and IDM as model drugs. However, based on the patented drug carrier, it is known that this carrier is mainly enriched in the intestine and cannot target the colon for colon-targeted drug delivery.

[0004] Chinese patent CN111265670A discloses a mesoporous silica carrier coated with galactosylated chitosan and its application. Based on the mesoporous silica carrier, and combined with galactosylated chitosan (GC), it achieves 5... A combined targeted therapy for colorectal cancer using fluorouracil (5-FU) and leucovorin (LV). While this patented treatment can also target the colon, it targets tumor cells in the colon and cannot target CD44 macrophages at sites of colon inflammation.

[0005] Chinese Patent CN 118806914 A discloses a drug delivery system, its preparation method, and its application. The drug delivery system comprises mucin, genipin, an oil phase, and an emulsifier. The preparation method includes: (1) mixing the oil phase and the emulsifier to obtain a mixture 1; (2) emulsifying a mixture 1 with an aqueous solution containing mucin to obtain a mixture 2; and (3) performing a cross-linking reaction between mixture 2 and a solution containing genipin to obtain a mixture containing the drug delivery system. Genipin is a metabolite of genipin and is used as part of the drug delivery system. However, genipin cannot be cross-linked with this drug delivery system, thus preventing genipin from targeting the colonic portion for efficacy. Therefore, a new colonic drug delivery system capable of targeting genipin-loaded drugs needs to be found. Summary of the Invention

[0006] Therefore, this invention provides an oral targeted nanodelivery system containing geniposide, its preparation method, and its applications to address the problems in the prior art. The oral colon-targeted delivery system of this invention, as an alternative to enemas, aims to improve compliance and targeting.

[0007] This invention utilizes the Stobol method to synthesize MSNs and construct a nano-targeted delivery system. Its advantages include: a highly efficient and simple preparation process under mild conditions, suitable for large-scale production; bio-inert silica excipients that meet FDA pharmaceutical excipient standards and are suitable for in vivo application; and an internal mesoporous framework that imparts an ultra-high specific surface area (954.37 m² / g) to the carrier, significantly increasing drug loading. Both the outer shell (hyaluronic acid layer) and the sodium alginate / chitosan layer are natural polysaccharides with good biocompatibility. The prepared Chi / Alg-GE@MSN-HA NPs exhibit uniform particle size, high encapsulation efficiency, and high drug loading. After oral administration, they overcome the physiological barriers of the upper gastrointestinal tract, sequentially targeting colonic lesions and nearby macrophages, significantly improving retention at the colonic target site and effectively enhancing the efficacy of geniposide in treating ulcerative colitis. This provides a new strategy for improving the bioavailability of geniposide and for precise treatment of UC.

[0008] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a method for preparing an oral targeted nanodelivery system containing geniposide is provided, the method comprising: Step 1: Fabrication of the targeted nanodelivery system MSNs were synthesized by the Stober method; after template removal, amino groups were further modified on the surface of MSNs to obtain MSN@NH2; HA activated by NHS / EDC was added to the MSN@NH2 suspension to obtain MSN-HA; Chi / Alg@MSN-HANPs were prepared using MSN-HA, chitosan solution and sodium alginate. Step 2: Preparation method of encapsulating geniposide Chi / Alg-GE@MSN-HANPs Gardenoside was loaded with Chi / Alg@MSN-HANPs in a certain ratio to prepare Chi / Alg-GE@MSN-HANPs loaded with gardenoside.

[0009] Furthermore, in step one, the method for synthesizing MSNs using the Stober method includes using CTAB as a template, TMB as a pore-forming and expanding agent, and TEOS as a silicon source.

[0010] Specifically, CTAB is dissolved in deionized water, and sodium hydroxide is added to the solution. Once CTAB is completely dissolved, an appropriate amount of TMB is rapidly added while stirring continuously. Then, an appropriate amount of tetraethyl orthosilicate is added dropwise using a separatory funnel. The particles are collected by centrifugation and washed with methanol. To remove the template, the synthesized material (CTAB@MSN) is dispersed in a hydrochloric acid / methanol mixture and stirred overnight. The particles are collected by centrifugation, washed with methanol, and dried. Preferably, the overnight stirring temperature is 55-65°C; the drying temperature is 75-85°C.

[0011] Furthermore, in step one, the method for further modifying the MSNs surface with amino groups includes: first dispersing the MSNs in methanol, then adding APTES, reacting overnight at room temperature, centrifuging the obtained product, washing it with methanol, and drying it to obtain MSN@NH2.

[0012] Furthermore, the MSNs:methanol:APTES ratio is 0.8-1.2g:0.8-1.2mL:0.8-1.2μL. As an example, the MSNs:methanol:APTES ratio is preferably 1g:1mL:1μL.

[0013] Furthermore, in step one, the MSN@NH2 suspension is obtained by dispersing MSN@NH2 in MES buffer.

[0014] More specifically: 50 mg of the obtained MSN-NH2 was weighed and added to MES buffer (adjusted to pH 5.5), stirred at 400 rpm for 2 hours at room temperature to obtain solution A. Separately, 30 mg of hyaluronic acid was weighed and added to 6 mL of MES buffer (adjusted to pH 5.5), stirred at 300 rpm for 1 hour at room temperature until fully dissolved. 144 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 86.4 mg of N-hydroxysuccinimide (NHS) were added, and stirred at room temperature for 1 hour to activate the carboxyl group of hyaluronic acid to obtain solution B. Solution A and solution B were mixed at a volume ratio of 1:1 and stirred at 300 rpm at room temperature for 24 hours. The obtained MSN-HA NPs were washed three times with deionized water, freeze-dried in a lyophilizer for 48 hours, and then collected and stored at -20℃.

[0015] Furthermore, in step one, the preparation method of Chi / Alg@MSN-HANPs includes first dispersing MSN-HA in a chitosan solution, stirring and reacting at room temperature, suspending the product after reaction in a sodium alginate solution, stirring and reacting, and centrifuging to obtain Chi / Alg@MSN-HANPs.

[0016] Furthermore, in step two, the preparation method of Chi / Alg-GE@MSN-HANPs loaded with geniposide includes: adding geniposide and nanocarrier Chi / Alg@MSN-HA NPs in proportion, dispersing them in anhydrous ethanol, sonicating, stirring magnetically overnight at room temperature, centrifuging, washing repeatedly with deionized water and anhydrous ethanol, and freeze-drying to obtain Chi / Alg-GE@MSN-HA NPs containing geniposide.

[0017] Furthermore, in step two, the ratio of geniposide to Chi / Alg@MSN-HANPs is 1:10-10:1.

[0018] This invention utilizes a modified Stober method to synthesize mesoporous silica nanoparticles, combined with natural polysaccharide hyaluronic acid modification and a chitosan / sodium alginate hydrogel coating, to prepare an oral drug delivery system that actively targets and adheres to the site of colonic inflammation to release drugs while avoiding degradation in the upper gastrointestinal tract after oral administration.

[0019] According to a second aspect of the present invention, an oral targeted nanodelivery system containing geniposide is provided, wherein the system is prepared by the method described above.

[0020] The application of an oral targeted nanodelivery system containing geniposide provided by a third aspect of the present invention in the preparation of a treatment for ulcerative colitis.

[0021] The present invention has the following advantages: This invention constructs a nano-targeted delivery system to alter the drug delivery site, thereby achieving localized release of geniposide in the colon, improving the efficacy of anti-UC drugs, and providing a solution to address the safety issues of geniposide.

[0022] This study utilizes a modified Stober method to synthesize mesoporous silica nanoparticles, combined with natural polysaccharide hyaluronic acid modification and a chitosan / sodium alginate hydrogel coating, to prepare an oral drug delivery system that actively targets and adheres to the site of colonic inflammation to release drugs while avoiding degradation in the upper gastrointestinal tract after oral administration.

[0023] The oral targeted nanodelivery system containing geniposide prepared in this invention can replace traditional enema administration and can greatly improve the compliance of oral administration of geniposide and the targeting of colonic therapy.

[0024] The nanodelivery system of this invention can achieve slow drug release, maintain effective drug concentration in the body, and prolong the duration of drug action. This is of great significance for the treatment of chronic inflammatory diseases such as ulcerative colitis, and can better control the progression of the disease.

[0025] The geniposide of the present invention is well protected in the nanodelivery system, which can prevent its degradation and inactivation in the gastrointestinal tract, improve the stability and bioavailability of the drug, and ensure that the drug can effectively exert its therapeutic effect.

[0026] The present invention exhibits significant efficacy and is safe and non-toxic. It can improve the physical signs of mice, reduce DAI scores, and significantly improve UC symptoms such as colonic shortening, hematochezia, and diarrhea. HE results also show that it can alleviate colonic inflammation. No significant toxic pathological changes were observed in its major organs. It has high targeting efficiency in vitro and in vivo, and demonstrates good anti-UC effects in the in vivo efficacy evaluation of DSS-induced UC mouse models. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0028] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0029] Figure 1 Transmission electron microscopy (TEM) characterization and particle size statistics of mesoporous silica at different reaction temperatures provided by this invention; Figure 2 Transmission electron microscopy (TEM) characterization and particle size statistics of mesoporous silica under different molar amounts of pore-expanding agent provided by the present invention. Figure 3 The present invention provides BET analysis of the adsorption-desorption curves and pore size distribution curves of MSN in various nanoparticles under different reaction temperatures and pore-expanding agent dosages; wherein, A-60℃; B-70℃; C-80℃; D-90℃; E-T1: E-TMB / MSN=1:1; F-T2: E-TMB / MSN=2:1; G-T4: E-TMB / MSN=4:1; Figure 4 The SEM, particle size, and zeta potential of the nano-drug delivery system provided by this invention are shown in Figure C. A - SEM results of the nanocarrier Chi / Alg-MSN-HA; B - particle size results of MSN, MSN-HA, and Chi / Alg-MSN-HA; C - zeta potential results of different intermediates during the preparation of the nanocarrier. In Figure C, the letter C on the horizontal axis represents the chitosan coating, A represents the sodium alginate coating, and the subscript numbers represent the number of self-assembled coating layers. Figure 5 This is a schematic diagram of Chi / Alg-GE@MSN-HA NPs provided by the present invention; Figure 6 Fourier transform infrared spectral characterization of MSN, MSN-NH2, MSN-HA, Chi / Alg-MSN-HA, Chi / Alg-GE@MSN-HANPs and GE provided for this invention; Figure 7 Test results of drug loading rate and encapsulation rate of Chi / Alg-GE@MSN-HA with different feed ratios provided in Example 1 of the present invention (n=3). Figure 8 The particle size and zeta potential changes (n=6) of Chi / Alg-GE@MSN-HA NPs sampled and detected at different time points during long-term cryopreservation of the Chi / Alg-GE@MSN-HA NPs provided in Example 2 of the present invention. Figure 9 The concentration standard curve of geniposide solution provided in Example 2 of this invention; Figure 10 The liquid chromatogram provided for Example 2 of this invention; A - blank solvent; B - standard geniposide solution; Figure 11The cumulative release curves (n=6) of GE@MSN-HANPs and Chi / Alg-GE@MSN-HA NPs were detected under in vitro simulated gastrointestinal environment conditions provided in Example 2 of the present invention. Figure 12 Cytotoxicity evaluation of MSN NPs, MSN-HA NPs and Chi / Alg-MSN-HA NPs provided in Example 2 of this invention (n=6); Figure 13 Example 2 of this invention provides an in vitro fluorescence confocal analysis of the uptake and distribution of free C6, C6@MSN-HA NPs and Chi / Alg-C6@MSN-HA NPs in an LPS-induced RAW264.7 inflammatory cell model after incubation for 30 min, 1 h, 2 h and 4 h (n=6). Figure 14 The colon-targeting verification results of the oral nano-drug delivery system Chi / Alg-Dir@MSN-HA provided in Example 2 of this invention in mice: (A) In vivo imaging images of mice after gavage administration of free coumarin 6, Dir@MSN-HA, and Chi / Alg-Dir@MSN-HA within 0-24 h; and (B) In vivo imaging fluorescence statistics at different time points (n=6); (Compared with the Free Dir group, p <0.05, and p <0.001); Figure 15 In vitro imaging images (n=3) of the colon isolated from mice after oral administration of free coumarin 6, Dir@MSN-HA and Chi / Alg-Dir@MSN-HA for 24 hours, provided in Example 2 of this invention. Figure 16 The in vivo efficacy evaluation of Chi / Alg-GE@MSN-HA provided in Example 2 of this invention includes: A - body weight change curve of mice during the administration observation period; B - disease activity index (DAI) score of mice; C - colon length statistics of mice in each group; D - colon images (n=6); (compared with Control group, p <0.05, p <0.01, and p <0.001; Compared with the Model, #p <0.05, ## p <0.01, and ### p <0.001); Figure 17 Organ coefficient evaluation of UC mice after administration of drugs in each treatment group provided in Example 2 of the present invention (n=6). Figure 18 Pathological paraffin sections of the colons of mice in each treatment group provided in Example 2 of this invention, wherein (A) H&E staining and (B) pathological scores (n=6) (compared with Control), p <0.001; compared with Model, # p <0.05, ## p <0.01, ### p <0.001. Detailed Implementation

[0030] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] 1. Optimization Results and Analysis of Materials Mesoporous silica (MSN) is a high-quality drug delivery matrix material. Its drug loading properties and in vivo drug release process are closely related to its structural characteristics, including the structural arrangement regularity of the material matrix, particle size, polydispersity index (PDI), zeta potential, pore size distribution, specific surface area, and pore volume. Reaction temperature and the amount of pore expander are key factors affecting the structural characteristics of mesoporous silica. The PDI index is the uniformity index of particle size; the more uniform the particle size, the more stable the properties. Zeta potential is an important indicator reflecting the surface charge of nanoparticles and has a significant impact on dispersibility and subsequent modification. The larger the negative zeta potential of MSN, the more stable its dispersion in solution, which is more conducive to subsequent modification.

[0032] Using reaction temperature and pore-expanding agent dosage as conditional variables, the preparation process of mesoporous silica was optimized by observing morphology, particle size, and zeta potential using transmission electron microscopy (TEM). TEM results are shown below. Figure 1 The results showed that at a reaction temperature of 60℃, the mesoporous silica framework was indistinct, and crystal growth was still in its early stages, without a regular mesoporous arrangement and framework structure, which could not meet the requirements for efficient preparation. As the reaction temperature increased, the particle size of the mesoporous silica gradually increased. When the reaction temperature was 70-90℃, the mesoporous arrangement of the silica became clearer, and the mesopore size increased significantly. TEM particle size statistics showed that at reaction temperatures of 60, 70, 80, and 90℃, the average particle sizes were 64.21±19.13 nm, 166.83±32.96 nm, 133.54±20.65 nm, and 134.85±27.90 nm, respectively. At a reaction temperature of 70℃, although the internal framework of mesopores is regularly arranged and has good roundness, the presence of spiky edges may be detrimental to subsequent modification. At a reaction temperature of 90℃, the surface mesopores are significantly enlarged and uniformly arranged, but some structures in the outer layer collapse. This may be because higher temperatures exacerbate the Brownian motion and collisions of the reacting molecules, which may also be detrimental to the final stage of surface growth of the mesoporous material. Conversely, at a temperature of 80℃, the mesopores are uniformly arranged, and the final stage of surface growth of the mesoporous material is relatively complete, without local structural collapse. Meanwhile, as shown in Table 1, the results of Malvern particle size, zeta potential, and PDI value indicate that when the reaction temperature is 80℃, the obtained nanoparticle size is 120.34nm, which is less than 200nm, which is beneficial for overcoming drug delivery barriers. The surface zeta potential is -35.39mV, and the absolute value is greater than 30mV, which is beneficial for dispersion and stability in the solution system. The PDI is 0.18, which is less than 0.20, indicating that the nanoparticle size is well uniform, which is beneficial for achieving more stable drug loading and release. Therefore, based on the TEM characterization results of the synthesis of mesoporous silica, the morphology and structure, as well as the Malvern particle size, potential, and PDI, the optimal reaction temperature is selected as 80℃.

[0033] Table 1

[0034] The amount of pore expander TMB can, on the one hand, alter the mesoporous pore size of mesoporous silica, thus affecting drug loading and release behavior; on the other hand, it may influence the structural properties of mesoporous silica. Results for different TMB / MSN ratios are shown in Table 2.

[0035] Table 2

[0036] TEM results showed that when the pore-expanding agent TMB / MSN (mol / mol) ratio was 1:2, the mesoporous channels of MSN were blurred, and their shape was between long columnar and elliptical, with a major axis to minor axis length ratio of approximately 2:1. As the amount of pore-expanding agent continued to increase, the roundness gradually improved. This indicates that the amount of pore-expanding agent not only affects the mesopore size but also alters the shape of the mesoporous nanoparticles, which will enrich the preparation requirements for different drug delivery functions. When the pore-expanding agent ratio TMB / MSN (mol / mol) was 1:1, 2:1, and 4:1, the mesopores were regularly arranged and had good roundness. The average particle sizes under TEM were 85.92±7.76 nm, 163.80±21.92 nm, 109.92±18.31 nm, and 119.46±20.37 nm, respectively. The microscopic results showed that the uniformity of the particles was in the order T4 < T1 / 2 < T1 < T2. Figure 2 Meanwhile, the Malvern particle size, zeta potential, and PDI values ​​showed that when the TMB / MSN (mol / mol) ratio was 1:1 and 2:1, the PDI value was less than 0.20, and at 1:2, the particle size was even smaller at 125.17±1.18 nm, which is similar to the results under TEM. After using the pore-expanding agent, the absolute value of the zeta potential of the nanoparticles increased, indicating that the surface charge distribution of the expanded MSN was more uniform and the charge was greater, which is beneficial to the dispersion stability in the solvent system. When the TMB / MSN (mol / mol) ratio was 1:1 and 2:1, the absolute value of the zeta potential was >30 mV, especially when the ratio was 1:2, the zeta potential was -38.17±0.61 mV (as shown in Table 2). Therefore, the optimal condition for the amount of pore-expanding agent was a TMB / MSN (mol / mol) ratio of 2:1.

[0037] Currently reported MSN types mainly include the MCM series (such as MCM-41, MCM-48, etc.) and the SBA series (SBA-15, SBA-16, etc.). The mesoporous silica synthesized using the Stöber method in this study differs from these types, exhibiting short-range ordering in its internal pore size distribution and channel shape rather than long-range ordering. Therefore, this study further explored the drug-loading potential of MSN nanoparticles under optimized conditions: a TMB / MSN (mol / mol) ratio of 2:1 and a synthesis temperature of 80℃. In this experiment, BET technology was used to measure the specific surface area, pore volume, and average pore size of the nanoparticles under different initial conditions, and adsorption-desorption curves were plotted to compare their effects on drug loading properties. Since the MSN nanoparticles exhibited irregular shapes between elongated columnar and elliptical forms under a TMB / MSN (mol / mol) ratio of 1:2, which are unsuitable for later drug loading applications, BET testing was only performed on MSN under other conditions. Figure 3AG shows the BET adsorption-desorption curves and pore size distribution curves of MSN under different conditions.

[0038] BET results show that the adsorption-desorption curves and pore size distribution curves at a reaction temperature of 60℃ exhibit significant low relative pressure hysteresis, with steep curves and narrow hysteresis loops, belonging to the H4 type hysteresis loop. This indicates that the particles have a mixed structure of mesoporous and microporous structures. The steep curves, the absence of obvious plateau regions, and the relatively narrow hysteresis loops reflect the narrowness and non-uniformity of the material's pore structure, which is consistent with the results observed by TEM. Figure 3 The hysteresis loop of C belongs to the H2 type, indicating that the particles have an ink bottle-like mesoporous structure with a large internal space. This provides numerous storage sites for drug molecules, enabling the particle to accommodate more molecules and thus achieve a higher drug loading capacity. This helps improve the therapeutic effect of the drug delivery system and reduce the frequency of dosing. Furthermore, materials with this mesoporous structure generally exhibit good biocompatibility. Simultaneously, its stable porous structure does not rapidly decompose or release large amounts of drug in vivo, but rather exerts its effect in a sustained-release manner, further improving the safety and reliability of the drug loading system. Figure 3 The F-hysteresis ring belongs to the H3 type, indicating that it possesses slit-like mesopores. These particles typically exhibit high drug loading potential, good sustained-release properties, and drug stability. BET analysis of the specific surface area, pore volume, and average pore size of each nanoparticle (MSN) at different reaction temperatures and pore expander dosages showed that the specific surface area first increased and then decreased with increasing reaction temperature. At a reaction temperature of 80℃, the BET (m 2 / g) reached a maximum of 745.98m 2 / g, and the pore volume also reaches a maximum of 1.07m. 3 The specific surface area is 5.98 nm / g, and the pore size is 5.98 nm. Regarding the amount of pore expander, the specific surface area first increases and then decreases with increasing TMB dosage. The maximum specific surface area reaches 954.37 m² / g when the reaction temperature is 80℃ and the TMB / MSN ratio is 2:1. 2 / g, with a pore volume of 1.16m³. 3 / g, with a pore size of 4.13nm, indicates that this condition is most favorable for drug loading. A large specific surface area and appropriate pore volume and pore size, typically mesoporous materials with pore sizes of 2-7nm have good drug loading characteristics (as shown in Table 3).

[0039] Table 3

[0040] SEM results showed that the Chi / Alg-MSN-HA nanocarrier consisted of spherical nanoparticles with good monodispersity and no obvious agglomeration. The particle size was approximately 122.18 nm, with uniformly distributed dot-like protrusions on the surface and a relatively uniform size distribution. Figure 4 A). Particle size analysis results showed that MSN, MSN-HA, and Chi / Alg-MSN-HA had particle sizes of 128.15 nm, 148.11 nm, and 158.17 nm, respectively. This indicates that the modification of the outer hyaluronic acid layer and sodium alginate / chitosan layer of MSN was successful and had an effect on increasing the particle size, but it still remained within the <200 nm range, thus retaining a good nanoscale effect, which is beneficial for overcoming biological barriers to drug delivery. Figure 4 B). Zeta potential results showed that the initial surface zeta potential of MSN was approximately -33.03 mV, and after surface amination, it was approximately 31.4 mV. The zeta potential of MSN-HA after hyaluronic acid functionalization was approximately -30.5 mV. To prevent degradation of the nanoparticles in the upper digestive tract after oral administration, sodium alginate and chitosan were added to the outer coating. During three cycles of coating, the surface potential also changed accordingly, from... Figure 4 As can be seen from the results in C, the absolute value of the surface potential also gradually increases, and the zeta potential of the Chi / Alg-MSN-HA surface after the final coating reaches about -34.13±1.35mv, which is beneficial to the dispersion stability in the solvent system.

[0041] 2. Fourier transform infrared spectroscopy characterization and analysis of the nanodelivery system The modification and functional groups of the nanocarrier were confirmed by FTIR. Figure 6 The FTIR spectra of MSN-NH2, MSN-HA, Chi / Alg-MSN-HA, Chi / Alg-GE@MSN-HA NPs, and GE were compared. (1089 cm⁻¹) 1 804cm 1 The absorption peak at 804 cm⁻¹ is attributed to the framework vibration peak of Si-O-Si. 1 The short peak is Si-OH, and all of the above are characteristic peaks of mesoporous silica. Located at 2929 cm⁻¹. 1 The absorption peaks at 1645 and 1470 cm⁻¹ are inferred to be due to the CH stretching of the propylamide group. 1 The absorption peak at 1552 cm⁻¹ is attributed to the NH bending vibration of the amino group. The MSN-NH₂ sample shows an absorption peak at 1552 cm⁻¹. 1 There is a band at point N, which disappears after Chi functionalization and is reclassified as N. H-curvature (δN) (H), the above proves that the amino group has been modified into MSN, and MSN-NH2 has been successfully synthesized. Located at 3395 cm⁻¹ 1The strong peaks around 945 cm⁻¹ are attributed to the hydroxyl absorption peaks of hyaluronic acid. 1 The absorption peak at 1632 cm⁻¹ is attributed to the antisymmetric out-plane vibration of the hyaluronic acid sugar ring. 1 1406cm 1 The antisymmetric and symmetric stretching vibration peaks of the -COO- group, attributed to hyaluronic acid, confirm the functionalization of hyaluronic acid. Located at 3422 cm⁻¹ 1 The broad peak at 2925 and 2851 cm⁻¹ is attributed to the large number of hydroxyl groups on the surface of the nanoparticles grafted with sodium alginate and hyaluronic acid. The absorption peaks at 2925 and 2851 cm⁻¹ are attributed to the vibrational absorption peaks of the methylene groups in sodium alginate. 1 The absorption peak at 1422 cm⁻¹ is likely due to the C=O vibration of sodium alginate carboxylic acid -COO-. 1 The absorption peak at 3520 cm⁻¹ is the vibrational absorption peak of the methyl groups in chitosan. This peak information confirms that the sodium alginate-chitosan outer shell was successfully modified onto the surface of the nanoparticles. 1 The sharp peaks around the left and right are attributed to the stretching vibrations of the intramolecular hydrogen bond -OH in geniposide, located at 1710 cm⁻¹. 1 The strong peaks around 1641 cm⁻¹ are attributed to the stretching vibration of the carbonyl group of geniposide. 1 The peaks on the left and right are attributed to the -C=C- stretching vibration of geniposide. Meanwhile, the characteristic peaks of the carrier are slightly shifted due to the interaction with geniposide. The above peak information confirms that the drug geniposide was successfully loaded into the nanoparticles.

[0042] 3. Establishment of a method for determining geniposide content Following the method for determining geniposide in the Chinese Pharmacopoeia, the chromatographic conditions used were as follows: octadecylsilane-bonded silica gel as the stationary phase (CAPCELLPAK C18MGⅡ, 4.6 mm × 100 mm column); water as mobile phase A and acetonitrile as mobile phase B, with a water:acetonitrile ratio of 15:85; flow rate of 0.6 mL / min; detection wavelength of 238 nm; and injection volume of 10 μL. The theoretical plate number was set at a peak value of at least 1500 for geniposide. Methodological investigations were conducted according to the Chinese Pharmacopoeia, including linearity, precision, stability, repeatability, and recovery.

[0043] Example 1 This embodiment provides a method for preparing an oral targeted nanodelivery system containing geniposide: 1. Preparation of mesoporous silica MSN Dissolve 750 mg cetyltrimethylammonium bromide (CTAB) (2.05 mmol) in 360 mL of deionized water and stir magnetically at 400 rpm at room temperature (using a 50 mm diameter rotor). Use a 17mm Type A (olive-shaped) stir bar for 20 minutes until completely dissolved, and then add 0.21g of sodium hydroxide to the solution. After CTAB is completely dissolved, quickly add an appropriate amount of 1,3,5-trimethylbenzene (TMB) and stir continuously. Adjust the stirring speed to the target temperature of 80°C, and continue heating with magnetic stirring for 2 hours. Then, allow it to cool naturally to room temperature.

[0044] Subsequently, 3.75 mL of tetraethyl orthosilicate was added dropwise at 1 drop / 5 s using a separatory funnel. The mixture was centrifuged at 12000 rpm at 4°C using a low-temperature high-speed centrifuge, the supernatant was discarded, deionized water was added, and the mixture was vortexed at 100 rpm for 5 min. After another centrifugation at 12000 rpm for 15 min, the supernatant was discarded. The mixture was then repeatedly washed three times with deionized water and methanol to remove residual reactants. The resulting lower precipitate was MSN NPs, which was vacuum dried at 60°C for 24 h. The MSN NPs were then baked in a muffle furnace at 550°C for 6 h, allowed to cool naturally to room temperature, resuspended in deionized water, and vortexed at 1000 rpm for 5 min each time. After centrifugation at 12000 rpm for 10 min, the supernatant containing impurities was discarded. This process was repeated three times to obtain mesoporous silica MSN (MSN NPs).

[0045] 2. Preparation of MSN-NH2 MSN NPs were dispersed in methanol, and then 3-aminopropyltriethoxysilane (APTES) was added to the mixture (MSNs / methanol / APTES = 1 g : 1 mL : 10 μL), and the mixture was magnetically stirred overnight at 300 rpm at room temperature. The resulting MSN-NH2NPs were centrifuged at 12000 rpm for 10 min, washed with methanol, and dried under reduced pressure at 60 °C overnight.

[0046] 3. Preparation of MSN-HA 50 mg of the obtained MSN-NH2 was weighed and added to MES buffer (adjusted to pH 5.5). After stirring at 400 rpm for 2 hours at room temperature, solution A was obtained. Separately, 30 mg of hyaluronic acid was weighed and added to 6 mL of MES buffer (adjusted to pH 5.5). After stirring at 300 rpm for 1 hour at room temperature until fully dissolved, 144 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 86.4 mg of N-hydroxysuccinimide (NHS) were added. After stirring at room temperature for 1 hour, the carboxyl groups of hyaluronic acid were activated, resulting in solution B. Solution A and solution B were mixed at a volume ratio of 1:1 and stirred at 300 rpm at room temperature for 24 hours. The obtained MSN-HA NPs were washed three times with deionized water, freeze-dried in a lyophilizer for 48 hours, and then collected and stored at -20°C.

[0047] 4. Preparation of Chi / Alg@MSN-HANPs 100 mg of the obtained MSN-HA NPs were weighed and dispersed in 20 mL of 2 mg / mL chitosan solution (pH=6, PBS buffer). The mixture was stirred at 500 rpm for 30 min at room temperature, and then washed three times with PBS. Subsequently, the obtained nanoparticles were suspended in 25 mL of 2 mg / mL sodium alginate solution and stirred at 500 rpm for 30 min at room temperature. After centrifugation (11000 rpm, 4℃, 15 min), the nanoparticles were washed three times with PBS. The above steps were repeated three times for alternating coating. After freeze-drying for 48 h, the Chi / Alg@MSN-HA NPs were collected and stored at -20℃.

[0048] 5. Chi / Alg-GE@MSN-HANPs After adding geniposide and the nanocarrier Chi / Alg@MSN-HA NPs in anhydrous ethanol according to the specified ratio, the mixture was sonicated for 10 min, magnetically stirred overnight at 400 rpm at room temperature, centrifuged at 12000 rpm for 10 min, washed three times with deionized water and anhydrous ethanol, and freeze-dried for 48 h to obtain Chi / Alg-GE@MSN-HA NPs containing geniposide. (See schematic diagram below.) Figure 5 As shown, the Fourier transform infrared spectrum is as follows: Figure 6 As shown.

[0049] Using the same method, geniposide and the nanocarrier Chi / Alg-MSN-HA were loaded with drugs at ratios of 1:10, 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, and 10:1, as shown in Table 4. The loading rate and release rate were used as indicators for optimization, and the optimal loading ratio of 1:1 (W(GE) / W(Chi / Alg-MSN-HA)) was selected. Subsequent experiments were conducted at a 1:1 ratio.

[0050] Based on the detection data under various optimization conditions, the results showed that with the increase of geniposide dosage, when W(GE) / W(Chi / Alg-MSN-HA) was 1:10~1:2, the nanoparticle size did not change significantly. This indicates that the main drug loading form may be through van der Waals forces and adsorption forces of the internal cavity of the mesoporous structure. The silanol groups inside the mesoporous silica may form hydrogen bonds with the hydroxyl groups of geniposide, thereby enhancing the interaction force of the mesoporous cavity sites on geniposide. It is inferred that most of the drug is mainly located inside the cavity of the carrier. When W(GE) / W(Chi / Alg-MSN-HA) was 1:2~10:1, the particle size gradually increased with the continued increase of drug dosage, and the particle size increased significantly. This indicates that the surface adsorption of the carrier may rely on the mutual attraction of van der Waals forces to adsorb drug molecules on the pore walls and surface of the mesoporous silica. The electrostatic interaction caused by the surface charge and the residual silanol groups may play a major role. At this stage, the drug gradually adheres to the particle surface, thus affecting the nanoparticle size. Meanwhile, the drug loading and encapsulation efficiency curves in the figure show that with the increase of W(GE) / W(Chi / Alg-MSN-HA), the drug loading increases while the encapsulation efficiency fluctuates little. When the drug loading ratio is 2:1 to 10:1, the drug loading is basically stable, while the encapsulation efficiency gradually decreases. This indicates that when the drug loading ratio is 1:1, the drug loading capacity of the carrier is basically saturated. The zeta potential results show that with a drug loading ratio of 1:10 to 1:1, the zeta potential does not change significantly. However, when the drug loading ratio is 1:1 to 10:1, the zeta potential increases, which may be related to the fact that increasing the loading of the weakly acidic geniposide to a certain extent affects the surface electron cloud density of the nanocarrier. Figure 7 According to the guidelines for microparticle formulations in the 2020 edition of the Chinese Pharmacopoeia (9014), the encapsulation efficiency should generally not be less than 80%. Therefore, based on the encapsulation efficiency requirement, only W(GE) / W(Chi / Alg-MSN-HA) ratios of 1:2, 1:1, and 2:1 met the requirements among the different drug loading ratios screened. Among these, only when W(GE) / W(Chi / Alg-MSN-HA) was 1:1 did the drug loading reach its highest level of 40.84% ​​± 0.53% (as shown in Table 4). Therefore, considering the four indicators of particle size, potential, drug loading efficiency, and encapsulation efficiency, the optimized drug loading ratio W(GE) / W(Chi / Alg-MSN-HA) of 1:1 was used to prepare the nanomedicine Chi / Alg-GE@MSN-HA for subsequent research.

[0051] Table 4

[0052] Example 2 1. Evaluation of drug loading, encapsulation efficiency, and stability of Chi / Alg@MSN-HANPs This embodiment investigates the stability of Chi / Alg@MSN-HANPs after long-term cryogenic storage. Dry Chi / Alg-GE@MSN-HANPs were placed at 4°C for 0, 7, 14, 21, 30, 45, and 60 days. Small amounts of the sample were then redispersed in deionized water, and particle size, PDI, and Zeta potential were measured. The measurement results were recorded on days 0, 7, 14, 21, 30, 45, and 60. The stability after long-term cryogenic storage was analyzed based on the measurement results.

[0053] This embodiment examines the changes in the physicochemical properties of Chi / Alg-GE@MSN-HANPs under long-term storage conditions, selecting particle size, potential, and dispersibility as indicators to evaluate the stability of DHA@MOF-TPP. Figure 8 As shown, with the extension of long-term cryopreservation time, the particle size and potential of Chi / Alg-GE@MSN-HANPs were sampled and detected at different time points during the 60-day period. After redispersing in deionized water, the particle size stabilized at 130-135 nm, and the PDI was less than 0.3, indicating good stability. The carrier skeleton and size did not change significantly due to long-term storage, and the mesopores and outer shell did not change significantly. It can be inferred that its drug loading capacity and drug release behavior should be largely preserved. Moreover, the drug is encapsulated inside the carrier and protected by the coating layer, which helps to avoid decomposition or oxidative deterioration caused by poor drug stability during long-term storage. At the same time, the absolute value of the surface charge zeta potential remained stable above -30 mV for a long time, indicating that its surface charge properties were largely preserved. Therefore, the performance of the nanocarrier was well maintained during the long-term storage test.

[0054] 2. Evaluation of drug release from nanocarriers The release profiles of Chi / Alg-GE@MSN-HANPs and free GE were determined using dialysis. 3 mL of Chi / Alg-GE@MSN-HANPs and free GE were added to a dialysis bag, which was then suspended in simulated gastric fluid (SGF, pH 1.2, 40 mL, 1% Tween 80) at 37°C and stirred at 100 rpm for 2 hours to simulate gastric conditions. The dialysis bag was then suspended in simulated small intestinal fluid (SIF, pH 6.8, 40 mL, 1% Tween 80) at 37°C and stirred at 100 rpm for 4 hours to simulate small intestinal conditions. Finally, the dialysis bag was further incubated in simulated colonic fluid (SCF, pH 7.4, 40 mL, 1% Tween 80) at 37°C and stirred at 100 rpm for 8 hours. At predetermined time points, 1 mL of sample was removed and 1 mL of fresh dissolving medium was added. This method overcomes some measurement errors mainly caused by the short duration of reaction in SGF, SIF, and SCF. Simultaneously, Chi / Alg-GE@MSN-NH2NPs and free GE were placed in the SGF (Self-Generating Fluid), and the concentration of the drug in the filtrate was determined by high-performance liquid chromatography (HPLC) at preset time points. The SIF (Self-Generating Fluid) and SCF (Self-Generating Fluid) methods were the same as those for SGF. The operating procedure is as described above.

[0055] Drug loading rate (%) = (mass of geniposide in nanocarrier / total mass of nanodrug) × 100%; Encapsulation efficiency (%) = Mass of geniposide in nanocarrier / Total amount of geniposide × 100%; 2.1 Results and Analysis of Linearity Study of the geniposide Detection Method Results of linear relationship examination Figure 9 As shown, the peak area values ​​of geniposide at concentrations ranging from 5 to 100 μg / mL were recorded. A standard curve was plotted with concentration (μg / mL) on the x-axis and peak area on the y-axis, yielding the linear regression equation: y = 13700x + 4860 (R² / mL). 2 =0.999), indicating good linearity within the concentration range of 5–100 μg / mL. The liquid chromatogram of geniposide is shown below. Figure 10 As shown, the retention time of the chromatographic peak is 7.101 min.

[0056] 2.2 Results and Analysis of In Vitro Drug Release Different parts of the human gastrointestinal tract have different pH values. The stomach is typically strongly acidic (pH approximately 1.5-3.5), the small intestine is weakly alkaline (pH approximately 6-7.5), while the colon has a relatively high pH, ​​generally between 7 and 8. The Chi / Alg-GE@MSN-HA NPs prepared in this experiment primarily respond to changes in acidity, triggering a drug release mechanism. By exploring the drug release behavior of the Chi / Alg-GE@MSN-HA NPs nanoparticles, an in vitro simulation of the fluid environment from the stomach to the small intestine to the colon was conducted (gastric juice containing SGF 0... 2 hours later, the SIF in the small intestinal fluid contained 2 6h, colonic fluid 6 The release curves of GE in GE@MSN-HA NPs and Chi / Alg-GE@MSN-HANPs were measured at 48 h. The results showed that, within 0–2 h, in a gastric fluid simulated environment, the cumulative GE release rates of GE@MSN-HA NPs and Chi / Alg-GE@MSN-HA NPs reached 20.58±1.74% and 9.20±1.73%, respectively, at 2 h. At 6 h, in a small intestinal fluid simulated environment, the cumulative GE release rates reached 51.30±2.18% and 23.32±1.88%, respectively. At 48 h, in a colonic fluid simulated environment, the cumulative GE release rates reached 78.32±0.69% and 72.76±0.65%, respectively. Based on the cumulative release curves, GE@MSN-HA NPs were almost completely released at 24 hours, with a cumulative release rate of 77.90±1.49%, while Chi / Alg-GE@MSN-HA NPs were almost completely released only at 48 hours. Figure 11 Due to the different biodegradability of the outer polymers, especially the swelling and degradation behavior of sodium alginate and chitosan hydrogels, which are sensitive to pH, GE@MSN-HA NPs, lacking the outer coating of sodium alginate and chitosan, are not resistant to gastric microenvironment. Different parts of the human gastrointestinal tract have different pH values. The stomach is generally strongly acidic (pH about 1.5-3.5), the small intestine is weakly alkaline (pH about 6-7.5), and the colon has a relatively high pH value, generally between 7 and 8. The Chi / Alg-GE@MSN-HA NPs prepared in this experiment mainly respond to changes in acidity to trigger drug release. By exploring the drug release behavior of the nanoparticles Chi / Alg-GE@MSN-HA NPs, the fluid environment from the stomach to the small intestine to the colon was simulated in vitro (gastric juice SGF 0 2 hours later, the SIF in the small intestinal fluid contained 2 6h, colonic fluid 6 The release curves of GE in GE@MSN-HANPs and Chi / Alg-GE@MSN-HA NPs were determined at 48h. The results showed that, within 0–2h, in a gastric fluid simulated environment, the cumulative GE release rates of GE@MSN-HA NPs and Chi / Alg-GE@MSN-HA NPs reached 20.58±1.74% and 9.20±1.73%, respectively, by 2h. From 2–6h, in a small intestinal fluid simulated environment, the cumulative GE release rates reached 51.30±2.18% and 23.32±1.88%, respectively. From 6h to 48h, in a colonic fluid simulated environment, the cumulative GE release rates reached 78.32±0.69% and 72.76±0.65%, respectively, at 48h. Based on the cumulative release curves, GE@MSN-HA NPs were almost completely released at 24 hours, with a cumulative release rate of 77.90±1.49%, while Chi / Alg-GE@MSN-HA NPs were almost completely released only at 48 hours. Figure 11 Due to the varying biodegradability of the outer polymers, especially the swelling and degradation behavior of sodium alginate and chitosan hydrogels, which are sensitive to pH, GE@MSN-HA NPs, lacking an outer coating of sodium alginate and chitosan, are intolerant of the acidity of the simulated gastric environment. Therefore, they release rapidly upon entering the simulated gastric and small intestinal environment, reaching their cumulative release saturation point prematurely. In contrast, Chi / Alg-GE@MSN-HA NPs, with their relatively stable outer coating structure, tolerate the acidity of the environment and release the drug slowly in the simulated gastric and small intestinal environment. Only upon reaching the colon does the outer shell disintegrate in response to the simulated environmental conditions, releasing the inner layer. Further disintegration of the hyaluronic acid layer then releases GE. Therefore, the in vitro simulated release results indicate that Chi / Alg-GE@MSN-HA NPs have a certain sustained-release effect on GE.

[0057] 2.3 In vivo evaluation of geniposide-containing oral targeted delivery system for the anti-UC effect of geniposide 2.3.1 Oral targeted delivery system without geniposide ① To test the cytotoxicity of the nanocarriers, two cell lines, NCM460 and RAW264.7, were selected. The treatment groups included MSN NPs, MSN-HA NPs, and Chi / Alg-MSN-HA NPs. The safety was assessed at the cellular level using the CCK-8 assay. Cell viability was detected by the CCK-8 assay, combined with cell morphology observation. The results showed that MSN NPs, MSN-HA NPs, and Chi / Alg-MSN-HA NPs maintained cell viability above 90% and intact cell morphology within a concentration range of 5–500 μg / ml. No obvious apoptosis was observed. Figure 12The results indicate that, within a concentration range below 500 μg / ml, mesoporous silica nanoparticles did not exhibit significant cytotoxicity against NCM460 and RAW264.7 cells. This outcome provides important safety data to support the potential applications of Chi / Alg-MSN-HA NPs in drug delivery and bioimaging, and also lays the foundation for further research on their targeting performance and efficacy evaluation after drug delivery.

[0058] ② To explore the targeting performance of nanocarriers, confocal fluorescence imaging was used to analyze the uptake and distribution of free coumarin 6 (C6), C6@MSN-HA NPs and Chi / Alg-C6@MSN-HA NPs after incubation for 30 min, 1 h, 2 h and 4 h in an LPS-induced RAW264.7 cell inflammation model.

[0059] According to existing literature, the expression level of CD44 receptor in RAW264.7 cells is significantly increased after LPS induction, which will facilitate the specific targeting and tracking of hyaluronic acid modified nanoparticles C6@MSN-HA NPs.

[0060] from Figure 13 The CLSM images show that free C6 in cells exhibits weak fluorescence and is mainly present in the cell periphery. Cellular fluorescence analysis indicates that RAW264.7 cells take up very little Chi / Alg-C6@MSN-HA NPs within 4 hours. However, after loading C6 into C6@MSN-HA NPs, the uptake of C6 by RAW264.7 cells significantly increases, and the uptake intensity is time-dependent within 4 hours. After 4 hours of incubation, the accumulation of C6@MSN-HA NPs in RAW264.7 cells is significantly higher than that of Chi / Alg-C6@MSN-HA NPs. pThe value <0.05% may be related to the stability of the outer sodium alginate / chitosan capsid layer in the culture environment. Conversely, the lack of a sodium alginate / chitosan (Chi / Alg) capsid layer on the outer surface of C6@MSN-HA NPs exposes the HA on the surface to the cellular environment, allowing it to directly bind to the CD44 receptor on the macrophage surface, thereby enhancing the targeting ability of C6@MSN-HA NPs. The results indicate that C6@MSN-HA NPs can actively target the CD44 receptor and enter RAW264.7 cells via endocytosis. Since UC patients have a large number of inflammatory and activated macrophages at the colonic lesion site, the ability of GE@MSN-HA NPs to target macrophages provides an effective pathway for the targeted delivery of GE to the inflammatory site to exert its therapeutic effect. Meanwhile, the protective effect of the sodium alginate / chitosan (Chi / Alg) shell layer allows the final product Chi / Alg-C6@MSN-HA NPs to bypass the damage in the upper digestive tract and reach the colon. Combined with the macrophage-targeting properties of MSN-HA NPs, it enables local release of drugs for treatment at the lesion site.

[0061] ③ To explore the effect of Chi / Alg-GE@MSN-HA NPs on targeting colonic lesions in UC mice, Chi / Alg-Dir@MSN-HANPs were obtained by using small animal in vivo imaging technology and replacing drugs with Dir fluorescent probes in the nanodelivery system. The dynamic process of in vivo delivery of nanoparticles was observed and analyzed.

[0062] like Figure 14 As shown, free Dir fluorescent probes reach their peak fluorescence intensity approximately 3 hours after entering the body and are then rapidly excreted without exhibiting any retention in the intestines. In contrast, Dir@MSN-HA NPs maintain their peak fluorescence intensity in vivo for up to 6 hours before being rapidly excreted. This may be due to the protective effect of the nanoparticles on the Dir probes, which slows down the premature release of the Dir fluorescent probes. Additionally, although the surface hyaluronic acid layer is damaged in the upper digestive tract, it provides some protection on the outer surface of the particles, delaying the contact between Dir and adverse conditions in the in vivo environment. Even at 24 hours, a small amount of fluorescence was still present in UC mice. In in vivo imaging, the fluorescence of Chi / Alg-Dir@MSN-HA NPs peaked at 3 hours and maintained a high fluorescence intensity until 24 hours, showing a significant difference compared to the Free Dir group (p<0.001), indicating strong retention. These results suggest that Chi / Alg-Dir@MSN-HA NPs may achieve targeted localization at the lesion site of colonic inflammation and possess strong retention, which is beneficial for the local treatment of colonic inflammation by GE drug and improves the drug's in vivo targeting and safety.

[0063] ④ To observe whether Chi / Alg-Dir@MSN-HA achieves targeted accumulation in UC mice, colon tissues of UC mice were separated and in vitro imaged 24 hours after oral administration of free coumarin 6, Dir@MSN-HA and Chi / Alg-Dir@MSN-HA.

[0064] The results are as follows Figure 15 The results showed that the free Dir fluorescence in the intestines of mice was widely distributed and did not accumulate in specific sites. In contrast, the Dir@MSN-HA group showed relatively stronger fluorescence intensity in the small intestine, which may be related to the lack of protection from the sodium alginate / chitosan (Chi / Alg) shell layer on the outer layer of Dir@MSN-HA particles. This allowed the Dir@MSN-HA particles to disintegrate in the upper digestive tract earlier, releasing the probe bound to the outer layer of the nanoparticles, which provided some protection and delayed the in vivo excretion of Dir. In contrast, 24 hours after entering UC mice, the Dir probe carried by Chi / Alg-Dir@MSN-HA NPs showed higher fluorescence intensity in the colon, which was significantly stronger than in other parts of the intestine. This indicates that Chi / Alg-Dir@MSN-HA is targeted and accumulates in the colonic inflammatory lesions of UC mice. This provides a better treatment method for delivering GE to treat UC symptoms and is beneficial to improving the targeting, efficacy and safety of GE in the treatment of UC.

[0065] 2.3.2 In vivo efficacy evaluation of Chi / Alg-GE@MSN-HA To verify the effectiveness of Chi / Alg-GE@MSN-HA in targeted GE delivery for the treatment of ulcerative colitis (UC), the general physical signs and behavior, weight change, DAI score, and colon length of UC mice were selected as evaluation indicators to comprehensively assess the therapeutic effect. General physical observation revealed that the indicators did not change significantly in the first three days after modeling, but on the fourth and fifth days, the mice experienced a sharp drop in weight and rectal bleeding. Furthermore, by the eighth day, most mice in the model group were curled up, immobile, and weak. Compared with the model group, the treatment groups showed improvement in these conditions. Especially by the end of the experiment, the mice in the Chi / Alg-GE@MSN-HA group were in good condition, with normal motor response, normal defecation, and no obvious rectal bleeding. The weight changes of the mice in each group during the observation period are shown below. Figure 16 As shown in Figure A, the average body weight of mice in the model group decreased by 6.1g, which severely affected their quality of life, while the body weight of mice in the positive control group (5-ASA) was significantly improved (vs. Control). p <0.001), gavage showed slight improvement in the GE and GE@MSN-HA groups, while Chi / Alg-GE@MSN-HA showed more significant recovery of body weight loss in UC mice (vs. Model, p<0.001), indicating that Chi / Alg-GE@MSN-HA, by targeting the colon to release GE, is more effective than direct gavage in exerting its anti-UC therapeutic effect, significantly alleviating weight loss in UC mice, which is related to improved local distribution of the drug in the colonic lesions. The DAI score mainly assesses the severity of UC mice from three aspects: percentage of weight loss, stool viscosity, and stool bleeding. The DAI score results are as follows: Figure 16 As shown in Figure B, by the end of the experiment, the average DAI score of the model group mice was 3.40, indicating that their bloody stools, weight loss, and defecation symptoms were more severe. The Chi / Alg-GE@MSN-HA group showed a more significant improvement in DAI score compared to the GE@MSN-HA groups administered by gavage, with an average DAI score of 1.11 (vs. Model). p <0.01). Furthermore, the results regarding the effect on colon length are as follows: Figure 16 As shown in CD, the average colon lengths of mice in the Control group, Model group, 5-ASA group, gavage GE group, GE@MSN-HA group, and Chi / Alg-GE@MSN-HA group were 7.72 cm, 5.36 cm, 6.83 cm, 6.16 cm, 6.55 cm, and 7.13 cm, respectively. The Chi / Alg-GE@MSN-HA group significantly improved colonic shortening symptoms compared to the GE and GE@MSN-HA groups. Combined with the results of previous in vivo imaging localization experiments, this indicates that the outer shell of the Chi / Alg-GE@MSN-HA nanoparticles effectively protects GE from release after reaching the UC lesion, thus improving the anti-UC effect of GE.

[0066] Meanwhile, the in vivo safety of Chi / Alg-GE@MSN-HA in UC mice was explored, and its safety was preliminarily evaluated using the organ coefficient of major organs as an indicator. Results are as follows: Figure 17 As shown, compared with normal mice in the Control group, the indices of the major organs liver, heart, spleen, lungs and kidneys of mice in the other groups did not change significantly after different drug treatments (P>0.05). Therefore, the preliminary safety evaluation results indicate that the developed oral colon-targeting nano-formulation Chi / Alg-GE@MSN-HA NPs has no significant in vivo toxicity in UC mice.

[0067] HE staining technique was used to stain the colons of mice in each group, and the morphology of the colon was observed. Figure 18As shown in Figure A, the colonic structure of the normal group mice remained intact. The model group mice exhibited extensive mucosal damage, with varying degrees of inflammatory infiltration in the mucosa and muscular layer, and irregular damage to the crypts. The GE oral administration group showed mucosal repair and some recovery of epithelial cells. The Chi / Alg-GE@MSN-HA group showed the best recovery of colonic mucosa and epithelial cells. The colonic pathological characteristics of each group were scored using epithelial cell integrity and the degree of inflammatory infiltration as indicators, and the results are as follows: Figure 18 As shown in B, the mice in the Chi / Alg-GE@MSN-HA group showed the best colonic recovery, which was significantly different from the GE oral administration group. p <0.05).

[0068] The optimal drug loading ratio of the delivery system of the present invention is 1:1 for W(GE) / W(Chi / Alg-MSN-HA), with a maximum drug loading of 40.84%±0.53% and an encapsulation efficiency of 81.68±1.06, which meets the pharmacopoeia requirements.

[0069] The present invention exhibits significant efficacy and is safe and non-toxic. It can improve the physical signs of mice, reduce DAI scores, and significantly improve UC symptoms such as colonic shortening, hematochezia, and diarrhea. HE results also show that it can alleviate colonic inflammation. No significant toxic pathological changes were observed in its major organs. It has high targeting efficiency in vitro and in vivo, and demonstrates good anti-UC effects in the in vivo efficacy evaluation of DSS-induced UC mouse models.

[0070] This invention constructs a nano-targeted delivery system to alter the drug delivery site, thereby achieving localized release of geniposide in the colon, improving the efficacy of anti-UC drugs, and providing a solution to address the safety issues of geniposide.

[0071] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing an oral targeted nanodelivery system containing geniposide, characterized in that, The method includes: Step 1: Fabrication of the targeted nanodelivery system MSNs were synthesized via the Stobol method; after template removal, amino groups were further modified on the surface of MSNs to obtain MSN@NH2; hyaluronic acid (HA) activated by NHS / EDC was added to the MSN@NH2 suspension to react and obtain MSN-HA; Chi / Alg@MSN-HA NPs were prepared using MSN-HA, chitosan solution, and sodium alginate. Step 2: Preparation method of Chi / Alg-GE@MSN-HA NPs loaded with geniposide Gardenoside was loaded with Chi / Alg@MSN-HA NPs in a certain ratio to prepare Chi / Alg-GE@MSN-HA NPs loaded with gardenoside. In step one, the method of synthesizing MSNs by the Stober method includes using CTAB as a template, TMB as a pore-forming and expanding agent, and TEOS as a silicon source. The molar ratio of TMB to MSN is 2:1, and the reaction temperature is 80°C. In step one, the preparation method of Chi / Alg@MSN-HA NPs includes first dispersing MSN-HA in a chitosan solution, stirring and reacting at room temperature, suspending the product after reaction in a sodium alginate solution, stirring and reacting and centrifuging to obtain Chi / Alg@MSN-HA NPs; In step two, the mass ratio of geniposide to Chi / Alg@MSN-HA NPs is 1:

1.

2. The method for preparing an oral targeted nanodelivery system containing geniposide according to claim 1, characterized in that, In step one, the method for further modifying the MSNs surface with amino groups includes: first dispersing the MSNs in methanol, then adding APTES, reacting overnight at room temperature, centrifuging the obtained product, washing it with methanol, and drying it to obtain MSN@NH2.

3. The method for preparing an oral targeted nanodelivery system containing geniposide according to claim 2, characterized in that, The MSNs: Methanol: APTES = 0.8-1.2g: 0.8-1.2mL: 0.8-1.2μL.

4. The method for preparing an oral targeted nanodelivery system containing geniposide according to claim 1, characterized in that, In step one, the MSN@NH2 suspension is obtained by dispersing MSN@NH2 in MES buffer.

5. The method for preparing an oral targeted nanodelivery system containing geniposide according to claim 1, characterized in that, In step two, the preparation method of Chi / Alg-GE@MSN-HA NPs loaded with geniposide includes adding geniposide and the nanocarrier Chi / Alg@MSN-HA NPs in anhydrous ethanol in proportion, sonicating, stirring overnight at room temperature, centrifuging, washing repeatedly with deionized water and anhydrous ethanol, and freeze-drying to obtain Chi / Alg-GE@MSN-HA NPs containing geniposide.

6. An oral targeted nanodelivery system containing geniposide, characterized in that, The system is prepared by any one of the methods of claims 1-5.

7. The application of the oral targeted nanodelivery system containing geniposide as described in claim 6 in the preparation of a drug for treating ulcerative colitis.

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