Drug-loaded hydrogel as well as preparation method and application thereof

Abstract

The invention provides drug-loaded hydrogel as well as a preparation method and application thereof, the preparation method comprises the following steps: dissolving HA and NB in dimethyl sulfoxide, adding a 2-morpholine ethanesulfonic acid solution and 4-(4, 6-dimethoxy-1, 3, 5-triazine-2-yl)-4-methylmorpholine chloride, stirring for 10-12 hours under a dark condition, dialyzing and freeze-drying to obtain HANB; the preparation method comprises the following steps: dissolving HA in deionized water, adding a methacrylic anhydride solution, then dropwise adding NaOH until the pH value is maintained at 8-11, continuing the reaction in the dark for 4-6 hours, then dropwise adding HCl to adjust the pH value of the solution to 7, centrifuging the solution, then collecting supernate, and dialyzing and freeze-drying the supernate to obtain HAMA; the preparation method comprises the following steps: fully dissolving a drug, adding HANB, HAMA and LAP at 37 DEG C to form an emulsion, carrying out ultrasonic and evaporation treatment on the emulsion, and finally carrying out gelation under LED light irradiation to obtain the drug-loaded hydrogel. The drug-loaded hydrogel is delivered to intestinal tracts through an endoscope and then is subjected to light curing, ulcerative colitis is treated, inflammatory response can be relieved, and intestinal barrier repair is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of drug sustained-release carrier materials, and particularly relates to a drug-loaded hydrogel and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) mainly includes Crohn's disease (CD) and ulcerative colitis (UC), and the pathophysiology of IBD involves complex genetic, environmental, microbial and immune factors. Ulcerative colitis is a chronic inflammatory bowel disease. The treatment of UC is limited due to significant adverse reactions associated with nonspecific drug delivery, such as systemic suppression of the host immune system. Although the understanding of the etiology of the disease has continued to increase with the approval of several new biological and small molecule drugs in the past few decades, which has promoted the progress of drug development, the remission rate of UC patients given new therapeutic agents in clinical trials remains at 20-30%. This also highlights the urgent need for alternative strategies to design more effective treatments for UC.

[0003] The therapeutic potential of conventional UC treatments is limited by significant adverse reactions caused by nonspecific drug delivery, such as systemic suppression of the host immune system. Therefore, the success of UC treatment depends largely on whether the drug can be effectively delivered to the target tissue, which can improve the therapeutic effect while reducing side effects. Endoscopy is a routine procedure for the diagnosis, management, and treatment of UC. With the advancement of endoscopic technology, new tools such as endoscopic ultrasound and balloon-assisted enteroscopy have been developed and widely used in the treatment of UC. Endoscopy can also be used as a potential drug delivery platform. Relevant technologies have been attempted to inject steroids and TNF-α antibodies under endoscopy. However, the clinical benefits of direct drug injection using endoscopy remain unclear.

[0004] Over the past few decades, the development of novel biomaterials, including liposomes, nanoparticles, organic carriers, and hydrogels, has facilitated the development of different drug carriers. Compared with traditional therapies, new drug delivery platforms have demonstrated the potential to overcome the challenges of treating different diseases by effectively changing the biodistribution, pharmacokinetics, and systemic toxicity of drugs. However, despite extensive preclinical studies, only a few targeted carriers have been approved for clinical use, such as Doxil and Abraxane for cancer treatment. Biocompatibility, production challenges, and the complexity of the tissue microenvironment limit the clinical benefits of different drug delivery systems.

[0005] In view of the above problems, the present invention provides a synthetic hydrogel material with biocompatible gelation, which can effectively adhere to and cover irregular tissue surfaces in the body, providing an effective way for endoscopically guided drug delivery. The ideal integration of synthetic hydrogel materials with the intestinal epithelium requires an intimate and preferred chemical bonding interface between the hydrogel and the mucosal surface. Although various dual-reactive macromolecules that can react with both biological surfaces and pre-gel polymers have been tested, they usually require multi-step operations to gel or harsh oxidative conditions that can impair the biocompatibility of the material. To address these issues, we developed a photo-crosslinked hyaluronic acid (HANB / HAMA) hydrogel for gastrointestinal controlled drug release using a light-triggered transient radical and persistent radical coupling (PTPC) reaction. The HANB / HAMA hydrogel is biocompatible and can quickly achieve in situ gelation under ultraviolet irradiation. Summary of the invention

[0006] The present invention provides a drug-loaded hydrogel and a preparation method and application thereof. The prepared HANB / HAMA hydrogel is delivered to the intestine through an endoscope and then photocured to treat ulcerative colitis, thereby relieving inflammatory response and promoting intestinal barrier repair.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: a method for preparing a drug-loaded hydrogel, characterized in that it comprises the following steps:

[0008] (1) HA and NB were dissolved in dimethyl sulfoxide, 2-morpholineethanesulfonic acid solution and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride were added, and stirred for 10-12 hours in the dark, and HANB was obtained after dialysis and freeze-drying;

[0009] (2) HA was dissolved in deionized water, methacrylic anhydride solution was added, and then 5 M NaOH was added dropwise until the pH was maintained between 8 and 11. The reaction was continued in the dark for 4 to 6 hours, and then 1 M HCl was added dropwise to adjust the pH of the solution to 7. The solution was centrifuged and the supernatant was collected. The supernatant was dialyzed and freeze-dried to obtain HAMA;

[0010] (3) After the drug is fully dissolved, HANB obtained in step (1), HAMA obtained in step (2) and photoinitiator LAP are added at 37° C. to form an emulsion, the emulsion is subjected to ultrasound and evaporation treatment, and finally gelled under LED light irradiation to obtain the drug-loaded hydrogel.

[0011] Furthermore, in the step (1), the mass volume ratio of HA, NB, dimethyl sulfoxide, 2-morpholineethanesulfonic acid solution and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride is 1 g:37 mg:2 mL:50 mL:0.2 g.

[0012] Furthermore, in the step (2), the mass volume ratio of HA, deionized water and methacrylic anhydride solution is 4 g:100 mL:2.4 mL.

[0013] Furthermore, in the step (3), the total content of HANB, HAMA and LAP in the emulsion is 2.37-7.11 w / v%.

[0014] Furthermore, the total content of HANB, HAMA and LAP in the emulsion is 4.74 w / v%, wherein the mass ratio of HANB, HAMA and LAP is 4:0.25:0.24.

[0015] Furthermore, in step (3), the ultrasonic conditions are 40% amplitude, 3s on, 3s off, and a total of 20s.

[0016] Furthermore, in step (3), the LED light irradiation conditions are 395 nm, 50 mW / cm 2 Irradiate for 10 s.

[0017] Furthermore, the drug in step (3) includes mesalazine and budesonide; when the drug is mesalazine, the specific operation of step (3) is as follows: mesalazine is dissolved in PBS with a pH of 7.4 at a concentration of 50 mg / mL, and then HANB, HAMA and LAP are added at 37° C. to form an emulsion, the emulsion is subjected to ultrasound and evaporation treatment, and finally gelled under LED light irradiation to obtain a mesalazine-loaded HANB / HAMA hydrogel;

[0018] When the drug is budesonide, the specific operation of step (3) is as follows: first, HANB, HAMA and LAP are dissolved in PBS with a pH of 7.4 to form a precursor solution, then 500 μg of budesonide is dissolved in 0.1 mL of dichloromethane, and the solution is added dropwise to 1 mL of the precursor solution under vigorous vortexing to form an emulsion, the emulsion is subjected to ultrasound and evaporation treatment, and finally gelled under LED light irradiation to obtain a HANB / HAMA hydrogel loaded with budesonide.

[0019] The invention discloses a drug-loaded hydrogel obtained by the preparation method.

[0020] The invention also discloses the application of the drug-loaded hydrogel in preparing a drug delivery medium for treating ulcerative colitis.

[0021] Furthermore, the drug-loaded hydrogel is delivered to the intestine via an endoscope and then photocured to treat ulcerative colitis.

[0022] Compared with the prior art, the at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects:

[0023] (1) The present invention utilizes a light-triggered coupling reaction as a cross-linking platform to develop a drug-loaded hydrogel that is biocompatible and can achieve rapid in situ gelation under ultraviolet irradiation. Under irradiation, the aldehyde group generated by o-nitrobenzyl (NB) can rapidly react with the amino group on the surface of the intestinal tissue mucosa, ensuring good tissue adhesion through covalent anchoring in vivo. The drug-loaded HANB / HAMA hydrogel can be well adhered to the inflamed colon epithelium through an endoscopic system and ultraviolet induction;

[0024] (2) The present invention provides a targeted drug delivery platform for the treatment of UC. In a rat colitis model, our research results show that the drug delivery platform can significantly improve the efficacy of different UC drugs, including budesonide and mesalazine. Endoscopically guided administration can alleviate inflammatory responses and promote intestinal barrier repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without paying creative work.

[0026] Figure 1 Schematic diagram of the synthesis mechanism of HANB / HAMA hydrogel provided by the present invention;

[0027] Figure 2 The mechanical properties analysis diagram of the HANB / HAMA hydrogel prepared in Examples 1-3, wherein (A) is the tensile stress of the HANB / HAMA hydrogel prepared in Examples 1-3, (B) is the elastic modulus of the HANB / HAMA hydrogel prepared in Examples 1-3, (C) is the rheological test curve of the HANB / HAMA hydrogel prepared in Example 2, the intersection of G' (storage modulus) and G" (loss modulus) in the figure is the gelation point of the hydrogel during the illumination process, (D) is the compressive stress of the HANB / HAMA hydrogel prepared in Example 2, and (E) is the strain of the HANB / HAMA hydrogel prepared in Example 2;

[0028] Figure 3The in vitro tissue adhesion analysis diagram of the HANB / HAMA hydrogel prepared in Example 2, wherein (A) is a fluorescence image of the hydrogel on a wet pig skin structure, and (B) is a tissue adhesion photograph of the hydrogel on wet fresh pig skin;

[0029] Figure 4 The adhesion analysis diagram of HANB / HAMA hydrogel prepared in Example 2 on the colon mucosa, wherein (A) is a representative HE staining of HANB / HAMA (left, 3 days) and GelMA (right, 1 day) hydrogels adhered to the colon mucosa, and the black arrows respectively mark the positions where HANB / HAMA or GelMA hydrogels adhered to the mucosa (scale bar: 50 μm), (B) is a fluorescence image of different ICG-labeled hydrogels detected by IVIS imaging system, and (C) is the corresponding average fluorescence intensity of ICG-labeled hydrogels at different time points;

[0030] Figure 5 The in vitro drug release analysis diagram of the drug-loaded hydrogel, wherein (A) is budesonide and (B) is mesalamine;

[0031] Figure 6 In vivo pharmacokinetic analysis of drug-loaded hydrogel, wherein (A) is the concentration analysis of budesonide in the colon, (B) is the area under the curve AUC of budesonide in the colon, (C) is the concentration analysis of budesonide in plasma, and (D) is the area under the curve AUC of budesonide in plasma;

[0032] Figure 7 The figures are the research results of the therapeutic effect of drug-loaded hydrogel in UC, where (A) is the change of rat body weight, (B) is the change of rat disease activity index (DAI), (C) is the quantitative colon length of each group at the end of the experiment, and (D) is the colon injury score of each group at the end of the experiment. DETAILED DESCRIPTION

[0033] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0034] The following describes the implementation methods of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0035] Example 1

[0036] (1) HA (1 g, molecular weight 340 kDa) and NB (37 mg, 0.1 mmol) were dissolved in 2 mL of dimethyl sulfoxide, 50 mL of 2-morpholineethanesulfonic acid solution (0.1 M, pH = 5.2) and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM, 0.2 g, 0.68 mmol) were added, and the mixture was stirred at 37 ° C for 10 h in the dark. HANB was obtained after dialysis and freeze-drying;

[0037] (2) HA (4 g, molecular weight 44 kDa) was dissolved in 100 mL of deionized water, 2.4 mL of methacrylic anhydride solution was added at 4°C, and then 5 M NaOH was added dropwise until the pH was maintained between 8 and 11. The reaction was continued at 4°C in the dark for 5 h, and then 1 M HCl was added dropwise to adjust the pH of the solution to 7. After the solution was centrifuged, the supernatant was collected, dialyzed and freeze-dried to obtain HAMA;

[0038] (3) HANB, HAMA and LAP were dissolved in PBS at pH 7.4, wherein the total content of HANB, HAMA and LAP in PBS was 2.37 w / v%, and the mass ratio of HANB, HAMA and LAP was 2:0.25:0.12, and then the HANB, HAMA and LAP were measured at 395 nm and 50 mW / cm 2 Under the above conditions, LED light was irradiated for 10 s for gelation to obtain HANB / HAMA hydrogel.

[0039] Example 2

[0040] (1) HA (1 g, molecular weight 340 kDa) and NB (37 mg, 0.1 mmol) were dissolved in 2 mL of dimethyl sulfoxide, 50 mL of 2-morpholineethanesulfonic acid solution (0.1 M, pH = 5.2) and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM, 0.2 g, 0.68 mmol) were added, and the mixture was stirred at 37 ° C for 12 h in the dark. HANB was obtained after dialysis and freeze-drying;

[0041] (2) HA (4 g, molecular weight 44 kDa) was dissolved in 100 mL of deionized water, 2.4 mL of methacrylic anhydride solution was added at 4°C, and then 5 M NaOH was added dropwise until the pH was maintained between 8 and 11. The reaction was continued at 4°C in the dark for 4 h, and then 1 M HCl was added dropwise to adjust the pH of the solution to 7. After the solution was centrifuged, the supernatant was collected, dialyzed and freeze-dried to obtain HAMA;

[0042] (3) HANB, HAMA and LAP were dissolved in PBS at pH 7.4, wherein the total content of HANB, HAMA and LAP in PBS was 4.74 w / v%, and the mass ratio of HANB, HAMA and LAP was 4:0.25:0.24, and then the HANB, HAMA and LAP were measured at 395 nm and 50 mW / cm 2 Under the above conditions, LED light was used for 10 s for gelation to obtain HANB / HAMA hydrogel.

[0043] Example 3

[0044] (1) HA (1 g, molecular weight 340 kDa) and NB (37 mg, 0.1 mmol) were dissolved in 2 mL of dimethyl sulfoxide, 50 mL of 2-morpholineethanesulfonic acid solution (0.1 M, pH = 5.2) and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM, 0.2 g, 0.68 mmol) were added, and the mixture was stirred at 37 ° C for 11 h in the dark. HANB was obtained after dialysis and freeze-drying;

[0045] (2) HA (4 g, molecular weight 44 kDa) was dissolved in 100 mL of deionized water, 2.4 mL of methacrylic anhydride solution was added at 4°C, and then 5 M NaOH was added dropwise until the pH was maintained between 8 and 11. The reaction was continued in the dark at 4°C for 6 h, and then 1 M HCl was added dropwise to adjust the pH of the solution to 7. After the solution was centrifuged, the supernatant was collected, dialyzed and freeze-dried to obtain HAMA;

[0046] (3) HANB, HAMA and LAP were dissolved in PBS at pH 7.4, wherein the total content of HANB, HAMA and LAP in PBS was 7.11 w / v%, and the mass ratio of HANB, HAMA and LAP was 6:0.75:0.36, and then the HANB, HAMA and LAP were measured at 395 nm and 50 mW / cm 2 Under the above conditions, LED light was irradiated for 10 s for gelation to obtain HANB / HAMA hydrogel.

[0047] The mechanical properties of the HANB / HAMA hydrogels prepared in Examples 1-3 were analyzed by an INSTRON 68SC-1 tensile testing machine. For the tensile test, the hydrogel samples were prepared in a dumbbell-shaped mold (3.3×20×1.9 mm), and the samples were stretched to failure at a test rate of 5 mm / min. For the compression test, the hydrogel samples were prepared in a cylindrical mold (10 mm in diameter, 3 mm in height). Ten cycles of compression tests were performed at 10%, 20%, and 30% for each strain, and the speed was set to 1 mm / min. The above hydrogels were exposed to light (395 nm LED, 50 mW / cm 2 ) completely gelled. Figure 2 A and 2B show that the HANB / HAMA hydrogels prepared in Examples 1-3 all have good mechanical properties, excellent elasticity and tensile strength, and the HANB / HAMA hydrogel prepared in Example 2 (solid content of 4.74%) has the longest elongation and the highest tensile strength of 200.675±13.025 kPa. Through the PTPC reaction, the HANB / HAMA hydrogel prepared in Example 2 quickly gelled within 3.362±0.261s, and the final storage modulus was 14615±745 Pa ( Figure 2 C) from Figure 2 D and 2E, it can be seen that the compressive stress of the HANB / HAMA hydrogel prepared in Example 2 is 846.667±15.031 kPa, and in the repeated compression test with strains of 10, 20, and 30%, the mechanical properties of the HANB / HAMA hydrogel do not decrease significantly during the compression cycle.

[0048] Example 4 Preparation of Mesalazine-loaded HANB / HAMA Hydrogel

[0049] (2) HA (1 g, molecular weight 340 kDa) and NB (37 mg, 0.1 mmol) were dissolved in 2 mL of dimethyl sulfoxide, 50 mL of 2-morpholineethanesulfonic acid solution (0.1 M, pH = 5.2) and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM, 0.2 g, 0.68 mmol) were added, and the mixture was stirred at 37 ° C for 12 h in the dark. HANB was obtained after dialysis and freeze-drying;

[0050] (3) HA (4 g, molecular weight 44 kDa) was dissolved in 100 mL of deionized water, 2.4 mL of methacrylic anhydride solution was added at 4°C, and then 5 M NaOH was added dropwise until the pH was maintained between 8 and 11. The reaction was continued at 4°C in the dark for 4 h, and then 1 M HCl was added dropwise to adjust the pH of the solution to 7. After the solution was centrifuged, the supernatant was collected, dialyzed and freeze-dried to obtain HAMA;

[0051] (4) Mesalazine Mes was dissolved in PBS at a concentration of 50 mg / mL, and then HANB, HAMA and LAP were added at 37°C to form an emulsion, wherein the total content of HANB, HAMA and LAP in the emulsion was 4.74 w / v%, and the mass ratio of HANB, HAMA and LAP was 4:0.25:0.24. The emulsion was then sonicated using a probe sonicator (Scientz Ultrasonic Homogenizer JY92-IIN) (40% amplitude, 3 s on, 3 s off, a total of 20 s) to further emulsify the solution. Subsequently, the organic phase was evaporated under mild vacuum conditions, and finally the emulsion was stirred at 395 nm, 50 mW / cm 2 Under the above conditions, LED light was used for 10 s to perform gelation to obtain a mesalazine-loaded hydrogel.

[0052] Example 5 Preparation of budesonide-loaded HANB / HAMA hydrogel

[0053] (1) HA (1 g, molecular weight 340 kDa) and NB (37 mg, 0.1 mmol) were dissolved in 2 mL of dimethyl sulfoxide, 50 mL of 2-morpholineethanesulfonic acid solution (0.1 M, pH = 5.2) and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM, 0.2 g, 0.68 mmol) were added, and the mixture was stirred at 37 ° C for 12 h in the dark. HANB was obtained after dialysis and freeze-drying;

[0054] (2) HA (4 g, molecular weight 44 kDa) was dissolved in 100 mL of deionized water, 2.4 mL of methacrylic anhydride solution was added at 4°C, and then 5 M NaOH was added dropwise until the pH was maintained between 8 and 11. The reaction was continued at 4°C in the dark for 4 h, and then 1 M HCl was added dropwise to adjust the pH of the solution to 7. After the solution was centrifuged, the supernatant was collected, dialyzed and freeze-dried to obtain HAMA;

[0055] (3) HANB, HAMA and LAP were first dissolved in PBS with a pH of 7.4 to form a HANB / HAMA hydrogel precursor solution. Then, 500 μg of budesonide Bud was dissolved in 0.1 mL of dichloromethane and added dropwise to 1 mL of the HANB / HAMA hydrogel precursor solution under vigorous vortexing to form an emulsion, wherein the total content of HANB, HAMA and LAP in the emulsion was 4.74 w / v%, and the mass ratio of HANB, HAMA and LAP was 4:0.25:0.24. The emulsion was then transferred to an ice bath and sonicated using a probe sonicator (Scientz Ultrasonic Homogenizer JY92-IIN) (40% amplitude, 3 s on, 3 s off, for a total of 20 s) to further emulsify the emulsion. The organic phase was then evaporated under mild vacuum conditions and finally irradiated at 395 nm, 50 mW / cm 2 Under the above conditions, LED light was used for 10 s to perform gelation to obtain a budesonide-loaded hydrogel.

[0056] Example 6 Analysis of the adhesion ability of HANB / HAMA hydrogel on tissue surface

[0057] To evaluate the adhesion ability of HANB / HAMA hydrogel on tissue surfaces, we deposited indocyanine green (ICG)-labeled HANB / HAMA hydrogel precursor on wet porcine skin and exposed it to ultraviolet light (395 nm LED, 50 mW / cm 2 ) in situ gelation. After repeated stretching, twisting, bending and washing with water, the HANB / HAMA hydrogel still adhered tightly to the wet tissue surface ( Figure 3 A). To further verify the strong tissue adhesion of HANB / HAMA hydrogel on pig skin, we used fluorescence microscopy to examine the intimate interface between the hydrogel and the tissue surface. The fluorescence images revealed the tight interaction between pig skin and HAN B / HAMA hydrogel ( Figure 3 B).

[0058] To further illustrate the adhesion of HANB / HAMA hydrogel on the tissue surface, the tightness between the hydrogel and the tissue surface was examined using a fluorescence microscope. HANB / HAMA precursors (HANB (4 w / v%), HAMA (5 w / v‰), and LAP (2.4 w / v‰)) were delivered to the rat colon using a small animal endoscope with a working channel from KARL STORZ. Subsequently, 395 nm UV light (50 mW / cm 2 ) for 10 s to form HANB / HAMA hydrogel in situ. Figure 4 The results of A showed that HANB / HAMA hydrogels could firmly adhere to the mucosa 3 days after delivery, in contrast, the control group hydrogel gelatin methacrylate (GelMA (20 w / v%)) containing 0.2 w / v% LAP) showed obvious detachment 1 day after delivery.

[0059] In order to monitor the retention of HANB / HAMA hydrogel in the colon, fluorescent ICG-HANB / HAMA hydrogel was delivered to the rat colon using an endoscope. Specifically, indocyanine green ICG was incorporated into the HANB / HAMA hydrogel as a fluorescent indicator, and ICG-labeled HANB / HAMA hydrogel was prepared according to the method of Example 2 by replacing HANB with ICG-labeled HANB, which was synthesized by condensation of ICG amine groups with carboxylic acid groups on HANB, and ICG-GelMA was synthesized by the same method, and the ICG-HANB / HAMA precursor was dissolved at a solid content of 1% (w / v).

[0060] Rats with DSS-induced colitis were fasted for 24 h and their abdominal hair was shaved before imaging. Rats were given 1 mL of ICG solution (denoted as Ctrl), ICG-GelMA, and ICG-HANB / HAMA through endoscopy, and fluorescence was detected using IVIS Spectrum (AniView, China) on days 0, 1, and 3. The results showed that HANB / HAMA hydrogel showed significant adhesion ability in rat colon compared with ICG-GelMA and free ICG solution ( Figure 4 B and C).

[0061] Example 7 In vitro drug release analysis of drug-loaded hydrogel

[0062] 0.2 g of hydrogel loaded with budesonide (Bud) and mesalamine (Mes) were dissolved in 1 mL of PBS with a pH of 7.4, respectively, and shaken at 100 rpm and 37°C. The amount of budesonide and mesalamine released from the hydrogel was determined using a UV / vis spectrometer and a Waters e2695 high performance liquid chromatograph (HPLC). The results are as follows: Figure 5As shown. The detector of HPLC is Waters 2489 UV / vis detector; mobile phase A is 42% acetonitrile, mobile phase B is 58% water (containing 1‰ trifluoroacetic acid); the chromatographic column model is XBridge C18, the chromatographic column specification is 5μm, 4.6×250mm; the flow rate is 1mL / min; the injection volume is 10μL; the column temperature is 35℃; and the detection wavelength is 246nm.

[0063] The drug release characteristics of hydrogels depend on cross-linking density, degradation and microenvironment. Budesonide and mesalamine are both commonly used drugs for the treatment of UC. In vitro drug release studies have shown that ( Figure 5 A and B), budesonide exhibited a slow and sustained release from the hydrogel, whereas mesalazine was released significantly faster from the hydrogel, which may be due to more extensive hydrogen bonding and cross-linking between budesonide and the hydrogel.

[0064] Example 8 In vivo pharmacokinetic analysis of drug-loaded hydrogel

[0065] Experimental animals: Adult male Sprague-Dawley rats (200-220 g) were used to establish a chronic colitis model. All animal experiments were approved by the Experimental Animal Center of Shanghai Cancer Center, Fudan University. Rats were raised in a specific pathogen-free (SPF) animal barrier and placed in a room with a humidity of 55±15% and a temperature of 22±5°C, with sufficient space for activities and sufficient food and drinking water.

[0066] Dextran sodium sulfate-induced colitis model in rats: After a one-week adaptation period, SD rats (n=60) drank 5% DSS for one week, and normal rats (n=6) drank purified water for one week, and then replaced it with ordinary water for one week, for a total of three cycles to induce chronic colitis. Endoscopic evaluation was performed at the end of weeks 1, 3, and 5 to simulate the course of chronic ulcerative colitis. After chronic colitis was confirmed at the end of the first cycle of intervention, the DSS-induced model rats were randomly divided into different treatment groups.

[0067] According to the above scheme, after DSS-induced ulcerative colitis was formed in rats, equal doses of drugs were administered to rats by different delivery methods, drug-loaded hydrogel (1.5 mg / kg) (Bud (hydrogel)-E group) was perfused by endoscopy, and the corresponding drug solution was administered by gavage (Bud-G group) or enema (Bud-E group). Blood samples were collected at different time points (0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48 h), colon tissue was collected at time points (0, 0.5, 1, 2, 4, 8, 12, 24, 48 h) in the Bud-G group, Bud-E group and Bud (hydrogel)-E group, and whole blood samples were centrifuged at 3500 rpm for 10 min to obtain plasma. All collected plasma and colon tissue were stored at -80 ° C. The concentration of budesonide in plasma and colon was then analyzed by mass spectrometry.

[0068] The results showed that although enema and hydrogel-mediated delivery achieved similar maximum budesonide concentrations (C max ) However, the release of drugs delivered by hydrogels in the colon was more sustained ( Figure 6 A), the cumulative drug exposure of hydrogel delivery was 9.59 times that of enema delivery ( Figure 6 B), In contrast, delivery with HANB / HAMA hydrogel significantly reduced plasma C max and cumulative drug exposure (6C and D). Hydrogel delivery resulted in plasma C max The total drug release was reduced by 70.3% and 42.7%. In summary, it was proved that the HANB / HAMA hydrogel of the present invention can effectively promote targeted drug release and improve the colonic bioavailability of budesonide in the treatment of UC.

[0069] Example 9 Study on the therapeutic effect of drug-loaded hydrogel in UC

[0070] The DSS-induced chronic enteritis model established in Example 8 was used to study the therapeutic effect of the drug-loaded hydrogel on UC. The colitis rats were divided into groups, including healthy mice fed with water only as a control (n=6, Ctrl), PBS-treated DSS-induced chronic colitis rat group (n=10, DSS), HANB / HAMA-treated DSS-induced chronic colitis rat group (n=10, DSS / Hydrogel), budesonide solution-treated DSS-induced chronic colitis rat group (n=10, DSS / Bud), and budesonide-treated DSS-induced chronic colitis mouse group-hydrogel group (n=10, DSS / Bud (Hydrogel)). Considering the sustained release effect of the drug-loaded hydrogel, in order to reduce the damage and irritation caused by endoscopic administration in rats, and considering the patient's compliance with subsequent clinical transformation, DSS / Bud (Hydrogel) was administered once every three days, DSS / Bud was administered once a day, and the budesonide dosage was 0.5 mg / kg / d.

[0071] from Figure 7 It can be seen that the DSS / Bud (Hydrogel) treatment group significantly improved the weight gain of rats and reduced the disease activity index (DAI) ( Figure 7 A and B) When hydrogel-mediated drug delivery was used, mice showed increased colon length and reduced colon injury scores (a quantification of inflammation severity, depth of inflammation, and extent of glandular damage) ( Figure 7 C and D), in summary, the drug-loaded hydrogel delivery method of the present invention can significantly improve the therapeutic effect of UC in rats.

[0072] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a drug-loaded hydrogel, characterized in that: The following steps are involved: (1) HA and NB were dissolved in dimethyl sulfoxide, 2-morpholineethanesulfonic acid solution and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride were added, and stirred for 10-12 hours in the dark, and HANB was obtained after dialysis and freeze-drying; (2) HA was dissolved in deionized water, methacrylic anhydride solution was added, and then 5 M NaOH was added dropwise until the pH was maintained between 8 and 11. The reaction was continued in the dark for 4 to 6 hours, and then 1 M HCl was added dropwise to adjust the pH of the solution to 7. The solution was centrifuged and the supernatant was collected. The supernatant was dialyzed and freeze-dried to obtain HAMA; (3) After the drug is fully dissolved, HANB obtained in step (1), HAMA obtained in step (2) and photoinitiator LAP are added at 37° C. to form an emulsion, the emulsion is subjected to ultrasound and evaporation treatment, and finally gelled under LED light irradiation to obtain the drug-loaded hydrogel.

2. The preparation method according to claim 1, characterized in that In the step (1), the mass volume ratio of HA, NB, dimethyl sulfoxide, 2-morpholineethanesulfonic acid solution and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride is 1 g:37 mg:2 mL:50 mL:0.2 g.

3. The preparation method according to claim 1, characterized in that: The mass volume ratio of HA, deionized water and methacrylic anhydride solution in step (2) is 4 g:100 mL:2.4 mL.

4. The preparation method according to claim 1, characterized in that: In the step (3), the total content of HANB, HAMA and LAP in the emulsion is 2.37-7.11 w / v%.

5. The preparation method according to claim 4, characterized in that: The total content of HANB, HAMA and LAP in the emulsion is 4.74 w / v%, wherein the mass ratio of HANB, HAMA and LAP is 4:0.25:0.

24.

6. The preparation method according to claim 1, characterized in that: The LED light irradiation conditions in step (3) are 395nm, 50mW / cm 2 Irradiate for 10 s.

7. The preparation method according to claim 1, characterized in that: The drugs in step (3) include mesalazine and budesonide; when the drug is mesalazine, the specific operation of step (3) is as follows: mesalazine is dissolved in PBS with a pH of 7.4 at a concentration of 50 mg / mL, and then HANB, HAMA and LAP are added at 37° C. to form an emulsion, the emulsion is subjected to ultrasound and evaporation treatment, and finally gelled under LED light irradiation to obtain a mesalazine-loaded HANB / HAMA hydrogel; When the drug is budesonide, the specific operation of step (3) is as follows: first, HANB, HAMA and LAP are dissolved in PBS with a pH of 7.4 to form a precursor solution, then 500 μg of budesonide is dissolved in 0.1 mL of dichloromethane, and the solution is added dropwise to 1 mL of the precursor solution under vigorous vortexing to form an emulsion, the emulsion is subjected to ultrasound and evaporation treatment, and finally gelled under LED light irradiation to obtain a HANB / HAMA hydrogel loaded with budesonide.

8. The drug-loaded hydrogel obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the drug-loaded hydrogel according to claim 8 in preparing a drug delivery medium for treating ulcerative colitis.

10. The use according to claim 9, characterized in that The drug-loaded hydrogel is delivered to the intestine through an endoscope and then photocured to treat ulcerative colitis.

Images