Gastric retention drug-loading magnetic micro-bomb as well as preparation method and application thereof

Abstract

The invention discloses a gastric retention drug-loading magnetic micro-bomb as well as a preparation method and application thereof. Relates to the technical field of biological medicine. Comprising a micro-cartridge case, a magnet block and medicine. Under the guidance of an external magnetic field, positioning and long-time retention in the stomach can be achieved, meanwhile, medicine is slowly and continuously released in the stomach, stomach diseases are treated, the problem that the stomach diseases are difficult to cure due to the fact that the concentration of the medicine at the focus position is low is solved, and the medicine release rate is adjusted by adjusting the number and the aperture size of the small holes in the bottom of the micro-cartridge case. Personalized design can be carried out according to different disease requirements, the residence time of the medicine-carrying magnetic micro-bomb in the stomach is controlled, after medicine release of the medicine-carrying magnetic micro-bomb is completed, the external magnet block is removed, and the medicine-carrying magnetic micro-bomb is discharged out of the body through peristalsis of the gastrointestinal tract.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more particularly to a gastric retention drug-loaded magnetic microprojectile and a preparation method and application thereof. Background Art

[0002] Helicobacter pylori is a microaerophilic bacterium that parasitizes the lower layer of gastric mucus and can cause diseases such as gastritis, gastric ulcers and even gastric cancer. At present, the main means of clinical treatment of Helicobacter pylori infection is oral antibiotics, such as amoxicillin, clarithromycin and furazolidone, combined with proton pump inhibitors and bismuth agents. However, the main disadvantage of traditional oral preparations is that the drug stays in the stomach for a short time and the effective concentration in the stomach is difficult to maintain. The purpose of anti-Helicobacter pylori can only be achieved by taking multiple drugs frequently and in large doses. In addition, some patients who take large amounts of antibiotics for a long time may experience adverse reactions such as nausea, vomiting, and diarrhea, which may lead to patients stopping the drug without authorization, interrupting the course of treatment, and increasing the drug resistance of Helicobacter pylori. Therefore, the development of safe and efficient anti-Helicobacter pylori preparations is of great clinical significance.

[0003] Prolonging the gastric retention time of traditional oral antibiotics is a key issue in improving the efficacy of drugs against Helicobacter pylori. Currently, the most commonly used gastric retention drug delivery system in clinical practice is the floating tablet form, but the ratio of drugs to floating materials is strict and cannot be adjusted in time according to the needs of disease treatment and the patient's own characteristics. In addition, the floating ability of the tablet is reduced after dissolution, the gastric retention time is difficult to control, and the drug utilization is reduced.

[0004] Therefore, whether or not a gastric retention drug delivery system with controllable gastric retention time can be developed is the key to achieving efficient elimination of Helicobacter pylori, and is also a problem that those skilled in the art need to solve urgently. Summary of the invention

[0005] In view of this, the present invention provides a gastric retention drug-loaded magnetic microprojectile and its preparation method and application. A drug-loaded magnetic microprojectile is designed and prepared, which can be positioned and retained in the stomach for a long time under the guidance of an external magnetic field, while slowly and continuously releasing drugs in the stomach to treat gastric diseases, avoiding the problem that gastric diseases are difficult to cure due to low drug concentration in the lesion site.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A gastric retention drug-loaded magnetic micro-bullet comprises a micro-bullet shell, a magnet block and a drug; the main body of the micro-bullet shell is a hollow cylindrical structure, the bottom of the cylinder is a closed thin sheet, the thin sheet has through holes, and micro-needles with needle tips facing upward are evenly distributed on the top ring of the wall of the hollow cylindrical structure;

[0008] The cavity part of the hollow cylindrical structure is loaded with the medicine; and the magnet block is horizontally placed on the medicine.

[0009] Preferably: the length of the micro-bullet shell is selected from 0.2 cm to 2.5 cm; the inner diameter of the micro-bullet shell is selected from 0.1 cm to 1 cm; the outer diameter of the micro-bullet shell is selected from 0.12 cm to 1.02 cm;

[0010] The thickness of the sheet is 0.05mm~0.1mm;

[0011] The number of the through holes is selected from 1 to 4, and the diameter of the through holes is 0.1 mm to 0.5 mm;

[0012] The shape of the microneedles is selected from cone, cylinder, triangular or quadrangular, and the number is selected from 3 to 15;

[0013] The drugs include: clarithromycin, amoxicillin, tetracycline, metronidazole, rifamycin, furazolidone, levofloxacin, ciprofloxacin, preferably any one or a combination of clarithromycin, amoxicillin and levofloxacin.

[0014] Preferably: the length of the micro-shell is selected from 0.2 cm to 1 cm;

[0015] The inner diameter of the micro-shell is selected from 0.1 cm to 0.6 cm;

[0016] The outer diameter of the micro-bullet shell is selected from 0.12 cm to 0.62 cm.

[0017] The microneedle is in the form of a conical microneedle; the height of the conical microneedle is selected from 0.2 mm to 1.5 mm; the bottom circular diameter of the conical microneedle is selected from 0.2 mm to 3 mm;

[0018] Drug: Clarithromycin and 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid were mixed uniformly at a mass ratio of 2.2:1 and ground for 30 minutes to obtain a ground mixture of clarithromycin and 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid.

[0019] The present invention also provides a method for preparing any of the above-mentioned gastric-retaining drug-loaded magnetic microprojectiles, which are prepared by a mold infusion curing method or a 3D printing method.

[0020] Preferred: 3D printing method: fused deposition modeling 3D printing method or photo-curing 3D printing method.

[0021] Preferred: Photocuring 3D printing method: According to the model parameters of the micro-bullet shell designed by computer, the printing solution in the material tank of the molding equipment is cured layer by layer on the printer platform to prepare the micro-bullet shell.

[0022] Preferably: the photocuring 3D printing method is specifically as follows: according to the model parameters of the micro-bullet designed by the computer, the printing solution in the material tank of the molding equipment is solidified layer by layer on the printer platform to prepare the micro-bullet; the cavity part of the hollow cylindrical structure of the micro-bullet is loaded with drugs; the drugs are filled into the micro-bullet through the opening at the top of the micro-bullet. After the drugs are filled into the micro-bullet, a magnet block is placed horizontally above the drugs, and the micro-bullet 3D printing solution is used as a bonding material, which is dripped into the gap between the magnet block and the inner wall of the hollow cylinder of the micro-bullet, and is quickly placed under a 405nm laser light source for irradiation and curing, and the drugs and the magnet block are encapsulated into the micro-bullet to form a complete drug-loaded magnetic micro-bullet.

[0023] Preferably, the printing solution is selected from acrylic acid, methyl acrylate, polyacrylic fumarate, polyethylene glycol diacrylate, N-vinyl pyrrolidone, and siloxane.

[0024] Preferred: any one of the printing solutions Dental SG, Dental LT Clear V2 resin, BioMed Clear resin, and BioMed Amber resin.

[0025] The present invention also provides the use of any of the above-mentioned gastric retention drug-loaded magnetic microprojectiles, or any of the above-mentioned preparation methods in the preparation of drugs or diagnostic and therapeutic equipment.

[0026] Further: the drug filling volume is set according to the disease treatment needs, but does not exceed the top edge of the micro-shell.

[0027] The small holes at the bottom of the microshell can control the release of loaded drugs. The larger the diameter of the small holes and the more the number of small holes, the faster the drug is released.

[0028] After the drug-loaded magnetic micro-bullet is swallowed, it reaches the position in the stomach corresponding to the magnet block under the attraction of the magnetic field of the magnet block placed in advance at the corresponding skin of the stomach. By adjusting the position of the external magnet block, the specific position of the stomach where the drug-loaded magnetic micro-bullet reaches can be controlled. Generally, it is chosen to stay near the pylorus after reaching the pylorus as much as possible. Since the N pole and S pole of the magnet block inside the drug-loaded magnetic micro-bullet are known in advance, by adjusting the direction of the N pole and S pole of the external magnet block, the microneedle of the drug-loaded magnetic micro-bullet can be directed toward the stomach wall and inserted into the gastric mucosa layer.

[0029] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a gastric retention drug-loaded magnetic microprojectile and a preparation method and application thereof, and the technical effects achieved are:

[0030] Under the guidance of the external magnetic field, it can be positioned and retained in the stomach for a long time, while slowly and continuously releasing drugs in the stomach to treat gastric diseases, avoiding the problem that gastric diseases are difficult to cure due to low drug concentration in the lesion site, and can effectively treat various gastric diseases, including gastric cancer, gastric ulcer, gastritis, and gastric Helicobacter pylori infection. In particular, when drug-loaded magnetic microprojectiles are used to treat gastric Helicobacter pylori infection, the eradication rate of Helicobacter pylori can be improved, gastric tissue damage can be reduced, and the recurrence rate of Helicobacter pylori infection can be reduced.

[0031] By adjusting the number and size of the small holes at the bottom of the microbullet shell, the drug release rate can be adjusted, and personalized design can be made for different disease needs. The retention time of the drug-loaded magnetic microbullet in the stomach can be controlled. After the drug-loaded magnetic microbullet has released the drug, the external magnet block is removed, and the drug-loaded magnetic microbullet is excreted from the body through the peristalsis of the gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0033] Figure 1 The attached figure is a 3D printing design diagram of the micro cartridge case provided by the present invention.

[0034] Figure 2 The attached figure is a physical picture of the 3D printed micro cartridge case provided by the present invention.

[0035] Figure 3 The accompanying drawing is a schematic diagram of the drug-loaded magnetic microprojectile provided by the present invention.

[0036] Figure 4 The attached drawing is a schematic diagram of the appearance of the drug-loaded magnetic microprojectile provided by the present invention.

[0037] Figure 5 The attached drawings are scanning electron microscope images of 3D printed micro-shells with different aperture combinations provided by the present invention.

[0038] Figure 6 The accompanying drawing is a schematic diagram of the force-displacement curve of the tip of the 3D printed micro-bullet shell (type 300 / 4) provided by the present invention.

[0039] Figure 7 The accompanying drawings are scanning electron microscope images provided by the present invention, wherein CAM: clarithromycin; AA-2G: 2-O-α-D-pyranose glucopyranosyl-L-ascorbic acid; CPM: clarithromycin physical mixture; CGM: clarithromycin grinding mixture, the same below.

[0040] Figure 8 The accompanying drawing is an infrared spectrum diagram provided by the present invention.

[0041] Fig. 9 The attached figure is an X-ray diffraction diagram provided by the present invention.

[0042] Fig.10 The accompanying drawing is a differential scanning calorimetry diagram provided by the present invention.

[0043] Fig.11 The accompanying drawing is a schematic diagram of the solubility of clarithromycin before and after solubilization provided by the present invention.

[0044] Fig.12 The accompanying drawings are graphs showing the inhibition of Helicobacter pylori growth by clarithromycin at different concentrations and a grinding mixture thereof provided by the present invention, wherein BHI: brain heart infusion medium, blank control.

[0045] Fig.13 The accompanying drawings are schematic diagrams of the cumulative release curves provided by the present invention, wherein A: clarithromycin-loaded microbullets; B: clarithromycin-loaded ground mixture microbullets; C: cumulative release rate of clarithromycin from clarithromycin-loaded microbullets and clarithromycin-loaded ground mixture microbullets at 24 hours.

[0046] Fig.14 The attached drawings are X-ray images of the micro-projectiles provided by the present invention, wherein MG: magnetic guidance group; NMG: non-magnetic guidance group; the circle represents the stomach, the arrow represents the micro-projectile, and the square represents the ear tag.

[0047] Fig.15 The attached drawings are diagrams showing the retention of microprojectiles in mice at different time points in the magnetic guidance group (MG) and the non-magnetic guidance group (NMG) provided by the present invention. The black block in the abdomen represents the external magnet, the circle represents the stomach, and the arrow points to the microprojectile.

[0048] Fig.16 The attached drawings show the depth of the mouse gastric tissue punctured by the microneedle at the top of the magnetic microbullet provided by the present invention and the H&E stained sections of the gastric tissue after healing.

[0049] Fig.17 The attached figure is a complete shell diagram of the magnetic microprojectile excreted with feces provided by the present invention.

[0050] Fig.18 The accompanying drawings are schematic diagrams of the micro-projectiles provided by the present invention being injected into an agarose gel block (A) and injected into the pyloric position (B) under magnetic guidance; the squares represent the micro-projectiles, and the arrows represent the micro-holes left after the micro-needles of the micro-projectiles are removed from the gel block.

[0051] Fig.19The accompanying drawings are H&E stained sections of gastric tissue of mice infected with Helicobacter pylori treated with the drug-loaded magnetic microbullets provided by the present invention, wherein Healthy: healthy group; Model: model group (infected with Helicobacter pylori, no treatment); CAM: clarithromycin group (infected with Helicobacter pylori, gavage of clarithromycin suspension); CGM: clarithromycin grinding mixture group (infected with Helicobacter pylori, gavage of clarithromycin grinding mixture solution); CMMB: drug-loaded magnetic microbullet group (infected with Helicobacter pylori, gavage of one CMMB), the same below.

[0052] Fig. 20 The attached figure is a WS-stained section of the gastric tissue of mice infected with Helicobacter pylori treated with clarithromycin magnetic microprojectiles provided by the present invention.

[0053] Fig.21 The accompanying drawing is a schematic diagram of the content of Helicobacter pylori in gastric tissue provided by the present invention. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The embodiment of the invention discloses a gastric retention drug-loaded magnetic microprojectile and a preparation method and application thereof.

[0056] The preparation processes not mentioned in the examples are conventional processes, and the sources of raw materials not mentioned are all commercially available conventional sources, which will not be described in detail here.

[0057] Example 1

[0058] Gastric retention drug loading (clarithromycin, CAM) magnetic microprojectiles

[0059] Preparation of micro-bullet shells: The micro-bullet shells were prepared by conventional light-curing 3D printing methods: 3D design software was used to model a device consisting of a hollow cylindrical structure, a thin sheet structure with holes at the bottom of the cylinder, and microneedles with needle tips evenly distributed on the top ring of the cylinder wall, and the device was converted into a parameter file recognizable by a 3D printer; (at room temperature) the Dental SG resin solution that can be used for dental surgical guides was poured into the resin tank of the light-curing printer, the target file was selected, and the micro-bullet shells were printed; the micro-bullet shells were removed from the platform, the residual printing solution on the surface was cleaned, and after drying, they were placed under a 405nm light source for post-curing to obtain a hard micro-bullet shell;

[0060] The micro-bullet includes: a micro-bullet shell, a magnet block and medicine;

[0061] The main body of the micro-bullet is a hollow cylindrical structure. The bottom of the cylinder is a closed sheet with small holes running through it. Micro-needles with their needle tips pointing upward are evenly distributed on the top ring of the cylinder wall.

[0062] To further optimize the technical solution: the thickness of the sheet is 0.05 mm to 0.1 mm (0.08 mm in this embodiment), the number of through holes is selected from 1 to 4 (4 in this embodiment), and the diameter of the through holes is 0.1 mm to 0.5 mm (0.3 mm in this embodiment);

[0063] The number of microneedles is selected from 3 to 15 (8 in this embodiment);

[0064] The length of the micro-shell is selected from 0.2 cm to 2.5 cm, and can be individually designed and prepared according to the patient's esophagus and disease treatment needs (3.4 mm in this embodiment), the inner diameter of the micro-shell is selected from 0.1 cm to 1 cm (1.6 mm in this embodiment), and the outer diameter of the micro-shell is selected from 0.12 cm to 1.02 cm (2 mm in this embodiment).

[0065] The microneedle of this embodiment is conical in shape, and the microneedle can be inserted into the gastric mucosal tissue under the action of magnetic force, so that the microprojectile is fixed at a specific position to prevent the microprojectile from moving under the action of gastric mucus;

[0066] The height of the conical microneedle is selected from 0.2 mm to 1.5 mm, preferably from 0.5 mm to 1 mm (0.9 mm in this embodiment); the bottom circular diameter of the conical microneedle is selected from 0.2 mm to 3 mm (0.3 mm in this embodiment). The height and bottom area of ​​other types of microneedles can be designed accordingly with reference to the conical microneedle.

[0067] Magnet block: round built-in magnet (1350GS in this embodiment, 1mm in diameter, 0.5mm in height);

[0068] Assembly of drug-loaded (clarithromycin) magnetic microbullets: Fill the cavity of the microbullet shell with drug (clarithromycin powder), then place the built-in magnet block horizontally above the drug powder, take a small amount of printing solution (Dental SG resin solution) and drip it into the gap between the built-in magnet block and the microbullet shell, and quickly place it under a 405nm light source for curing, and encapsulate the drug and magnet into the microbullet shell to obtain a drug-loaded (clarithromycin) magnetic microbullet;

[0069] To further optimize the technical solution: different modeling can obtain micro-shells with different numbers of bottom drug release holes and pore diameters. When the number of holes is 2 and the pore diameter is 200μm, a 200 / 2 micro-shell can be obtained; when the number of holes is 4 and the pore diameter is 200μm, a 200 / 4 micro-shell can be obtained; when the number of holes is 2 and the pore diameter is 300μm, a 300 / 2 micro-shell can be obtained; when the number of holes is 4 and the pore diameter is 300μm, a 300 / 4 micro-shell can be obtained.

[0070] The gastric retention drug-carrying (clarithromycin) magnetic micro-projectile of this embodiment is type 300 / 4.

[0071] To further optimize the technical solution: the above-mentioned printing materials can be replaced with other photopolymerizable substances, and the drug (clarithromycin) can be replaced with other drugs.

[0072] Figure 1 This is a 3D printed design of the micro-bullet shell (specifically 300 / 4 type micro-bullet shell) of this embodiment. See the actual photo for details. Figure 2 (The rice grains are on the right to illustrate the size).

[0073] Example 2

[0074] Preparation method of gastric retention drug-loaded (clarithromycin and 2-O-α-D-pyranose glucopyranosyl-L-ascorbic acid, i.e., AA-2G, the same below) magnetic microprojectile

[0075] Preparation of micro-bullet shells: Micro-bullet shells are prepared by light-curing 3D printing: 3D design software is used to model and design a device consisting of a hollow cylindrical structure, a thin sheet structure with holes at the bottom of the cylinder, and microneedles with needle tips evenly distributed on the top ring of the cylinder wall, and converted into a parameter file recognizable by a 3D printer; at room temperature, the (Dental) SG resin solution that can be used for dental surgical guides is poured into the resin tank of the light-curing printer, the target file is selected, and the micro-bullet shell is printed. The micro-bullet shell is removed from the platform, the residual printing solution on the surface is cleaned, and after drying, it is placed under a 405nm light source for post-curing to obtain a hard micro-bullet shell;

[0076] Assembly of drug-loaded (clarithromycin and 2-O-α-D-glucopyranosyl-L-ascorbic acid) magnetic microprojectiles: Clarithromycin and 2-O-α-D-glucopyranosyl-L-ascorbic acid were uniformly mixed at a mass ratio of 2.2:1 and ground for 30 minutes to obtain a ground mixture CGM of clarithromycin and 2-O-α-D-glucopyranosyl-L-ascorbic acid;

[0077] The micro-bullet includes: a micro-bullet shell, a magnet block and medicine;

[0078] The main body of the micro-bullet is a hollow cylindrical structure. The bottom of the cylinder is a closed sheet with small holes running through it. Micro-needles with their needle tips pointing upward are evenly distributed on the top ring of the cylinder wall.

[0079] To further optimize the technical solution: the thickness of the sheet is 0.05 mm to 1 mm (0.08 mm in this embodiment), the number of through holes is selected from 1 to 4 (4 in this embodiment), and the diameter of the through holes is 0.1 mm to 0.5 mm (0.3 mm in this embodiment);

[0080] The number of microneedles is selected from 3 to 15 (8 in this embodiment);

[0081] The length of the micro-shell is selected from 0.2cm to 2.5cm (3.4mm in this embodiment), the inner diameter of the micro-shell is selected from 0.1cm to 1cm (1.6mm in this embodiment), and the outer diameter of the micro-shell is selected from 0.12cm to 1.02cm (2mm in this embodiment).

[0082] The microneedle is conical in shape; the height of the conical microneedle is selected from 0.2 mm to 1.5 mm (0.9 mm in this embodiment); the bottom circular diameter of the conical microneedle is selected from 0.2 mm to 3 mm (0.3 mm in this embodiment).

[0083] Magnet block: round built-in magnet (1350GS in this embodiment, 1mm in diameter, 0.5mm in height);

[0084] Assembly of drug-loaded (clarithromycin) magnetic microbullets: Fill the cavity of the microbullet shell with drug (clarithromycin powder), then place the built-in magnet block horizontally above the drug powder, take a small amount of printing solution (Dental SG resin solution) and drip it into the gap between the built-in magnet block and the microbullet shell, and quickly place it under a 405nm light source for curing, and encapsulate the drug and magnet into the microbullet shell to obtain a drug-loaded (clarithromycin) magnetic microbullet;

[0085] To further optimize the technical solution: different modeling can obtain micro-shells with different numbers of bottom drug release holes and pore diameters. When the number of holes is 2 and the pore diameter is 200μm, a 200 / 2 micro-shell can be obtained; when the number of holes is 4 and the pore diameter is 200μm, a 200 / 4 micro-shell can be obtained; when the number of holes is 2 and the pore diameter is 300μm, a 300 / 2 micro-shell can be obtained; when the number of holes is 4 and the pore diameter is 300μm, a 300 / 4 micro-shell can be obtained.

[0086] The CGM powder was filled into the cavity of the micro-bullet, and then the built-in magnet block was placed horizontally above the drug powder. A small amount of printing solution was dripped into the gap between the built-in magnet block and the micro-bullet, and then quickly placed under a 405nm light source for curing. The drug and magnet were encapsulated into the micro-bullet to obtain drug-loaded (clarithromycin and 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid) magnetic micro-bullets.

[0087] Schematic diagram of the assembly of 300 / 4 type drug-loaded (clarithromycin and 2-O-α-D-pyranosyl-L-ascorbic acid) magnetic microprojectiles is shown in Figure 3 , see the appearance photos Figure 4 , referred to as CMMB.

[0088] Technical effect verification:

[0089] 1. Properties of micro cartridges

[0090] Materials: Micro-shell prepared according to Example 1.

[0091] Method: The sample was observed by scanning electron microscope (SEM) with an accelerating voltage of 20 kV. The sample was analyzed for mechanical strength of the needle tip by electronic universal testing machine with a compression rate of 0.15 mm / min. The test was stopped when the displacement reached 800 μm, and the force-displacement curve was plotted.

[0092] Results: SEM images are shown in Figure 5 As shown in Figure 1, the 3D printed micro-shell has a complete structure, the main body is hollow, and 8 microneedles of equal size are evenly distributed on the top of the hollow cylinder. From the top view, it can be observed that the microneedles have sharp tips and a distinct layered structure. Figure 5 Middle A. The hole structure at the bottom of different types of micro-shells is complete, see Figure 5 Middle B, illustrates that the embodiment uses light-curing 3D printing technology with sufficient precision to successfully print sharp microneedles to provide better puncture performance and penetration ability while reducing damage to tissues, while the micropores at the bottom provide a release channel for the drug.

[0093] After mechanical compression testing, the micro-bullet needle tip structure of the present invention showed excellent mechanical properties, and the compressive strength reached 56.83N. Figure 6 , indicating that the micro-bullet shell of the present invention not only has sufficient hardness to penetrate the gastric mucosa, but also has sufficient strength to withstand various stresses that may occur during the drug release process.

[0094] 2. Characterization of Clarithromycin Grinding Mixture

[0095] Materials: Clarithromycin (CAM); 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G); Helicobacter pylori.

[0096] Methods: CAM and AA-2G (CAM:AA-2G=2.2:1.0, w / w) were mixed and then the physical mixture of CAM and AA-2G (CPM, where CAM:AA-2G=2.2:1.0, w / w) was obtained. CAM and AA-2G (CAM:AA-2G=2.2:1.0, w / w) were mixed and then ground for 30 min to obtain the ground mixture of CAM and AA-2G (CGM, where CAM:AA-2G=2.2:1.0, w / w).

[0097] The samples were analyzed by SEM with an acceleration voltage of 20 kV and a Fourier transform infrared spectrometer with a scanning range of 4000 cm -1 ~650cm -1 , resolution 1cm -1 The samples were analyzed by X-ray diffractometer, with Cu Kα as the radiation source, wavelength 1.5418nm, voltage 40kV, current 40mA, scanning range (2θ) 5°~50°, scanning step size 0.02°, scanning speed 2° / min. The samples were measured by differential scanning calorimetry, under the conditions of N 2 Purge, flow rate 60mL / min, temperature range 30℃~400℃, temperature rise rate 10℃ / min. Take a sample and determine the solubility of CAM in it by sulfuric acid colorimetry.

[0098] CAM and CGM powders were accurately weighed, and the inhibitory effects of different concentrations of the drug solution on Helicobacter pylori were determined. The concentrations investigated were calculated based on the CAM content, which were 0.5, 0.25, 0.125, and 0.0625 μg / mL, respectively. 990 μL of the drug solution was mixed with 10 μL of fresh Helicobacter pylori solution (OD 600 =1) co-incubation, cultured at 37℃ microaerobic environment; negative control was co-incubated with brain heart infusion medium (containing 10% fetal bovine serum) and bacterial solution, taken out after 24h, 100μL bacterial solution was transferred to blood agar plate after 100-fold dilution, and cultured at 37℃ microaerobic environment for 48h and then observed for the growth of cultured colonies. MIC was the drug concentration contained in the plate without colony growth.

[0099] Results: SEM images showed that after grinding CAM and AA-2G, the columnar crystals of CGM disappeared, the particle size decreased, and it appeared as an amorphous powder. Figure 7 .

[0100] Infrared spectra show that the carbonyl stretching vibration peak of CAM in CGM shifts to higher wavenumbers, see Figure 8 , indicating that hydrogen bonding occurred between AA-2G and CAM during the grinding process.

[0101] The X-ray diffraction pattern shows that the characteristic peak of CAM in CGM disappears. Fig. 9 This indicates that CAM may be affected by hydrogen bonding, shear force and other factors during the grinding process, and the crystals are transformed into amorphous substances.

[0102] The differential scanning calorimetry graph shows that CAM has sharp endothermic peaks at 227.67℃ and 301.33℃. CPM only has an endothermic peak at 186.67℃, and CGM has an endothermic peak at 189.67℃. Compared with CPM, the endothermic peak shifts to the right, as shown in Figure 2. Fig.10 , indicating that CAM exists in an amorphous state in CGM, which is conducive to the increase of solubility.

[0103] The solubility results showed that the water solubility of CAM in CGM was greatly improved, reaching 2526 μg / mL, while the water solubility of pure CAM was 27 μg / mL. Fig.11 The high solubility of CGM is conducive to the release of CAM.

[0104] From the growth of Helicobacter pylori on blood agar plates, it can be seen that the minimum inhibitory concentration of CAM and CGM is 0.5 μg / mL (calculated by CAM). Fig.12 This indicates that the auxiliary materials in the grinding process have no effect on the anti-Helicobacter pylori ability of CAM in CGM.

[0105] 3. In vitro drug release of drug-loaded magnetic microprojectiles

[0106] Materials: CAM, CGM, 200 / 2 micro cartridge case; 200 / 4 micro cartridge case; 300 / 2 micro cartridge case; 300 / 4 micro cartridge case.

[0107] Methods: CAM and CGM (containing about 1 mg of CAM) were prepared into drug-loaded magnetic microprojectiles according to the methods of Examples 1 and 2 by weight loss method. The above microprojectiles were placed in 10 mL centrifuge tubes, 2 mL of sodium acetate buffer solution (pH 5.0) was added, and the microprojectiles were shaken at 37 ° C and 100 rpm for 24 h. Samples were taken at predetermined time points (1, 2, 4, 6, 12, 24 h), 600 μL each time, and an equal volume of acetate buffer was immediately added. The samples were centrifuged at 14800 rpm for 10 min, and the supernatant was collected. The OD value at 490 nm was determined by sulfuric acid colorimetry. After deducting the blank background, the amount of CAM in the sample was calculated by substituting it into the CAM standard curve, and the cumulative release curve of CAM in the drug-loaded magnetic microprojectile was plotted.

[0108] Results: With the improvement of solubility, the cumulative release rate of CAM in drug-loaded magnetic microprojectiles increased significantly. The design of the drug release hole at the bottom of the microprojectile shell cleverly ensures that the CAM in different microprojectiles can achieve long-term, stable and sustained release. Specifically, the CMMB (300 / 4 type CGM-loaded magnetic microprojectile) prepared in Example 2 showed excellent advantages in cumulative drug release performance, such as Fig.13 As shown in A, B, and C, they were therefore selected as the preferred drug-loaded magnetic microprojectiles for subsequent studies to optimize drug release.

[0109] 4. Retention time of drug-loaded magnetic microprojectiles in mice stomach

[0110] Materials: CMMB prepared according to Example 2, magnet block (3800GS, 5 mm in diameter, 3 mm in height).

[0111] Methods: Mice were anesthetized with isoflurane, and 0.3 mL of iohexol solution (20%) was orally administered on an empty stomach. The mice were placed under an X-ray machine (the shape and position of the stomach can be observed through iohexol). After glycerol was applied to the surface of CMMB, it was placed vertically on the mouse's throat and gently pushed into the mouse's stomach with the help of an oral administration needle. A magnet block (3800GS, 5 mm in diameter, 3 mm in height) was placed above the abdomen of the mice in the magnetic guidance group (MG) at the stomach position to control the trajectory of the microprojectile (wrap and fix it with 3M transparent dressing). Under a portable X-ray machine, the mice were dissected at predetermined time points (8, 12, 16, 20, and 24 h) to observe the position of the microprojectile in the gastrointestinal tract of the mice. At the same time, the non-magnetic guidance group (NMG) mice, which did not undergo other operations after the microprojectile was administered, were used as the control group. The mice were fasted but not watered during the detention period.

[0112] Results: Microbullets can be visualized by built-in magnets under X-ray (black dots indicated by arrows). Placing an external magnet near the pylorus can accurately guide the clarithromycin-loaded magnetic microbullets to the vicinity of the pylorus. Fig.14 , indicating that CMMB has good positioning function. Fig.15 As shown, with the extension of time, when there is no external magnetic field guidance, CMMB can be discharged along the intestine under the action of gastrointestinal peristalsis, and the gastric retention time of CMMB is only 8 to 12 hours; when the external magnetic field guidance is applied, the retention time of CMMB in the stomach exceeds 24 hours, which proves that CMMB has excellent gastric retention performance, which is its advantage.

[0113] 5. Investigation of gastric tissue puncture performance of drug-loaded magnetic microprojectiles

[0114] Materials: CMMB prepared according to Example 2, external circular magnet block (3800GS, 5 mm in diameter, 3 mm in height).

[0115] Methods: According to the method described in (4. Mouse gastric retention time of drug-loaded magnetic microprojectiles), CMMB was injected into the stomach of C57BL / 6J mice. An external circular magnet block (3800GS, 5 mm in diameter, 3 mm in height) was fixed on the abdominal skin outside the mouse stomach to magnetically guide the CMMB. To investigate whether the microneedle can quickly penetrate the gastric tissue, the external magnet block was removed after only 10 minutes. Ten minutes and seven days after the external magnetic field was removed, the gastric tissue of the mouse was dissected and stained with hematoxylin-eosin (H&E), and the puncture depth of the microprojectile needle tip and the subsequent recovery of the gastric mucosa were observed under a microscope.

[0116] Results: H&E staining results showed that the microneedle at the tip of the microbullet was successfully inserted into the gastric tissue to a depth of about 270 μm, which is about 1 / 3 of the length of the microneedle, confirming the accuracy and effectiveness of the microbullet design. On the 7th day after administration, the gastric tissue recovered well and the tissue structure was intact. Fig.16 More importantly, the microprojectiles can be excreted smoothly with feces after completing drug release. Fig.17 , without causing intestinal obstruction, indicating that it has good oral safety. This feature is of great significance for the clinical application of drug-loaded magnetic microprojectiles, ensuring their safety and controllability during in vivo operation.

[0117] 6. Investigation of the directional behavior of magnetic microprojectile tip under magnetic guidance

[0118] Materials: CMMB prepared according to Example 2, 3% agarose gel block, external magnet (3800GS, 5 mm in diameter, 3 mm in height).

[0119] Methods: After dissection according to the steps of (5. Investigation of gastric tissue puncture performance of drug-loaded magnetic microprojectiles), the position of the CMMB in the stomach and the direction of its needle tip were carefully observed. The magnetic microprojectiles were taken out with ceramic scissors and then released. The process of the microprojectiles being attracted and attached to the gastric wall under the guidance of the external magnetic field was observed and recorded. An external magnet (3800GS, 5 mm in diameter, 3 mm in height) was placed under the gel, and the microprojectile was suspended above the gel block with ceramic scissors, with the needle tip facing upward. The movement trajectory of the magnetic microprojectile was observed after the ceramic scissors were released, and the pinholes on the agarose gel were observed after 5 minutes.

[0120] Results: In the in vivo experiment, the magnetic microbullet was orally administered into the stomach and successfully located at the pyloric region under the precise guidance of the external magnetic field. The needle tip was perpendicular to the pyloric stomach wall, and after releasing the microbullet, the microbullet could vertically enter the pylorus again. Fig.18 A. In vitro experiments further confirmed that under the guidance of the magnetic field, the magnetic microprojectile quickly flipped upward from the initial needle tip and accurately inserted vertically into the agar block. After the external magnetic field was removed, 8 micropinholes were clearly visible on the agarose gel. Fig.18Figure B shows that the external magnetic field can effectively control the incident angle of the microneedle, and the needle tip has good penetration performance. The internal and external magnets of the magnetic microbullet are both NdFeB polar magnets, which means that when administering the drug, it is only necessary to ensure the direction of the magnetic poles of the external magnetic field to accurately position the microbullet to the pylorus, thereby achieving accurate drug release.

[0121] 7. Pharmacodynamics of drug-loaded magnetic microprojectiles in the treatment of Helicobacter pylori infection

[0122] Materials: CMMB prepared according to Example 2; CAM suspension (3 mg / mL); CGM solution (CAM concentration 3 mg / mL).

[0123] Methods: Mice were gavaged with fresh bacterial solution for 30 consecutive days to establish a mouse model of Helicobacter pylori. The healthy group (no Helicobacter pylori infection, Healthy), model group (infected with Helicobacter pylori, no treatment, Model), clarithromycin group (infected with Helicobacter pylori, gavaged with clarithromycin suspension, CAM), clarithromycin grinding mixture group (infected with Helicobacter pylori, gavaged with clarithromycin grinding mixture solution, CGM), and drug-loaded magnetic microbullet group (infected with Helicobacter pylori, gavaged with one CMMB). After CMMB was gavaged into the stomach of mice, it was guided to the pylorus by an external magnet block, and the external magnet block was removed after 36 hours. All mice fasted for 6 hours before administration, and ate egg white until the 36th hour after administration, and then resumed normal diet. After 7 days, the mice were killed, the whole stomach was taken, and it was cut into two parts longitudinally. One part was fixed in 4% paraformaldehyde solution for H&E staining and silver staining (WS) to observe the pathological morphology of tissues and the distribution of Helicobacter pylori in gastric tissues, and the other part was used for the detection of the content of Helicobacter pylori in gastric tissues.

[0124] Results: After Helicobacter pylori infection, the gastric mucosal tissue of the mice in the model group showed obvious erosion, which was deep into the lamina propria, and there was interstitial inflammatory cell infiltration. Fig.19 Compared with the model group, the gastric mucosa of mice in the CAM group showed no obvious erosion, but large pieces of exfoliated cells were observed in the gastric mucosal epithelium. Compared with the CAM group, the gastric parietal cells of mice in the CMMB group were neatly arranged, and the integrity of the gastric mucosal epithelium and lamina propria structure was restored, with significant mucosal repair effect, which was no different from the healthy group.

[0125] Helicobacter pylori usually colonizes in the gastric mucus layer. A large number of black rod-shaped Helicobacter pylori can be seen in the gastric pits at the pylorus of mice in the model group. After treatment with clarithromycin-loaded magnetic microprojectiles, the content of Helicobacter pylori in gastric tissue decreased significantly, and Helicobacter pylori was almost not observed in gastric tissue. Fig. 20 The qPCR quantitative results showed that the clearance rate of Helicobacter pylori was the highest in the CMMB group, with the content of Helicobacter pylori in the stomach being 10 0.69 CFU / mg, significantly lower than that of the model group (106.06 CFU / mg) and CAM group (10 4.10 CFU / mg) and CGM group (10 3.30 CFU / mg), the difference was statistically significant (P<0.05), see Fig.21 , which confirmed that CMMB has the ability to eradicate Helicobacter pylori in the body with a single oral dose, can reduce gastric damage, promote gastric mucosal repair, and has a positive effect on the treatment of gastric diseases such as chronic gastritis or ulcers caused by Helicobacter pylori.

[0126] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0127] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A gastric retention drug-loaded magnetic microprojectile, characterized in that: The invention comprises a micro-bullet, a magnet block and a drug; the main body of the micro-bullet is a hollow cylindrical structure, the bottom of the cylinder is a closed thin sheet, the thin sheet has through holes, and micro-needles with needle tips facing upward are evenly distributed on the top ring of the wall of the hollow cylindrical structure; the cavity part of the hollow cylindrical structure is loaded with the drug; the magnet block is horizontally placed on the drug.

2. The gastric-retaining drug-loaded magnetic microprojectile according to claim 1, characterized in that: The length of the micro-bullet shell is selected from 0.2 cm to 2.5 cm; the inner diameter of the micro-bullet shell is selected from 0.1 cm to 1 cm; the outer diameter of the micro-bullet shell is selected from 0.12 cm to 1.02 cm; The thickness of the sheet is 0.05 mm to 0.1 mm; The number of the through holes is selected from 1 to 4, and the diameter of the through holes is 0.1 mm to 0.5 mm; The shape of the microneedles is selected from cone, cylinder, triangular or quadrangular, and the number is selected from 3 to 15; The drugs include: clarithromycin, amoxicillin, tetracycline, metronidazole, rifamycin, furazolidone, levofloxacin, ciprofloxacin, preferably any one or a combination of clarithromycin, amoxicillin and levofloxacin.

3. The gastric-retaining drug-loaded magnetic microprojectile according to claim 2, characterized in that: The length of the micro-shell is selected from 0.2 cm to 1 cm; The inner diameter of the micro-shell is selected from 0.1 cm to 0.6 cm; The outer diameter of the micro-bullet shell is selected from 0.12 cm to 0.62 cm. The microneedle is in the form of a conical microneedle; the height of the conical microneedle is selected from 0.2 mm to 1.5 mm; the bottom circular diameter of the conical microneedle is selected from 0.2 mm to 3 mm; The drug: clarithromycin and 2-O-α-D-glucopyranosyl-L-ascorbic acid are uniformly mixed at a mass ratio of 2.2:1, and ground for 30 minutes to obtain a ground mixture of clarithromycin and 2-O-α-D-glucopyranosyl-L-ascorbic acid.

4. The method for preparing gastric-retaining drug-loaded magnetic microprojectiles according to any one of claims 1 to 3, characterized in that: It is prepared by mold infusion curing method or 3D printing method.

5. The preparation method according to claim 4, characterized in that: The 3D printing method is: a fused deposition modeling 3D printing method or a light-curing 3D printing method.

6. The preparation method according to claim 5, characterized in that: The light-curing 3D printing method: according to the model parameters of the micro-bullet shell designed by computer, the printing solution in the material tank of the molding device is cured layer by layer on the printer platform to prepare the micro-bullet shell.

7. The preparation method according to claim 6, characterized in that: The photocurable 3D printing method specifically comprises: according to the model parameters of the micro-bullet shell designed by the computer, the printing solution in the material tank of the molding device is cured layer by layer on the printer platform to prepare the micro-bullet shell; The hollow part of the hollow cylindrical structure of the micro-bullet is loaded with drugs; the drugs are filled into the micro-bullet through the opening at the top of the micro-bullet. After the drugs are filled into the micro-bullet, a magnet block is placed horizontally above the drugs, and the micro-bullet 3D printing solution is used as a bonding material, which is dripped into the gap between the magnet block and the inner wall of the hollow cylinder of the micro-bullet. The micro-bullet is quickly placed under a 405nm laser light source for irradiation and curing, and the drugs and the magnet block are encapsulated into the micro-bullet to form a complete drug-loaded magnetic micro-bullet.

8. The preparation method according to claim 7, characterized in that: The printing solution is selected from acrylic acid, methyl acrylate, polypropylene fumarate, polyethylene glycol diacrylate, N-vinyl pyrrolidone, and siloxane.

9. The preparation method according to claim 8, characterized in that: The printing solution is any one of Dental SG, Dental LTClear V2 resin, BioMed Clear resin, and BioMed Amber resin.

10. Use of the gastric-retaining drug-loaded magnetic microprojectile according to any one of claims 1 to 3, or the preparation method according to any one of claims 4 to 9 in the preparation of drugs or diagnostic and therapeutic equipment.

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