Oral nanonarray hydrogel and method of making same

Abstract

The application discloses an oral nano-array hydrogel and a preparation method thereof, and relates to the technical field of biological medicine. The hydrophobic drug is covalently grafted to the hydrophilic polysaccharide through an amide reaction, then the nano-particles are formed by using the self-assembly characteristics of the amphiphilic copolymer, and finally the metal ions with immunoregulatory activity are introduced based on the ionic crosslinking characteristics of the carboxyl of the hydrophilic polysaccharide and the metal ions, the crosslinking network is formed by the carboxyl on the surface of the nano-particles and the metal ions by regulating the metal ion concentration and the reaction conditions, and finally the hydrogel network with the nano-array structure is prepared. After oral administration, the hydrogel can successfully reach the intestinal tract through the harsh gastric acid environment, is degraded in the intestinal tract in response to the pH and enzymes, releases the nano-particles and free metal ions, the released nano-particles and free metal ions synergistically act, the oxidative stress state of a lesion site is improved, the immune cell homeostasis is regulated, and the local immune microenvironment is reshaped.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an oral nanoarray hydrogel and its preparation method. Background Technology

[0002] Inflammatory bowel disease (IBD) is a heterogeneous group of diseases characterized by chronic relapsing inflammation of the gastrointestinal tract, and its pathogenesis is closely related to the imbalance of intestinal macrophage polarization. Under physiological conditions, M2 type anti-inflammatory macrophages (CD206) + CD163 + Microbiota maintain immune tolerance and mediate intestinal mucosal repair by secreting IL-10 and TGF-β; however, under pathological conditions, microbial metabolites (such as lipopolysaccharide LPS) induce macrophages to adopt the M1 pro-inflammatory phenotype (CD80). + CD86 + The shift in M1 macrophages leads to the release of pro-inflammatory factors such as TNF-α and IL-6, activates the NF-κB signaling pathway, exacerbates damage to tight junctions in the intestinal epithelium, and increases intestinal permeability. Recent studies have found that although existing treatments can induce M1 to M2 phenotype polarization, polarized M2 macrophages have extremely low survival rates due to their high ferroptosis sensitivity. This is mainly reflected in the fact that M2 macrophages lack inducible nitric oxide synthase-mediated antioxidant protection and rely on fatty acid oxidation metabolism to accumulate iron ions. In the highly reactive oxygen species (ROS) microenvironment of IBD lesions, they are prone to ferroptosis characterized by glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) inactivation, and accumulation of malondialdehyde (MDA), a lipid peroxidation product, ultimately weakening anti-inflammatory and tissue repair functions.

[0003] As an endogenous antioxidant, bilirubin (BR) can directly scavenge reactive oxygen species (ROS) and reduce oxidative damage due to its molecular structure. Furthermore, it can activate the Nrf2 signaling pathway, upregulate the expression of various antioxidant enzymes and related substances, and enhance the body's endogenous antioxidant capacity, thus holding promise for the treatment of macrophage ferroptosis. However, BR is highly hydrophobic, making direct in vivo application difficult. It often requires combining BR with hydrophilic materials to mitigate the hydrophobicity. For example, Lee et al. developed a hyaluronic acid-bilirubin nanomedicine (HABN) in their study "Hyaluronic acid–bilirubin nanomedicine for targeted modulation of dysregulated intestinal barrier, microbiome and immune responses in colitis." This technology utilizes hyaluronic acid (HA) and bilirubin (BR) to self-assemble into nanoparticles, achieving multi-target treatment of colitis. HABN accumulates at the inflammatory site by targeting CD44 receptors on the surface of inflamed colonic epithelial cells and pro-inflammatory macrophages through HA; the BR core provides potent ROS scavenging capabilities, reducing oxidative damage. In a colitis model, HABN can repair intestinal barrier function, regulate gut microbiota structure, and balance immune responses, thereby achieving synergistic therapy. It is evident that combining BR with HA holds promise for overcoming the highly hydrophobic properties of BR itself; however, this technology still has the following drawbacks:

[0004] (1) Insufficient gastrointestinal tolerance, unable to withstand the harsh environment in the stomach, easily damaged or premature drug release;

[0005] (2) The intestinal residence time is short and it is easily cleared by gastrointestinal motility, making it difficult to maintain a long-term effective therapeutic concentration;

[0006] (3) The mechanism of action is singular and the therapeutic effect is poor.

[0007] Therefore, further research is needed to apply BR to the treatment of macrophage ferroptosis. Summary of the Invention

[0008] This invention provides an oral nanoarray hydrogel and its preparation method to solve many problems of existing IBD oral nanoparticle delivery systems, such as insufficient gastrointestinal tolerance, short intestinal residence time, and single mechanism of action.

[0009] The technical solution adopted in this invention is as follows:

[0010] In a first aspect, the present invention provides a method for preparing an oral nanoarray hydrogel, comprising the following steps:

[0011] (1) Activate the carboxyl group on hydrophobic drugs;

[0012] (2) Under nitrogen protection, ethylenediamine is reacted with the activated hydrophobic drug. After the reaction is completed, the drug is extracted, washed and concentrated with the organic phase, and then separated and extracted to obtain the aminated hydrophobic drug.

[0013] (3) The hydrophilic polysaccharide was purified by acidification and then freeze-dried to convert it into an acidic hydrophilic polysaccharide;

[0014] (4) Activate the carboxyl groups on acidic hydrophilic polysaccharides;

[0015] (5) The aminated hydrophobic drug is added to the activation reaction solution of the acidic hydrophilic polysaccharide to undergo chemical coupling. After the reaction is completed, the amphiphilic drug-polysaccharide conjugate is obtained by precipitation, dialysis purification and lyophilization.

[0016] (6) The amphiphilic drug-polysaccharide conjugate is dispersed in an aqueous system and induced to self-assemble by ultrasonic treatment. The assembly products are collected to obtain self-assembled nanoparticles.

[0017] (7) Select a solution of multivalent metal ions with physiological regulatory activity and mix it with the self-assembled nanoparticles. Induce ionic cross-linking under acidic conditions, and obtain oral nanoarray hydrogel after separation and drying.

[0018] Further, in step (1), the activation process of the carboxyl group on the hydrophobic drug is as follows: the hydrophobic drug, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a molar ratio of 1:0.7:0.45 are dissolved in a solvent, the pH of the reaction system is adjusted to be greater than 7 using triethylamine, the activation temperature is 10~40 ℃, and the activation time is 5~15 min; the hydrophobic drug is a drug that exerts a therapeutic effect in the colon or an anti-tumor drug that is targeted to the colon to improve the anti-cancer efficacy. In this technical solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide mainly acts as a condensing agent to promote the activation of the carboxyl group, N-hydroxysuccinimide acts as an activator to improve the stability of the intermediate, and triethylamine acts as an alkaline agent to neutralize the hydrochloric acid byproduct generated in the reaction. The mixture is stirred and activated at 10~40 ℃ for 5~15 min to ensure that the activation reaction is fully carried out and to avoid the occurrence of side reactions. The molar ratio of 1:0.7:0.45 can effectively improve the stability of the activated intermediate and promote the subsequent amidation reaction with ethylenediamine. This ratio system can ensure reaction efficiency, improve product yield, and effectively control the formation of byproducts.

[0019] Furthermore, in step (2), the molar ratio of the hydrophobic drug to ethylenediamine is 1:0.75, and ethylenediamine is slowly added dropwise to the activated mixture obtained in step (1). The reaction temperature is 10~40 ℃ and the reaction time is 3~5 h. In the extraction system, the volume ratio of chloroform to the initial reaction system is 6.7:1. In this technical solution, ethylenediamine, as a nucleophile, undergoes an amidation reaction with the carboxyl group of an activated hydrophobic drug. Chloroform extraction effectively separates byproducts and unreacted reagents in the aqueous phase. Sequential washing with acidic, alkaline, and neutral solutions removes unreacted ethylenediamine, neutralizes residual acidic byproducts in the reaction system, and eliminates ionic impurities. The reaction is carried out at 10–40 °C for 3–5 hours to ensure complete amidation and avoid over-reaction leading to byproduct formation. A 1:0.75 molar ratio of hydrophobic drug to ethylenediamine is used, which has the advantage of ensuring sufficient reaction with the activated hydrophobic drug while avoiding excessive substitution side reactions caused by excessive amine reagents. A stepwise washing purification scheme, using alternating acid and alkali washing, effectively removes various impurities from the reaction system, improving product purity. These process conditions ensure high synthesis efficiency and yield high-purity amidated products, providing stable intermediates for subsequent conjugation reactions.

[0020] Further, in step (3), the hydrophilic polysaccharide is acidified with a 0.01 M hydrochloric acid solution. The hydrophilic polysaccharide is a polysaccharide capable of cross-linking with metal ions, such as sodium alginate, pectin, carrageenan, and chitosan. In this technical solution, dialysis with hydrochloric acid is used to replace the metal ions in the hydrophilic polysaccharide, converting them into an acidic form, thereby exposing more free carboxyl groups and providing reaction sites for subsequent conjugation reactions with amine groups. Overnight dialysis ensures sufficient ion exchange, while subsequent lyophilization facilitates the dissolution of the acidic hydrophilic polysaccharide in the organic solvent dimethyl sulfoxide and subsequent reactions.

[0021] Further, the carboxyl activation process of the acidic hydrophilic polysaccharide in step (4) is as follows: An acidic hydrophilic polysaccharide, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a molar ratio of 1:0.5:1.75 are dissolved in a solvent. The activation temperature is 10~40 °C, and the activation time is 5~15 min. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide acts as a condensing agent, and N-hydroxysuccinimide acts as an activating agent, working together to generate an active N-hydroxysuccinimide ester intermediate. Short-term stirring and activation at 10~40 °C for 5~15 min ensures activation efficiency and prevents side reactions such as hydrolysis of the activated groups during excessively long waiting times.

[0022] Further, in step (5), the molar ratio of the carboxyl group of the hydrophilic polysaccharide to the aminated hydrophobic drug is 4:1, the reaction temperature is 10~40 ℃, and the reaction time is 10~15 h. In this technical solution, the aminated hydrophobic drug acts as a nucleophile and undergoes an amidation reaction with the activated hydrophilic polysaccharide ester to form a stable amphiphilic drug-polysaccharide block. The light-protected operation is to prevent the hydrophobic drug structure from photodegrading during the long-term reaction. Precipitation and preliminary dialysis using an alkaline solution can effectively neutralize the acidic byproducts in the reaction system and remove unreacted small molecules. Subsequent stepwise dialysis using solvents of different polarities can thoroughly remove organic byproducts and solvents, ensuring the purity of the block. This molar ratio is beneficial for controlling the conjugation density and avoiding excessive cross-linking of the hydrophilic polysaccharide molecular chains.

[0023] Further, in step (6), the ultrasonic treatment is performed under ice-water bath conditions, using a probe-type ultrasonic instrument for 8-15 min. The output power of the probe-type ultrasonic instrument is 140 W, and the working cycle is 2 s pulse on / 3 s pulse off; the filter membrane pore size is 0.45 μm. In this technical solution, the controllable ultrasonic treatment under ice-water bath conditions can provide uniform energy for the formation of nanoparticles. Through acoustic cavitation, the BR-Alg macromolecular chains are coiled and self-assembled to form nanoscale particles. The pulse working mode can prevent the solution from overheating and protect the drug activity. The subsequent filtration step is used to remove any small amount of large-particle aggregates or insufficiently assembled impurities, thereby ensuring that the final self-assembled nanoparticles have a uniform particle size distribution and good dispersion stability.

[0024] Further, in step (7), the final concentration of metal ions in the reaction system is 10-15 mM, and the final concentration of nanoparticles is 0.1-1.0 mg / mL. The metal ions are one of calcium, zinc, iron, copper, and magnesium ions. In this technical solution, metal ions act as crosslinking agents, specifically binding to guluronic acid units on the polysaccharide molecular chain to form a three-dimensional network structure through an "egg box" model, thereby constructing a hydrogel. Precisely adjusting the pH to 4.5±0.1 provides the optimal acid-base environment for the ion crosslinking reaction, promoting the gelation process and ensuring the stability of the gel structure. Moderate centrifugation (4,000×g for 5 minutes) can effectively collect the formed hydrogel particles while avoiding structural damage. The hydrogel prepared by this method combines the strength of ion crosslinking with the function of nanomedicine.

[0025] As a preferred embodiment, the hydrophilic polysaccharide is sodium alginate, the hydrophobic drug is bilirubin, and the metal ion is calcium ion. Sodium alginate (Alg) is a natural anionic polysaccharide extracted from brown algae. Its abundant carboxyl and hydroxyl groups on its molecular chain not only endow it with antioxidant activity but also allow it to interact with calcium ions. 2+Cross-linking forms a pH-sensitive hydrogel, which shrinks in the gastrointestinal environment to protect the drug from premature leakage. The mannuronic acid (M unit) structure of Alg can achieve active targeting by specifically binding to mannose receptors on the surface of M2 macrophages. Therefore, we first covalently grafted bilirubin molecules onto sodium alginate polysaccharide via an amide reaction, utilizing the self-assembly properties of this amphiphilic copolymer to form BAN nanoparticles. Based on this, based on the Alg carboxyl group and Ca... 2+ The ionic crosslinking properties of Ca2+ introduce Ca2+ with macrophage polarization regulation function. 2+ By regulating Ca 2+ Concentration and reaction conditions promote the reaction of carboxyl groups and Ca on the surface of BAN nanoparticles. 2+ A cross-linked network was formed, ultimately producing a BAN@Ca hydrogel network with a nanoarray structure. After oral administration, this hydrogel successfully reached the colon through the harsh acidic environment of the stomach, where it degraded in the colon in response to pH and enzymes, releasing BAN and free Ca. 2+ Ca 2+ It acts on macrophages, causing changes in macrophage phenotype. BAN targets M2 macrophages, continuously and efficiently clearing ROS within macrophages, reducing lipid peroxidation, inhibiting macrophage ferroptosis, and improving the survival rate of M2 macrophages.

[0026] In a second aspect, the present invention provides an oral nanoarray hydrogel prepared using any of the methods described in the first aspect.

[0027] In summary, compared with the prior art, the present invention has the following advantages and benefits:

[0028] 1. The oral nanoarray hydrogel prepared by this invention exhibits strong protective effects on the loaded drug in simulated gastric and small intestinal fluids. In in vitro simulated release experiments, the release in simulated gastrointestinal fluids was minimal, while in colonic fluid simulated by rat cecal contents solution, the release of calcium ions and nanoparticles was approximately 87.0% and 82.7%, respectively. Compared with existing technologies, this invention provides better gastrointestinal protection for the target drug.

[0029] 2. The oral hydrogel prepared by this invention is specifically degraded in the colon under the action of pH and enzymes after reaching the colon, realizing the targeted delivery and release of nanoparticles and calcium ions in the colon.

[0030] 3. The nanoparticles generated by the specific degradation of the nanoarray hydrogel in the colon region in this invention can target the mannose receptors on M2 macrophages through the mannuronic acid units on sodium alginate, thereby achieving drug enrichment.

[0031] 4. The oral hydrogel prepared by this invention can not only promote macrophage polarization, but also inhibit ferroptosis of polarized macrophages, improve the survival rate of M2 macrophages, and achieve synergistic therapeutic effects. Attached Figure Description

[0032] Figure 1 This is a synthetic route diagram for bilirubin-grafted sodium alginate. In the diagram: BR: bilirubin; Alg: sodium alginate; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS: N-hydroxysuccinimide; Ethylene diamine: ethylenediamine; DMSO: dimethyl sulfoxide; BR-Alg: bilirubin-grafted sodium alginate.

[0033] Figure 2 The image shows the transmission electron microscope (TEM) morphology of the BAN prepared in Example 1.

[0034] Figure 3 The image shows the transmission electron microscope (TEM) morphology of BAN@Ca prepared in Example 1.

[0035] Figure 4 The image shows the scanning electron microscope (SEM) morphology of BAN@Ca prepared in Example 1.

[0036] Figure 5 This is a simulated in vitro release curve of BAN@Ca.

[0037] Figure 6 This figure shows the intestinal retention of BAN@Ca in mice after gavage administration. Detailed Implementation

[0038] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0039] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0040] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0041] This invention provides a method for preparing an oral nanoarray hydrogel, which involves first covalently grafting a hydrophobic drug (e.g., bilirubin) onto a hydrophilic polysaccharide (e.g., sodium alginate) via an amide reaction. Figure 1 As shown, bilirubin is first amino-modified, followed by an acidification reaction of sodium alginate, and then an amide reaction between the two. The self-assembly properties of this amphiphilic copolymer are then utilized to form nanoparticles (BAN). Finally, based on the cross-linking properties of Alg carboxyl groups with metal ions (calcium ions as an example), the specific steps include the following:

[0042] (1) Under nitrogen protection, bilirubin and N-hydroxysuccinimide were dissolved in dimethyl sulfoxide containing triethylamine, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added. After stirring and activation, a mixture was obtained.

[0043] (2) Under nitrogen protection, ethylenediamine was added dropwise to the mixture. After the reaction was completed, it was extracted with chloroform and washed successively with hydrochloric acid solution, sodium bicarbonate solution and distilled water. The organic phase was evaporated under reduced pressure. The residue was redissolved with methanol, centrifuged to remove the supernatant and concentrated to obtain BR-NH2.

[0044] (3) Sodium alginate was dialyzed overnight with hydrochloric acid solution and then lyophilized to convert it into acidic sodium alginate;

[0045] (4) Under nitrogen protection, acidic sodium alginate and N-hydroxysuccinimide were dissolved in dimethyl sulfoxide, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added. After stirring and activation, the reaction solution was obtained.

[0046] (5) Under nitrogen protection and light-protected conditions, BR-NH2 was added dropwise to the reaction solution and reacted in the dark. After the reaction was completed, the crude product was precipitated into sodium hydroxide solution, purified by dialysis and lyophilized to obtain BR-Alg conjugate.

[0047] (6) The BR-Alg conjugate was dissolved in ultrapure water, ultrasonically treated and filtered, and the filtrate was collected and freeze-dried to obtain BAN nanoparticles;

[0048] (7) Mix BAN nanoparticles or BAN nanoparticle solution with calcium chloride solution evenly, adjust pH to 4.5±0.1, stir and centrifuge, wash the precipitate and freeze dry to obtain oral nanoarray hydrogel.

[0049] The present application will now be described in detail with reference to embodiments and experimental data.

[0050] Example 1

[0051] In this embodiment, a nanoarray hydrogel was prepared. The specific preparation steps are as follows:

[0052] (1) Amino modification of bilirubin (BR-NH2): Under nitrogen protection, 64.3 mg of bilirubin (750 μmol) and 59.9 mg of N-hydroxysuccinimide (520 μmol) were dissolved in 7.5 mL of dimethyl sulfoxide containing 0.225 μL of triethylamine. Then, 64.8 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (337.5 μmol) was added to the system, and the mixture was stirred and activated at room temperature for 10 min. Then, 33.8 μL of ethylenediamine (562.5 μmol) was slowly added dropwise, and the reaction was continued to be stirred at room temperature for 4 h under nitrogen protection. After the reaction was completed, 50 mL of chloroform was added for extraction, and the mixture was washed twice each with 50 mL of 0.1 M HCl solution, 50 mL of 0.1 M NaHCO3 solution, and 50 mL of distilled water. The organic phase was collected and the solvent was removed by vacuum evaporation. The residue was reconstituted with 45 mL of methanol, centrifuged at 3000 g for 10 min, and the supernatant was collected and concentrated to finally obtain BR-NH2.

[0053] (2) Sodium alginate grafted with bilirubin (BR-Alg): Sodium alginate was first dissolved in ultrapure water, dialyzed with 0.01 M HCl solution for 12 h, and then freeze-dried to obtain acidic sodium alginate. 16.0 mg of acidic sodium alginate (80 μmol, based on carboxyl groups) and 4.6 mg of NHS (40 μmol) were accurately weighed and dissolved in 4.8 mL of DMSO. 26.9 mg of EDC (140 μmol) was added and the mixture was stirred at room temperature for 10 min to activate it. Then, 11.2 mg of BR-NH2 (20 μmol) obtained in step (1) was dissolved in an appropriate amount of DMSO and slowly added dropwise to the reaction system. The reaction was carried out under nitrogen protection and in the dark, with stirring at room temperature for 12 h. The reaction solution was slowly poured into 30 mL of 0.01 M NaOH solution to precipitate the precipitate. The precipitate was first dialyzed against 0.01 M NaOH for 5 h, then dialyzed against a mixture of water / acetonitrile (1:1, v / v) three times (24 h each time), and finally dialyzed against distilled water three times (48 h each time). The purified solution was freeze-dried to obtain the BR-Alg conjugate.

[0054] (3) Preparation of BAN nanoparticles: Weigh 5 mg of BR-Alg conjugate, dissolve it in 5 mL of ultrapure water, and continuously sonicate it for 10 min under ice-water bath conditions using a probe-type ultrasonic disruptor (power 140 W, working / intermittent cycle of 2 s / 3 s). After sonication, filter through a 0.45 μm microporous membrane and freeze-dry to finally obtain BAN nanoparticles.

[0055] (4) Preparation of BAN@Ca nanoarray hydrogel: Mix 100 μL of BAN (5 mg / mL) and 900 μL of CaCl2 (13.9 mM) solution evenly, adjust the pH to 4.5, and stir for 10 min. Centrifuge the reaction solution at 4000 rpm for 5 min, wash three times with ultrapure water, and freeze-dry the precipitate to obtain the nanoarray hydrogel, which is denoted as BAN@Ca.

[0056] Experimental Example

[0057] The nanoparticles and nanoarray hydrogels prepared in Example 1 were subjected to microscopic characterization and functional tests, as detailed below:

[0058] (1) Transmission electron microscope (TEM) image

[0059] The prepared nanoparticles and nanoarray hydrogel were diluted appropriately, and 10 μL of the solution was dropped onto a 200-mesh copper grid coated with an ultrathin carbon film, ensuring that the solution uniformly covered the grid. After standing for 10 min, the residual liquid was removed. The copper grid was placed at room temperature until it was completely dry, and the morphology of the BAN nanoparticles and BAN@Ca was characterized using transmission electron microscopy.

[0060] like Figure 2 As shown, the obtained BAN nanoparticles are regular spherical, with smooth surfaces and uniform distribution. Their particle size is similar to that measured by DLS, indicating that the prepared BAN nanoparticles have uniform particle size.

[0061] like Figure 3 As shown, BAN and Ca 2+ After stirring, the Alg carboxyl groups on the surface of the nanoparticles react with Ca... 2+ Through cross-linking, Example 1 prepared a densely and regularly connected nanoarray hydrogel.

[0062] (2) Scanning electron microscope (SEM) image

[0063] BAN@Ca hydrogel was pretreated using a gradient ethanol dehydration method, followed by treatment in a critical point desiccator with liquid CO2 as the replacement medium for 1.5 h. It was then sputter-coated with gold for 150 s, and finally, electron signal images were acquired using a scanning electron microscope in low vacuum mode to observe the morphology of the sample.

[0064] The results are as follows Figure 4 As shown, Example 1 yielded a nanoarray hydrogel with a highly cross-linked structure.

[0065] (3) In vitro simulated degradation study

[0066] Five mL of BAN@Ca solution (BAN concentration 1 mg / mL) was injected into a dialysis bag (molecular weight cutoff 1000 Da). The dialysis bag was sealed at both ends and placed in simulated gastric fluid (SGF, pH 1.2, 50 mL) to ensure complete immersion. The bag was incubated on a shaker at 37°C and 100 rpm for 2 h to simulate a gastric retention environment. Subsequently, the dialysis bag was transferred to simulated small intestinal fluid (SIF, pH 6.8, 50 mL) and incubated for another 4 h under the same conditions to simulate small intestinal digestion. Finally, the dialysis bag was transferred to a freshly prepared mouse cecal contents suspension (pH 7.4, 50 mL, homogenized with sterile PBS at a 1:5 w / v ratio and centrifuged to obtain the supernatant) for an 8 h colonic environment simulation. Throughout the simulated digestion process, 1 mL of release medium sample was collected every 1 h, and isothermal and equal-volume fresh digestive fluid was added simultaneously to maintain a constant system volume. The calcium ion concentration in the sample was determined using a calcium ion detection kit. 2+ Concentration, BAN concentration was measured by ultraviolet light, and quantitative analysis was performed to calculate Ca. 2+ And the cumulative release rate of BAN.

[0067] The results are as follows Figure 5 As shown, BAN@Ca showed almost no degradation within the first 6 hours, indicating that the formulation has good stability and can avoid degradation of BAN and Ca. 2+ It is released prematurely in the stomach and small intestine. Upon reaching the colon, BAN and Ca... 2+ Both were released rapidly, and after 12 hours, the cumulative release reached 83% and 87% respectively, proving that BAN@Ca can be specifically degraded in the colon.

[0068] (4) Intestinal retention in mice

[0069] Male C57BL / 6J mice were randomly selected and acclimatized for 7 days, followed by free access to 3% (w / v) DSS aqueous solution for 7 days to establish an IBD pathological model. Mice were randomly divided into two groups, with the first group receiving free Cy5 solution via gavage and the second group receiving Cy5-modified BAN@Ca solution (Cy5 concentration 0.2 mg / kg). At 4, 8, 12, 24, and 48 h, three mice from each group were randomly selected and sacrificed. Stomach, small intestine, and colon tissues were collected, and the fluorescence signal distribution of the isolated mouse organs was detected using an in vivo imaging system.

[0070] The results are as follows Figure 6As shown, starting 4 hours after oral administration, the average fluorescence intensity in the colon of the formulation group was higher than that of the control group. After 12 hours, the average fluorescence intensity in the colon of the mice in the formulation group was the strongest, while the fluorescence of the free Cy5 group had already begun to decay. At 48 hours, the fluorescence of the free Cy5 group had almost completely disappeared, while the colon of the formulation group still retained significant fluorescence. These results indicate that BAN@Ca can target the site of colonic inflammation and specifically accumulate in the lesion area, effectively avoiding premature drug release in the gastric and small intestinal environments, and can also prolong the drug's residence time in the colon, improving drug bioavailability.

[0071] Finally, it should be noted that the terms "comprising," "including," or any other variations are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make further changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0072] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A method for preparing an oral nanoarray hydrogel, characterized in that, Includes the following steps: (1) Activate the carboxyl group on hydrophobic drugs; (2) Under nitrogen protection, ethylenediamine is reacted with the activated hydrophobic drug. After the reaction is completed, the drug is extracted, washed and concentrated with the organic phase, and then separated and extracted to obtain the aminated hydrophobic drug. (3) The hydrophilic polysaccharide was purified by acidification and then freeze-dried to convert it into an acidic hydrophilic polysaccharide; (4) Activate the carboxyl groups on acidic hydrophilic polysaccharides; (5) The aminated hydrophobic drug is added to the activation reaction solution of the acidic hydrophilic polysaccharide to undergo chemical coupling. After the reaction is completed, the amphiphilic drug-polysaccharide conjugate is obtained by precipitation, dialysis purification and lyophilization. (6) The amphiphilic drug-polysaccharide conjugate is dispersed in an aqueous system and induced to self-assemble by ultrasonic treatment. The assembly products are collected to obtain self-assembled nanoparticles. (7) Select a solution of multivalent metal ions with physiological regulatory activity and mix it with the self-assembled nanoparticles. Induce ionic cross-linking under acidic conditions, and obtain oral nanoarray hydrogel after separation and drying.

2. The method for preparing the oral nanoarray hydrogel as described in claim 1, characterized in that, In step (1), the activation process of the carboxyl group on the hydrophobic drug is as follows: the hydrophobic drug, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a molar ratio of 1:0.7:0.45 are dissolved in a solvent, the pH of the reaction system is adjusted to be greater than 7 using triethylamine, the activation temperature is 10~40 ℃, and the activation time is 5~15 min; the hydrophobic drug is a drug that exerts a therapeutic effect in the colon or an anti-tumor drug that is targeted to the colon to improve the anti-cancer efficacy.

3. The method for preparing the oral nanoarray hydrogel as described in claim 2, characterized in that, In step (2), the molar ratio of the hydrophobic drug to ethylenediamine is 1:0.75, and ethylenediamine is slowly added dropwise to the activated mixture obtained in step (1). The reaction temperature is 10~40 ℃ and the reaction time is 3~5 h. In the extraction system, the volume ratio of chloroform to the initial reaction system is 6.7:

1.

4. The method for preparing the oral nanoarray hydrogel as described in claim 1, characterized in that, In step (3), the hydrophilic polysaccharide is acidified with a 0.01 M hydrochloric acid solution. The hydrophilic polysaccharide is a polysaccharide that can crosslink with metal ions.

5. The method for preparing the oral nanoarray hydrogel as described in claim 1, characterized in that, The carboxyl activation process of the acidic hydrophilic polysaccharide in step (4) is as follows: acidic hydrophilic polysaccharide, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a molar ratio of 1:0.5:1.75 are dissolved in a solvent, the activation temperature is 10~40℃ and the activation time is 5~15min.

6. The method for preparing the oral nanoarray hydrogel as described in claim 1, characterized in that, In step (5), the molar ratio of the carboxyl group of the hydrophilic polysaccharide to the amino-modified hydrophobic drug is 4:1, the reaction temperature is 10~40 ℃, and the reaction time is 10~15 h.

7. The method for preparing the oral nanoarray hydrogel as described in claim 1, characterized in that, In step (6), the ultrasonic treatment is carried out under ice water bath conditions. The ultrasonic treatment is performed for 8~15 min using a probe ultrasonic instrument with an output power of 140 W and a working cycle of 2 s pulse on and 3 s pulse off. The filter membrane pore size is 0.45 μm.

8. The method for preparing the oral nanoarray hydrogel as described in claim 1, characterized in that, In step (7), the final concentration of metal ions in the reaction system is 10~15 mM, and the final concentration of nanoparticles is 0.1~1.0 mg / mL. The metal ions are one of calcium ions, zinc ions, iron ions, copper ions, and magnesium ions.

9. The method for preparing the oral nanoarray hydrogel as described in claim 1, characterized in that, The hydrophilic polysaccharide is sodium alginate, the hydrophobic drug is bilirubin, and the metal ion is calcium ion.

10. The drug-loaded microgel precursor prepared by the method for preparing oral nanoarray hydrogels according to any one of claims 1 to 9.

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