PH-responsive attapulgite microsphere-based drug loading system, preparation method thereof and application of pH-responsive attapulgite microsphere-based drug loading system in preparation of drugs for treating colitis

Abstract

The invention discloses a pH response type attapulgite microsphere-based drug loading system, a preparation method thereof and application of the pH response type attapulgite microsphere-based drug loading system in preparation of drugs for treating colitis, and belongs to the technical field of medicines and biological materials. The invention aims to construct a drug carrier with colon targeted drug release, anti-inflammatory treatment and flora regulation functions by optimizing an attapulgite loading process and introducing a colon environment-responsive coating material, and solves the problems of uncontrollable drug release, insufficient flora regulation and poor targeting in the prior art. The drug entrapment rate of the prepared pH response type attapulgite microsphere-based drug loading system is greater than or equal to 95%, the 8h release rate in simulated gastric fluid (pH 1.2) is less than or equal to 10%, and the 24h release rate in simulated colon fluid (pH 7.4) is greater than or equal to 60%.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and biomaterials, and particularly relates to a pH-responsive attapulgite microsphere-based drug-carrying system, a preparation method thereof, and an application thereof in preparing a drug for treating colitis. Background Art

[0002] Colitis is a common inflammatory bowel disease characterized by abdominal pain, diarrhea, and intestinal mucosal damage, which can lead to colon cancer in severe cases. Current treatments, such as aminosalicylic acids and glucocorticoids, suffer from significant side effects, low bioavailability, and high recurrence rates. Furthermore, intestinal dysbiosis is a key contributing factor to colitis, and traditional medications struggle to directly regulate the balance of the microbiota, limiting their effectiveness.

[0003] Berberine is a natural alkaloid with anti-inflammatory, antibacterial, and intestinal flora-modulating properties. However, its poor water solubility and rapid intestinal absorption make it difficult to effectively retain in the colon, and high doses can easily cause gastrointestinal irritation. While existing drug carriers such as liposomes and polymer microspheres can improve drug release, they suffer from complex preparation processes, insufficient colon targeting, and limited microbial modulatory effects.

[0004] Attapulgite, a natural nano-silicate material with a high specific surface area, strong adsorption, and biocompatibility, has been used for drug loading. However, berberine-loaded attapulgite tends to release the drug rapidly in the intestinal environment, failing to achieve colon-targeted sustained release. Furthermore, existing technologies fail to fully utilize the physicochemical properties of attapulgite to achieve pH-responsive drug release and achieve synergistic regulation between attapulgite-loaded drugs and intestinal flora.

[0005] In addition, although the prior art also discloses "Preparation and Evaluation of Dung Beetle Chitosan Microspheres Loaded with Berberine Hydrochloride", the release rate of the drug-loaded microspheres disclosed in the prior art in simulated gastric fluid is 49.05%, and a large amount of drug is wasted in the stomach; in addition, the encapsulation rate of the drug-loaded microspheres disclosed in the prior art can only reach 80.05%±5.73%, which also causes waste of drugs in the pharmaceutical process and low drug utilization rate.

[0006] In summary, the technical problems to be solved by the present invention are as follows:

[0007] (1) Direct loading of drugs with attapulgite causes severe burst release: The high adsorption of attapulgite leads to the initial burst release of berberine in the intestine (release rate > 85% within 8 hours), which induces side effects and reduces the therapeutic effect.

[0008] (2) Traditional berberine preparations are released too quickly in the colon: Due to the poor water solubility of berberine and its rapid intestinal absorption, existing carriers cannot achieve colon-targeted sustained release, resulting in low drug utilization.

[0009] (3) Existing drug carriers have limited effects on regulating intestinal flora: there is a lack of carrier design that can synergize with drugs, making it difficult to regulate the balance of the flora in the long term through sustained-release drugs. Summary of the Invention

[0010] Based on the above reasons, in response to the problems or defects existing in the prior art, the purpose of the present invention is to provide a pH-responsive attapulgite microsphere-based drug loading system, a preparation method thereof, and an application in the preparation of drugs for treating colitis, so as to solve or at least partially solve the above-mentioned technical defects existing in the prior art: the present invention aims to optimize the attapulgite loading process and introduce a colon environment-responsive coating material to construct a drug carrier with colon-targeted drug release, anti-inflammatory treatment and microflora regulation functions, so as to solve the problems of uncontrollable drug release, insufficient microflora regulation and poor targeting in the prior art.

[0011] In order to achieve one of the above purposes of the present invention, the technical solution adopted by the present invention is as follows:

[0012] A method for preparing a pH-responsive attapulgite microsphere-based drug-carrying system, the method specifically comprising the following steps:

[0013] (1) Preparation of attapulgite microspheres

[0014] The purified attapulgite (ATT) is mixed with a dispersant, a binder, and a solvent according to a ratio, and ball-milled to form a slurry; the slurry is then spray-dried to form microspheres, and the microspheres are then degreased and acid-activated to obtain attapulgite microspheres;

[0015] (2) Preparation of attapulgite microsphere-based drug-loaded particles

[0016] The attapulgite microspheres described in step (1) are dispersed in a berberine (BBR) solution in proportion and stirred for a period of time; after the stirring is completed, the resulting mixture is centrifuged to collect the precipitate to obtain an ATT-BBR complex; and the ATT-BBR complex is then freeze-dried to obtain attapulgite microsphere-based drug-loaded particles;

[0017] (3) Preparation of pH-responsive attapulgite microsphere-based drug delivery system

[0018] The attapulgite microsphere-based drug-loaded particles described in step (2) are mixed with the pH-responsive polymer according to a ratio, stirred evenly, and then vacuum-dried to obtain the pH-responsive attapulgite microsphere-based drug-loaded system.

[0019] Furthermore, in step (1) of the above technical solution, in a preferred embodiment of the present invention, the mass ratio of the purified attapulgite to the dispersant and binder is 25:2.5:3.

[0020] Furthermore, in step (1) of the above technical solution, the dispersant is at least one of ammonium polyacrylate, sodium polyacrylate, sodium hexametaphosphate, etc.

[0021] Specifically, in step (1) of the above technical solution, the main function of the dispersant is to form a double electric layer (Zeta potential enhancement) on the surface of the attapulgite particles through ionization, significantly improving the electrostatic repulsion energy between particles and preventing agglomeration.

[0022] Furthermore, in step (1) of the above technical solution, the adhesive is at least one of polyvinyl alcohol, polyethylene glycol (PEG), polylactic acid-glycolic acid copolymer (PLGA), etc.

[0023] Specifically, in step (1) of the above technical solution, the binder is mainly adsorbed on the surface of the attapulgite particles through hydrogen bonds and van der Waals forces, forming a "particle-polymer-particle" bridging structure, enhancing the binding force between particles and promoting the subsequent formation of complete microspheres in the spray drying process.

[0024] Furthermore, in step (1) of the above technical solution, the solvent may be an inorganic solvent or an organic solvent; the inorganic solvent is preferably deionized water; the organic solvent is preferably an alcohol solvent, for example, any one of anhydrous ethanol or isopropyl alcohol.

[0025] Furthermore, in step (1) of the above technical solution, the amount of the solvent used is not specifically limited, as long as the attapulgite, dispersant, and binder are uniformly mixed to form a slurry. For example, the mass ratio of the attapulgite, dispersant, binder, and solvent is 25:2.5:3:69.5.

[0026] Furthermore, in step (1) of the above technical solution, the ball milling time is 12-36 hours. In a preferred embodiment of the present invention, the ball milling time is 24 hours.

[0027] Furthermore, in step (1) of the above technical solution, the specific process parameters adopted for the spray drying are as follows: feed rate 3-8 mL / min, inlet temperature 130-170°C, outlet temperature 100-140°C, and air flow rate 10-20 L / min.

[0028] Furthermore, in step (1) of the above technical solution, the degreasing treatment temperature is 280-350° C. In a preferred embodiment of the present invention, the degreasing temperature is 330° C.

[0029] Furthermore, in step (1) of the above technical solution, the specific method of the acid activation treatment is as follows:

[0030] The defatted microspheres are soaked in dilute hydrochloric acid for 3-6 hours, then taken out and washed with alkaline solution until the pH value of the washing solution reaches 6.5-7.5, and finally freeze-dried.

[0031] Specifically, in step (1) of the above technical solution, the role of the acid activation is to increase the nanopore size and specific surface area of ​​the prepared attapulgite microspheres, change the surface charge, and enhance the adsorption capacity of the attapulgite microspheres.

[0032] Furthermore, in step (2) of the above technical solution, the concentration of the berberine solution is 5-25 mg / mL, for example, 6.29 mg / mL.

[0033] Furthermore, in step (2) of the above technical solution, the mass ratio of the attapulgite microspheres to the berberine in the berberine solution is 1:0.25-1:2. In a preferred embodiment of the present invention, the mass ratio of the attapulgite microspheres to the berberine in the berberine solution is 1:1.

[0034] Furthermore, in step (2) of the above technical solution, the stirring time is 6-10 hours.

[0035] Specifically, in step (2) of the above technical solution, the function of the stirring is to ensure that berberine is fully contacted and evenly dispersed with the attapulgite microspheres, and that berberine is adsorbed inside the pores and on the outer surface of the attapulgite microspheres.

[0036] Furthermore, in step (3) of the above technical solution, the mass ratio of the attapulgite microsphere-based drug-loaded particles to the pH-responsive polymer is 1:0.5 to 1:1.

[0037] Furthermore, in step (3) of the above technical solution, the pH-responsive polymer is a methacrylic acid copolymer, and its coating thickness is controlled by stirring time, so that the drug-loaded microspheres can achieve 60%-90% drug sustained release under a pH 7.4 environment.

[0038] Furthermore, in step (3) of the above technical solution, in a preferred embodiment of the present invention, the pH responsive polymer is FS 30D (abbreviated as FS).

[0039] Furthermore, in step (3) of the above technical solution, the stirring time is 8-16 hours.

[0040] The second object of the present invention is to provide a pH-responsive attapulgite microsphere-based drug-carrying system prepared by the above-mentioned method.

[0041] The third object of the present invention is to provide the use of the pH-responsive attapulgite microsphere-based drug-carrying system prepared by the above-mentioned method in the preparation of oral drugs for treating colitis.

[0042] The fourth object of the present invention is to provide an oral medicine for treating colitis, wherein the medicine comprises the pH-responsive attapulgite microsphere-based drug delivery system prepared by the method described above.

[0043] The present invention utilizes attapulgite as a drug delivery system for treating colitis and regulating intestinal flora balance through the sustained release of berberine. Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) The release rate of the pH-responsive attapulgite microsphere-based drug-loading system provided by the present invention in simulated gastric fluid is only 5.79%, which greatly reduces the release of drugs in the stomach and reduces the waste of drugs in the stomach.

[0045] (2) The encapsulation efficiency of the pH-responsive attapulgite microsphere-based drug-loading system provided by the present invention can reach 99.66±0.00%, reducing the waste of drugs during the preparation process.

[0046] (3) The present invention provides a pH-responsive oral drug delivery system based on attapulgite microspheres, which has multiple technical advantages and inventive merits, including pH-responsive drug release, prolonged drug retention time in the colon, reduced systemic drug exposure, improved intestinal microbiota, simple and efficient preparation process, broad application prospects, reduced inflammatory factor levels, and promoted macrophage M2 polarization. This invention has important application value in the development of oral formulations for inflammatory bowel disease (IBD) and other gastrointestinal diseases.

[0047] (4) The present invention uses a spray drying method to prepare attapulgite microspheres, which is simple and easy to scale up. The simplicity and efficiency of the preparation process make this drug delivery system highly promising for industrial production, reducing production costs and improving production efficiency.

[0048] (5) The attapulgite microspheres prepared by the present invention have good adhesion and can remain in the colon for a longer period of time, thereby prolonging the release time of the drug. The high drug encapsulation efficiency and prolonged drug retention time in the colon help improve drug bioavailability, reduce dosing frequency, and improve patient compliance.

[0049] (6) The pH-responsive attapulgite microsphere-based drug delivery system prepared by the present invention has a drug encapsulation efficiency of ≥95%, an 8-h release rate of ≤10% in simulated gastric fluid (pH 1.2), and a 24-h release rate of ≥60% in simulated colon fluid (pH 7.4). BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0051] Figure 1 This is the infrared spectrum of the raw material components, intermediate products and the finally synthesized drug carrier FS-ATT-BBR microspheres used in Example 1.

[0052] Figure 2 In Example 1: (A) scanning electron microscope image of ATT microspheres; (B) scanning electron microscope image of FS-ATT-BBR microspheres; (C) particle size distribution of ATT microspheres; (D) particle size distribution of FS-ATT microspheres.

[0053] Figure 3 Comparison of the in vitro release curves of the attapulgite microsphere-based drug-loaded particles ATT-BBR and FS-ATT-BBR microspheres prepared in Example 1 in simulated gastric fluid (pH 1.2), simulated small intestinal fluid (SIF Ph 6.8) and simulated colonic fluid (SCF, pH 7.4), respectively.

[0054] Figure 4 Colon appearance (A), colon length (B), and spleen index (C) of each group of mice in Example 2. n = 6. Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001. Compared with the DSS group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0055] Figure 5 This is a schematic diagram of the process flow for preparing FS-ATT-BBR microspheres according to Example 1 of the present invention.

[0056] Figure 6 This is the standard curve of berberine solution.

[0057] Figure 7 is the surface zeta potential of BBR, ATT-BBR and FS-ATT-BBR.

[0058] Figure 8 Schematic diagram of the mechanism of FS-ATT-BBR prepared in Example 1 in treating inflammatory bowel disease (IBD).

[0059] Figure 9 Schematic diagram of the construction and drug administration of ulcerative colitis model mice.

[0060] Figure 10The weight (A) and DAI score (B) of each group of mice in Example 2 are shown (n=6).

[0061] Figure 11 (A) Typical pathological examination results of colon tissue from each group of mice in Application Example 2. (B) Histological injury scores of colon tissue. n = 6. Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001. Compared with the DSS group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0062] Figure 12 Figure 2. Colonic TNF-α (A), IL-6 (B), IL-1β (C), MPO (D), and IL-10 (E) levels in each group of mice in Example 2. n = 6. Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001. Compared with the DSS group, *p < 0.05, **p < 0.01, ***p < 0.001.

[0063] Figure 13 Figure 2: (A) Immunohistochemical staining for iNOS / CD206, a key marker of macrophage M1 / M2 polarization, and (B, C) positive rates. n = 6. Compared with the control group, #p < 0.05, ##p < 0.01, ###p < 0.001. Compared with the DSS group, *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0064] The process innovation of the present invention lies in: integrating the triple process features of attapulgite microsphere preparation → drug loading → pH-responsive coating, and verifying the parameter range (such as acid activation time control and coating ratio) through examples.

[0065] The adsorption capacity of attapulgite is enhanced through acidification treatment to avoid excessive sudden release of drugs.

[0066] The present invention is further described in detail below through an implementation case. This implementation case is implemented based on the technology of the present invention. Detailed implementation methods and specific operating procedures are now given to illustrate the creativity of the present invention, but the protection scope of the present invention is not limited to the following implementation case.

[0067] Based on the information contained in this application, it will be readily apparent to those skilled in the art that various changes can be made to the precise description of the present invention. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are provided only to illustrate specific aspects of the present invention.

[0068] In order to better understand the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values ​​should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0069] The equipment and raw materials used in the present invention can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0070] The attapulgite used in the following examples was purchased from Xuyi Botu Attapulgite Co., Ltd.

[0071] The berberine used in the following examples was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with a specification of ≥99% and a CAS number of 2086-83-1.

[0072] The following examples use The specification of FS 30D is 1L and was purchased from Shanghai Changwei Pharmaceutical Excipients Technology Co., Ltd.

[0073] Example 1

[0074] The present embodiment provides a method for preparing a pH-responsive attapulgite microsphere-based drug-carrying system, the method specifically comprising the following steps:

[0075] (1) Preparation of attapulgite microspheres

[0076] (i) Purification of attapulgite

[0077] Mix attapulgite with deionized water at a mass ratio of 1:40. Stir thoroughly with a magnetic stirrer for 24 hours, then let it stand. Once the mixture is completely separated, pour off the supernatant liquid by pouring it off. Transfer the middle layer to a new beaker to remove the sand and gravel below. Add water to the original volume. Repeat this process three times. Centrifuge the mixture, remove the upper precipitate, and dry it in a freeze dryer for 24 hours. Grind it, pass it through a 200-mesh sieve, and continue drying it in a 70°C oven until ready for use.

[0078] (ii) Preparation of attapulgite microspheres

[0079] 25 g of the purified attapulgite from step (i), 2.5 g of ammonium polyacrylate, 3 g of polyvinyl alcohol, and 69.5 g of deionized water were mixed in a mass ratio of 25:2.5:3:69.5 and ball-milled for 24 hours to form a slurry. Microspheres were prepared by spray drying using the following process parameters: feed rate 5 mL / min, nozzle inner diameter 0.7 mm, inlet temperature 150°C, outlet temperature 120°C, and air flow rate 15 L / min. The microspheres were degreased at 330°C for 6 hours. Finally, the degreased attapulgite microspheres were acidified in 1 M dilute hydrochloric acid solution at 75°C for 4 hours, neutralized with NaOH solution to pH 7, and finally freeze-dried in a freeze dryer for 24 hours to obtain attapulgite microspheres.

[0080] (2) Preparation of attapulgite microsphere-based drug-loaded particles

[0081] Dissolve 250 mg of berberine in 20 mL of boiling water to obtain a 12.5 mg / mL berberine solution;

[0082] 250 mg of the attapulgite microspheres prepared in step (1) were mixed with the berberine solution described above and stirred for 8 hours to obtain a berberine-loaded attapulgite particle suspension. The suspension was centrifuged at 4000 revolutions per minute for 5 minutes, and the precipitate was collected to obtain an ATT-BBR complex. The supernatant obtained after centrifugation was used for subsequent UV spectrophotometric testing; finally, the ATT-BBR complex was freeze-dried for 24 hours to obtain attapulgite microsphere-based drug-loaded particles (ATP-BBR-NPs); wherein: the mass ratio of the attapulgite microspheres to berberine was 1:1;

[0083] (3) Preparation of pH-responsive attapulgite microsphere-based drug delivery system

[0084] ATP-BBR-NPs were mixed with FS 30D was combined and uniformly dispersed in an ultrasonic emulsifier (ultrasound 5 seconds, stop 5 seconds, a total of 10 minutes, 50%), followed by magnetic stirring for 12 hours and then drying under vacuum for 15 hours. Finally, the sample was crushed with liquid nitrogen to obtain FS 30D is the shell and attapulgite is the core of the microsphere FS-ATT-BBR; wherein: the ATP-BBR-NPs and The quality ratio of FS 30D is 1:1.

[0085] The test shows that the coating thickness of FS in FS-ATT-BBR microspheres is 2.18 μm.

[0086] Example 2

[0087] The present embodiment provides a method for preparing a pH-responsive attapulgite microsphere-based drug delivery system, which is substantially the same as that of Example 1, except that the mass ratio of the attapulgite microspheres to berberine in step (2) of the present embodiment is 1:0.25.

[0088] Example 3

[0089] The present embodiment provides a method for preparing a pH-responsive attapulgite microsphere-based drug delivery system, which is substantially the same as that of Example 1, except that the mass ratio of the attapulgite microspheres to berberine in step (2) of the present embodiment is 1:0.5.

[0090] Example 4

[0091] The present embodiment provides a method for preparing a pH-responsive attapulgite microsphere-based drug delivery system, which is substantially the same as that of Example 1, except that the mass ratio of the attapulgite microspheres to berberine in step (2) of the present embodiment is 1:2.

[0092] Example 5

[0093] The present embodiment provides a method for preparing a pH-responsive attapulgite microsphere-based drug delivery system, which is substantially the same as that of Example 1, except that the mass ratio of the attapulgite microspheres to berberine in step (2) of the present embodiment is 1:3.

[0094] Example 6

[0095] The preparation method of a pH-responsive attapulgite microsphere-based drug-loading system in this embodiment is basically the same as that in Example 1, except that the stirring time in step (3) of this embodiment is 8 hours.

[0096] Example 7

[0097] The preparation method of a pH-responsive attapulgite microsphere-based drug delivery system in this embodiment is basically the same as that in Example 1, except that the stirring time in step (3) of this embodiment is 16 hours.

[0098] Example 8

[0099] The present embodiment provides a method for preparing a pH-responsive attapulgite microsphere-based drug-loading system, which is substantially the same as that of Example 1, with the only difference being that the mass ratio of the attapulgite microsphere-based drug-loading particles to the pH-responsive polymer in step (3) of the present embodiment is 1:0.5.

[0100] Example 9

[0101] The present embodiment provides a method for preparing a pH-responsive attapulgite microsphere-based drug-loading system, which is substantially the same as that of Example 1, with the only difference being that the mass ratio of the attapulgite microsphere-based drug-loading particles to the pH-responsive polymer in step (3) of the present embodiment is 1:2.

[0102] Structural testing:

[0103] Figure 1 This is the infrared spectrum of the raw material components used in Example 1 and the final synthesized drug carrier FS-ATT-BBR microspheres. Figure 1 Infrared spectrum analysis showed that all components in the drug carrier were present.

[0104] Figure 1 From top to bottom are the infrared spectrum of berberine (BBR), the infrared spectrum of attapulgite microspheres (ATT), Infrared spectrum of FS 30D (FS), infrared spectrum of attapulgite microspheres-berberine complex (ATT-BBR) and infrared spectrum of FS-ATT-BBR. In order to illustrate that the model drug was successfully loaded into the drug delivery system and FS 30D is coated on the surface. The inventors used Fourier transform infrared spectroscopy (FTIR) to prove the chemical composition. The infrared spectrum of BBR (berberine) is 1502cm -1 : This peak usually corresponds to the bending vibration (δ(NH)) of the NH group in berberine, indicating the presence of an amino structure in the berberine molecule. Other characteristic peaks: The infrared spectrum of berberine may also show other characteristic peaks, such as CH stretching vibration (about 2900cm -1 ), C=O stretching vibration (about 1700cm -1 ), etc., depending on the molecular structure of berberine. The infrared spectrum of ATT (attapulgite) is 1030cm -1 :This peak usually corresponds to the stretching vibration of Si-O-Si, indicating the presence of silicon-oxygen bond structure in attapulgite. Other characteristic peaks: The infrared spectrum of attapulgite may also show OH stretching vibration (about 3400cm -1 ) and Si-OH vibration (about 900 cm -1 ), these peaks indicate the presence of hydroxyl and silanol structures in attapulgite. FS( FS 30D) infrared spectrum 1735cm -1 :This peak usually corresponds to the stretching vibration of C=O, indicating There are ester or carboxyl structures in FS 30D. Other characteristic peaks: The infrared spectrum of FS 30D may also show CH stretching vibration (about 2900 cm -1 ) and COC stretching vibration (about 1100 cm-1 ), these peaks indicate the presence of alkyl and ether bond structures in the polymer. Comprehensive analysis: Combination of BBR and ATT: In the infrared spectra of BBR and ATT, the 1502 cm -1 1030cm of peak and ATT -1 Peak, indicating that berberine was successfully loaded onto attapulgite. Changes after FS coating: In the infrared spectrum of FS-ATT-BBR, the peak of FS at 1735 cm -1 Peak, indicating FS 30D was successfully coated on the surface of attapulgite microspheres. Infrared spectrum analysis showed that berberine was successfully loaded onto attapulgite and FS 30D was successfully coated on the surface of attapulgite microspheres. These results confirmed the successful preparation of the drug delivery system, which was pH-responsive and capable of releasing drugs in the colon.

[0105] The micromorphology of ATT and FS-ATT was characterized by scanning electron microscopy (SEM). Figure 2 (A and B) are shown. The SEM images clearly show that the surface of the microspheres presents a significant irregular wrinkled structure. This unique rough surface structure may be mainly due to the mechanical force generated during the grinding process. Analysis shows that this rough texture on the surface of the microspheres can effectively improve their adhesion properties, which is conducive to enhancing the adhesion and retention of the microspheres in the intestinal mucosa, thereby potentially improving the bioavailability of the drug and prolonging its release time in the intestine. Further observation of the particle size distribution of the microspheres, such as Figure 2 As shown in (C and D), ATT-BBR still maintains complete structural morphology within the particle size range of 7 μm, which shows that the system has good stability at the micron level. After FS 30D coating treatment, the particle size of FS-ATT-BBR increased to 13 μm. The significant increase in particle size confirmed the successful combination of the coating material and attapulgite, which may provide new ideas for the optimized design of drug delivery systems.

[0106] To evaluate the surface potential of BBR, ATT-BBR, and FS-ATT-BBR, the inventors used a zeta potential analyzer. The procedure involved dispersing a small amount of sample powder in an appropriate solution to form a stable suspension. The suspension was then placed in the cell of a particle size analyzer for zeta potential measurement. To ensure data reliability, the measurement was repeated three times for each sample, and the average result was taken as the final result.

[0107] The results are as follows Figure 7As shown, the surface charges of BBR, ATT-BBR and FS-ATT-BBR in water are -7.23±0.31, -3.80±0.09 and -15.97±0.37, respectively.

[0108] Encapsulation efficiency test:

[0109] First, draw a BBR standard curve. Accurately weigh 20 mg of BBR in a beaker and dissolve in 150 mL of boiling water. Let cool and transfer to a 200 mL volumetric flask to volume to obtain a 100 μg / mL BBR reference stock solution. Measure 0.4, 0.8, 1.2, 2.4, 3.6, and 4.8 mL of this stock solution into a 10 mL volumetric flask and dilute to the mark with deionized water to obtain a series of BBR standard solutions at concentrations of 4, 8, 12, 24, 36, and 48 μg / mL. Scan the BBR standard solution using a UV spectrophotometer to determine its maximum absorption wavelength (345 nm). Measure the absorbance of the BBR standard solution at each concentration at this wavelength. Draw a regression curve for BBR, with the abscissa representing the BBR concentration and the ordinate representing the absorbance value corresponding to that concentration.

[0110] The BBR content in the supernatant was quantified at a wavelength of 345 nm. The drug loading rate (DL) and encapsulation efficiency (EE) were calculated using the following formula:

[0111]

[0112] The berberine solution was scanned at full wavelength, and the maximum absorption wavelength of BBR was 427nm. The BBR standard curve was drawn with this wavelength as the measurement wavelength. Figure 6 As shown in the figure, the standard curve equation of BBR is Y=0.0588X-0.0457, R 2 =0.9999 (n=3), indicating that there was a good linear relationship between the berberine concentration and the absorbance value in the concentration range of 4-48 μg / mL.

[0113] This study systematically investigated the loading performance of berberine (BBR) on attapulgite (ATT) for the first time. As shown in Table 1, when the mass ratio of ATT to BBR reached 1:1, the BBR encapsulation efficiency (EE) reached its maximum value (99.66% ± 0.00%). Based on the above research results, subsequent experiments all used an ATT-BBR mass ratio of 1:1 for sample preparation. The needle-like pore structure of ATT significantly improved the adsorption efficiency of drug molecules (especially small molecule drugs). After H + Acid-treated ATT can replace its internal Mg 2+ , Al3+ and Fe 3+The cation exchange capacity of ATT and other metal ions is enhanced, thereby more efficiently adsorbing alkaline drugs (such as BBR). The experimental results show that the solid-liquid ratio (ATT / solvent) is positively correlated with the drug loading. After spray drying, the surface of the microspheres still maintains the porous topology of ATT. Therefore, FS 30D shell wrapped in the outer layer. FS 30D enteric coating layer, the final drug loading rate is about 25%.

[0114] Table 1 Drug loading rate and encapsulation efficiency of ATT-BBR at different ATT to BBR mass ratios in Examples 1-4

[0115]

[0116] Calculation shows that the encapsulation efficiency of ATT-BBR in Example 1 is the highest, which is 99.66%.

[0117] The present invention tests the anti-colitis effect of the prepared pH-responsive attapulgite microsphere-based drug-carrying system by adopting a DSS-induced colitis model in mice and establishing a control experiment for comparison, thereby setting up Application Example 1.

[0118] Application Example 1

[0119] The drug release behavior of berberine-loaded microspheres in different pH environments was evaluated by a multi-stage in vitro dissolution experiment. The specific experimental process is as follows: the ATT-BBR and FS-ATT-BBR microsphere samples prepared in Example 1 were placed in a centrifuge tube containing 50 mL of artificial gastric fluid (SGF, pH 1.2) and incubated for 2 hours, then transferred to artificial intestinal fluid (SIF, pH 6.8) for 4 hours, and finally monitored for 42 hours of release in artificial colonic fluid (SCF, pH 7.4). The experiment was carried out at a constant temperature of 37 ° C throughout the experiment, and dynamic dissolution was performed using the dialysis bag method (100 rpm slow oscillation). Dissolution samples were collected at preset time points (1, 2, 3, 4, 6, 8, 12, 24, 36, 48h), and an equal volume of fresh medium was added after each sampling to maintain the sink condition. The drug concentration was determined by ultraviolet spectrophotometry (detection wavelength 345nm). All data points were obtained through three independent parallel experiments, and the cumulative drug release rate was calculated according to the following formula.

[0120] Q n =C n ×V0+(C1+C2+C3+...C n-1 )×V

[0121]

[0122] where Q nrepresents the total amount of berberine released in a given time; Q represents the total amount of berberine in the microspheres; Cn represents the concentration of berberine in the solution within a predetermined time; V0 represents the total volume of the simulated liquid in the dissolution container, and V represents the volume of liquid extracted each time.

[0123] The sustained release and burst release of drugs were tested and compared. Figure 3 It can be seen that the release rate of berberine-loaded attapulgite without microspheres is significantly faster than that of the drug carrier coated with microspheres. This drug carrier has certain performance improvements in both sustained drug release and burst drug release.

[0124] In vitro release performance test results:

[0125] Contrast wrapping FS 30D and unwrapped Burst release and sustained release properties of FS 30D microspheres. Figure 3 The dissolution behaviors of ATT-BBR and FS-ATT-BBR prepared in Example 1 in simulated gastric fluid (SGF, pH 1.2) and different intestinal fluid environments were systematically compared: the cumulative release rate of FS-ATT-BBR in SGF was only 5.97%, indicating that its gastric release inhibition effect was significant; in simulated small intestinal fluid (SIF, pH 6.8) and simulated colonic fluid (SCF, pH 7.4), the release rates were increased to 17.63% and 69.27%, respectively, showing pH-dependent release characteristics. The release rate of ATT-BBR in SGF was as high as 39.26%, and the cumulative release rate within 6 hours reached 73.39%, and it was almost completely released within 12 hours, indicating that its release kinetics was dominated by the concentration gradient and there was a burst release phenomenon. The pH-sensitive release characteristics of FS-ATT-BBR can be attributed to The molecular design of the FS 30D enteric coating layer includes: Low pH inhibited release: The carboxylic acid groups in the coating remain non-ionized in an acidic environment, maintaining membrane structural integrity and effectively blocking drug diffusion. High pH triggered release: In the neutral / weakly alkaline environment of the colon, the carboxylic acid groups ionize, causing coating erosion and rapid drug release through pores. The burst release rate of FS-ATT-BBR is lower than that of ATT-BBR, demonstrating that the coating significantly improves release kinetics.

[0126] Application Example 2

[0127] In this application example, a mouse acute colitis model was established by inducing DSS. A control group, a DSS model group, a free BBR treatment group, an ATT-BBR (adsorbed BBR microspheres) treatment group prepared in Example 1, and a FS-ATT-BBR (functionalized surface adsorbed BBR microspheres) treatment group prepared in Example 1 were set up. By comparing indicators such as weight change, disease activity index (DAI), colon length, and histological damage score of each group of mice, the in vivo therapeutic effect of BBR-loaded microspheres was comprehensively evaluated. Body weight and DAI are important parameters reflecting the overall health status of mice and disease progression; shortening of colon length is a direct reflection of the severity of colitis; and histological damage score can more carefully reveal pathological changes such as mucosal epithelial cell desquamation, erosion, and inflammatory cell infiltration.

[0128] The specific steps of the in vivo anti-inflammatory test of this application example are as follows:

[0129] (1) Grouping

[0130] SPF male Kunming (KM) mice, 6-8 weeks old, were fed adaptively for one week and then the dextran sulfate sodium (DSS) acute colitis model was established. The mice were randomly divided into five groups (n=7) according to their body weight, including: healthy control (Control) group, DSS model (DSS) group, berberine raw material (BBR) group, ATT-BBR group and FS-ATT-BBR group.

[0131] (2) Modeling and drug administration

[0132] Dextran sulfate sodium (DSS) was dissolved in deionized water to prepare a 4% w / v solution. The solution was sterilized through a 0.22 μm filter and dispensed into sterile drinking water dispensers. The experimental period lasted for 7 days. The DSS solution in the modeling group was replaced every 24 hours to maintain a constant concentration. The healthy control group was supplied with sterile deionized water throughout the experiment. The dosing schedule for each treatment group followed the modeling group standard.

[0133] A simultaneous drug administration method was used to treat different groups of mice. Specifically, the BBR group was dosed at 100 mg / kg. For the ATT-BBR and FS-ATT-BBR groups, the dose was calculated based on the same amount of BBR as in the BBR group to ensure fairness and accuracy of the experiment.

[0134] On days 1 to 7 of the experimental period, the healthy control group (Control group) was continuously supplied with sterile deionized water to maintain the basic physiological state, and the other five groups of experimental animals were given 4% w / v dextran sulfate sodium (DSS) solution through free drinking water.

[0135] From the start of the experiment to the 10th day, the control group and the disease model group (DSS group) were orally gavaged with 0.5% sodium carboxymethylcellulose aqueous solution daily, while the drug-treated groups were given BBR, ATT-BBR, and FS-ATT-BBR suspension preparations (constant dosing volume 10 mL / kg·d), respectively. All gavage operations were completed within a fixed time window to control circadian rhythm interference.

[0136] At the end of the experiment on day 10, the rats were fasted in metabolic cages for 12 hours to effectively remove gastrointestinal residues and ensure the accuracy of tissue sampling.

[0137] The next day, the animals were euthanized by cervical dislocation, and colon and spleen samples were collected.

[0138] The construction of ulcerative colitis model mice and the time of drug administration are shown in Figure 9 .

[0139] Result testing and analysis

[0140] (I) Mouse weight and DAI score

[0141] Starting from the day of dosing, mice were weighed daily to monitor weight changes. Fecal samples were collected for occult blood and hematochezia, and the stool characteristics and quantity were recorded in detail. Fecal occult blood was tested using a dedicated fecal occult blood qualitative test kit to ensure the accuracy of the results. Based on daily observations, a systematic score was assigned according to the DAI scoring standard.

[0142] DAI = (weight loss score + stool characteristics score + fecal occult blood / colonic bleeding score) / 3, scoring details see Figure 10 .

[0143] The daily body weight and DAI scores of mice in each group are shown in Figure 10 .

[0144] Body weight changes and disease activity index of colitis animals are the criteria for evaluating the colitis model and treatment efficacy. Although all treatment groups showed a significant reduction in DAI scores (DSS: 1.14 ± 0.30 vs. BBR: 0.67 ± 0.00, p < 0.01), the FS-ATT-BBR group showed a unique therapeutic advantage in weight restoration. Although the ATT-BBR group and the FS-ATT-BBR group used the same attapulgite carrier, the ATT-BBR group lacked The FS 30D coating failed to achieve colon-specific release. This resulted in premature drug release in the stomach, potentially explaining the similar weight recovery patterns between the ATT-BBR and BBR solution groups. In contrast, the sustained weight gain in the FS-ATT-BBR group from day 4 onward suggests that its sustained-release system was able to maintain therapeutic drug concentrations in the inflamed areas of the colon.

[0145] The importance of targeted drug delivery was further validated by the intergroup differences in DAI scores. Although the BBR solution group had the lowest DAI value (0.67 ± 0.00), this group failed to improve body weight, suggesting that simply increasing the dose may induce gastrointestinal irritation. The FS-ATT-BBR group achieved the best balance between DAI improvement (0.71 ± 0.12) and weight restoration, confirming the unique value of smart drug carriers in optimizing the therapeutic window.

[0146] (2) Sample collection and processing

[0147] In the mouse feces collection experiment, six healthy mice were randomly selected from each group and placed in clean, sterilized cages. The mice were then allowed to defecate naturally. Three to five fresh feces pellets were collected from each mouse and immediately placed in pre-labeled cryovials. Subsequently, the feces were quickly frozen in liquid nitrogen to maximize the activity and diversity of the fecal microbiota and prevent deterioration or degradation during subsequent storage. Finally, the cryovials were properly stored at -20°C for subsequent microbial diversity analysis.

[0148] Without dissecting the abdominal cavity, separate the spleen and colon using forceps, and remove surrounding intestinal fat. Immediately measure colon length and photograph the macroscopic appearance of the intestinal tract. Rinse the intestine three times with pre-chilled saline, remove moisture with filter paper, and accurately weigh. Fix the mid-segment of the colon in 4% paraformaldehyde for 24 hours. Aliquot the remaining tissue and quickly freeze at -80°C.

[0149] The spleens isolated synchronously were accurately weighed and the spleen index (= spleen mass (mg) / body weight (g)) was calculated.

[0150] The colon length and spleen index of mice in each group are shown in Figure 4 .

[0151] Colon length can be used as a macroscopic indicator to assess the severity of colitis, and shortened colon length indicates an aggravated inflammatory response. Experimental animals in the colitis model group induced by dextran sulfate sodium (DSS) showed significant colon shortening, with an average colon length of 7.8 cm, which was statistically significantly different from 11.3 cm in the normal control group, indicating that the intestinal inflammation model was successfully constructed. Intervention experiment data showed that the colon length of the FS-ATT-BBR group recovered to 10.8 cm, and the colon length of the BBR group recovered to 10.5 cm. It is worth noting that the colon length of the ATT-BBR group was 9.7 cm, which showed an improvement trend compared with the model group, but was not as good as the FS-ATT-BBR group and the BBR group.

[0152] The spleen index also reflects the degree of inflammatory response in the body. Experimental data showed that the average spleen index of healthy mice was 2.5. In sharp contrast, the index in the disease model group showed a statistically significant upward trend, reaching 3.4. Compared with the model group, the spleen index of mice in the BBR group, ATT-BBR group, and FS-ATT-BBR group was significantly reduced. This therapeutic advantage may come from a synergistic mechanism: (1) local high concentrations of BBR directly inhibit proinflammatory cytokines; (2) improving the anti-inflammatory microenvironment by restoring microbial diversity.

[0153] (III) Pathological examination of mouse colon and pathological changes of mouse colon

[0154] Colonic specimens were fixed in 4% paraformaldehyde for 24 hours and then embedded in graded paraffin. Ultrathin sections (5 μm thickness) were prepared using an ultrathin microtome. After dewaxing with xylene and hydration with graded ethanol, hematoxylin and eosin double staining (H&E) was performed. The staining process included key steps such as nuclear-cytoplasmic differentiation (treatment with 1% hydrochloric acid and ethanol for 15 seconds), bluing (immersion in saturated lithium carbonate solution for 30 seconds), and neutral gum sealing. Digital images were acquired using an optical microscope and histopathology scores (HS) were calculated. The scoring criteria for pathological tissue sections are shown in Table 2.

[0155] Table 2HS scoring criteria

[0156]

[0157] Typical hematoxylin-eosin (H&E) staining photos and HE scores of the colon sections of mice in each group are shown in Figure 2. Figure 11 .

[0158] H&E staining visually demonstrated that DSS-induced colitis in KM mice exhibited extensive epithelial erosion, reduced goblet cells, and pronounced inflammatory infiltrates (arrowheads), with an average histopathological score of 6.333. Notably, mucosal repair efficacy varied significantly between treatment groups: oral administration of the BBR solution partially alleviated the inflammatory infiltrate (score decreased to 3.333, p < 0.01), but goblet cell loss and localized epithelial sloughing persisted. In contrast, the FS-ATT-BBR microsphere group exhibited near-normal mucosal architecture, with significant recovery of goblet cells and minimal inflammatory infiltrates (score 1.333, p < 0.01). The FS-ATT-BBR group demonstrated the best therapeutic effect. This difference may be due to the colon-targeted delivery mechanism, which reduces gastric drug waste caused by direct oral administration of berberine hydrochloride through controlled and sustained release in the colon. Compared with the low bioavailability of BBR solution due to gastric degradation, the colon-specific release of FS-ATT-BBR significantly increased the local drug concentration and directly inhibited proinflammatory cytokines (such as TNF-α and IL-6), thereby protecting goblet cells from apoptosis and promoting mucosal barrier repair.

[0159] (IV) Determination of inflammatory factors and MPO levels in mouse colon tissue:

[0160] Solid-phase enzyme-linked immunosorbent assay (ELISA) was used for quantitative detection of inflammatory factors. The specific operation procedures strictly followed the manufacturer's instructions. Pre-experimental preparation: The colon specimens frozen at -80°C were placed in an ice box for slow thawing. The tissue blocks (100 mg ± 2%) were accurately weighed and placed in a sterile culture dish and quickly cut into 1 mm 3 The fragments were added with pre-chilled phosphate buffer (0.01 M, pH 7.3) at a mass-to-volume ratio of 1:9, and then transferred to a 2 mL centrifuge tube containing 4 zirconium oxide grinding beads (3 mm diameter). Intermittent homogenization was performed using a high-throughput tissue disruptor at 65 Hz amplitude (operating parameters: 25 seconds / time × 3 cycles, with a 10-second ice bath in between). The homogenate was centrifuged at low temperature (4°C, 12,000 × g, 15 minutes), and the supernatant was collected and aliquoted into centrifuge tubes and stored at -20°C for testing. TNF-α, IL-1β, IL-6, and MPO levels in the supernatant were quantitatively detected according to the ELISA kit operating procedures.

[0161] Inflammatory factors and MPO levels in the colon Figure 12 shown.

[0162] MPO is primarily secreted by activated neutrophils and is often used as a marker for assessing inflammation levels. Compared with untreated controls, colonic MPO activity was significantly reduced in mice with colitis treated with FS-ATT-BBR (p < 0.01). Furthermore, the intensity of inflammatory cytokines correlated with the number of proinflammatory cytokines. BBR inhibited proinflammatory polarization of macrophages and significantly reduced the release of proinflammatory cytokines, including TNF-α and IL-6, by activating the STAT6 signaling pathway. Compared with the DSS-induced colitis control group, mice treated with FS-ATT-BBR showed significantly reduced local levels of proinflammatory cytokines, including IL-1β (p < 0.01), IL-6 (p < 0.01), and TNF-α (p < 0.01). Notably, the anti-inflammatory efficacy of FS-ATT-BBR was superior to that of BBR monotherapy, especially in terms of TNF-α inhibition (p < 0.01).

[0163] (V) Immunohistochemical staining of mouse colon and the effect of immune response in mouse colon

[0164] Macrophage polarization was assessed by immunohistochemistry. The following protocol was used: 7-μm-thick paraffin sections of the distal colon were labeled with antibodies against CD206 (a marker for M2 phenotype, diluted 1:200) or iNOS (a marker for M1 phenotype, diluted 1:200). Sections were deparaffinized with xylene, rehydrated through a series of ethanol gradients, and subjected to microwave-assisted heat antigen retrieval using citrate buffer (pH 6.0). Nonspecific binding sites were then blocked with 10% horse serum for 30 minutes. Sections were then incubated with primary antibodies (diluted 1:200) overnight at 4°C and biotinylated secondary antibodies (diluted 1:500) at room temperature. Signal amplification was performed using the Vectastain ABC kit, and peroxidase activity was visualized using DAB substrate. Nuclei were counterstained with hematoxylin.

[0165] Macrophage activation is generally divided into two categories: pro-inflammatory M1 and anti-inflammatory M2. Macrophages maintain mucosal balance by secreting a variety of cytokines. Therefore, our subsequent research focused on the intestinal immune response of colitis mice and examined the polarization of macrophages in their colonic tissues on day 11. The results were as follows: Figure 13 shown.

[0166] BBR blocks the AKT1 / SOCS1 / NF-κB signaling pathway, hindering the polarization of M1 macrophages and enhancing the polarization of M2 macrophages, thereby ameliorating intestinal inflammation and alleviating colitis. Immunohistochemical analysis of CD206 (an M2 marker) and iNOS (an M1 marker) demonstrated enhanced macrophage polarization in colitis mice treated with FS-ATT-BBR. Furthermore, FS-ATT-BBR significantly reduced the release of proinflammatory cytokines IL-1β, IL-6, and TNF-α and increased the level of the anti-inflammatory IL-10. In summary, oral administration of FS-ATT-BBR to colitis mice promoted M2 polarization of intestinal macrophages. During mucosal inflammation, selected leukocytes directly contact epithelial cells, secreting inflammatory cytokines that target proteins at intercellular junctions, thereby altering barrier function. Some proinflammatory cytokines reduce tight protein levels, trigger cytoskeletal contraction, and lead to epithelial cell death, thereby weakening barrier function. FS-ATT-BBR can control the immune response and maintain the balance between inflammatory and anti-inflammatory cytokine secretion, thereby restoring mucosal homeostasis.

Claims

1. A method for preparing a pH-responsive attapulgite microsphere-based drug delivery system, characterized in that: The method specifically comprises the following steps: (1) Preparation of attapulgite microspheres Purified attapulgite (ATT) is mixed with a dispersant and a binder according to a ratio, and ball-milled to form a slurry; the slurry is then spray-dried to form microspheres, and the microspheres are then degreased and acid-activated to obtain attapulgite microspheres; (2) Preparation of attapulgite microsphere-based drug-loaded particles The attapulgite microspheres described in step (1) are dispersed in a berberine (BBR) solution in proportion and stirred for a period of time; after the stirring is completed, the resulting mixture is centrifuged to collect the precipitate to obtain an ATT-BBR complex; and the ATT-BBR complex is then freeze-dried to obtain attapulgite microsphere-based drug-loaded particles; (3) Preparation of pH-responsive attapulgite microsphere-based drug delivery system The attapulgite microsphere-based drug-loaded particles described in step (2) are mixed with the pH-responsive polymer according to a ratio, stirred evenly, and then vacuum-dried to obtain FS-ATT-BBR microspheres, namely the pH-responsive attapulgite microsphere-based drug-loaded system.

2. The preparation method according to claim 1, wherein: Step (1), the specific method of the acid activation treatment is as follows: The defatted microspheres are soaked in dilute hydrochloric acid for 3-6 hours, then taken out and washed with alkaline solution until the pH value of the washing solution reaches 6.5-7.5, and finally freeze-dried.

3. The preparation method according to claim 1, wherein: In step (2), the concentration of the berberine solution is 5-25 mg / mL.

4. The preparation method according to claim 1, wherein: In step (2), the mass ratio of the attapulgite microspheres to the berberine in the berberine solution is 1:0.25-1:

2.

5. The preparation method according to claim 1, wherein: In step (3), the mass ratio of the attapulgite microsphere-based drug-loaded particles to the pH-responsive polymer is 1:0.5 to 1:

1.

6. The preparation method according to claim 1, wherein: The pH responsive polymer is a methacrylic acid copolymer.

7. A pH-responsive attapulgite microsphere-based drug delivery system prepared by the preparation method according to any one of claims 1 to 6.

8. Use of a pH-responsive attapulgite microsphere-based drug delivery system prepared by the preparation method according to any one of claims 1 to 6 in the preparation of an oral drug for treating colitis.

9. An oral medication for treating colitis, characterized by: The drug comprises a pH-responsive attapulgite microsphere-based drug-carrying system prepared by the preparation method according to any one of claims 1 to 6.

Images