A driven oral nanoformulation targeting intracellular Helicobacter pylori

Abstract

The present invention discloses a driven oral nanoformulation for intracellular Helicobacter pylori, wherein the nanoformulation includes a nanocarrier and a loaded drug, and the surface of the nanoformulation is modified with a bacteria-targeting functional polymer A-PEG-B and a mucus layer penetrating functional inclusion compound X@CD-TPGS. The driven oral nanoformulation for intracellular Helicobacter pylori of the present invention can maintain good stability in a gastric acid environment, and can give the nanoformulation gastric mucus layer penetration performance through the catalytic gas production approach, and then reach the Helicobacter pylori infection site through the bacteria-targeting function of the nanoformulation, and activate host cell autophagy under the synergistic action of the nanoformulation and the loaded drug, thereby achieving the effect of enhancing the clearance of intracellular infected Helicobacter pylori. The present invention provides a new approach for the efficient delivery of chemical drugs for treating Helicobacter pylori infection and the treatment of intracellular Helicobacter pylori infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a driven oral nano preparation targeting intracellular Helicobacter pylori. Background Art

[0002] Helicobacter pylori is a Gram-negative bacterium that colonizes the submucosa of the human stomach. It is characterized by high infection rates, severe drug resistance, and a high risk of developing gastric cancer. In recent years, the increasing prevalence of Helicobacter pylori infection and its increasing drug resistance have made eradication of Helicobacter pylori infection a major global public health challenge.

[0003] There are three main factors that contribute to the poor treatment effect of Helicobacter pylori. First, when antibacterial drugs reach the stomach through the oral route, the acidic environment in the stomach destroys the stability of the drugs. Secondly, because Helicobacter pylori colonizes in the lower part of the gastric mucus layer, the steric hindrance, dynamic renewal and rapid clearance mechanism of the gastric mucus layer hinder the effective delivery of antibacterial drugs to the site of Helicobacter pylori infection. Most importantly, Helicobacter pylori immune escape hinders the treatment of intracellular infection of host cells. The autophagy ability of cells invaded by Helicobacter pylori is weakened, and it is more difficult for these cells to be recognized by the body's immune system. Unrecognized infected cells will lead to the inability to completely eliminate Helicobacter pylori, further aggravating the degree of infection in the body, and ultimately causing repeated and long-term infection.

[0004] Nanoformulations, through rational design, can have unique advantages such as increasing drug targeting, promoting drug-infected lesion permeability, and improving drug bioavailability. In the treatment of Helicobacter pylori, by encapsulating therapeutic drugs inside nanoformulations, the drugs can be protected from destruction by gastric acid. However, in view of the many common problems in the treatment of Helicobacter pylori, there are still many problems or shortcomings in the design process of multifunctional nanoformulations. The low pH gastric acid environment in the stomach is a major obstacle to the stability of nanoformulations. The surface properties (such as charge, hydrophilicity and surface modification), size and morphology of nanoformulations play a key role in the stability of the preparation in the gastric environment. By adjusting these properties, the aggregation, sedimentation or degradation of nanoformulations by gastric acid in the gastric environment can be avoided.

[0005] In the prior art, a variety of strategies have been proposed to enhance the ability of nanoformulations to penetrate the gastric mucus barrier. First, the chemical modification strategy introduces functional groups such as thiol or maleimide groups on the surface of nanoformulations to form stable covalent bonds, thereby enhancing the interaction between nanoformulations and mucin (e.g., Chinese Patent 2022105858333). However, the passive diffusion efficiency of this method is low, and the clearance rate of nanoformulations is high during the dynamic renewal of mucus, which limits the effective delivery of nanoformulations to the bottom of the gastric mucus layer. Secondly, penetration strategies such as loading proteases or disulfide bond reducing agents in carriers (e.g., Chinese Patent 2015106394016) can enhance drug penetration by degrading mucin amino acids, but this mechanism may destroy the structural integrity of the gastric mucus layer, thereby affecting the natural defense function of the gastric mucus layer. In addition, taking advantage of the hydrophilicity, three-dimensional network structure and negatively charged groups of the gastric mucus layer, the design of water-soluble, appropriately sized and negatively charged nanoformulations is also considered to be another way to promote the penetration of nanoformulations through the mucus layer (e.g., Chinese patent 202310903615.4). However, this technology can only reduce the retention of nanoparticles in the mucus layer, but lacks the power to accelerate their passage through the mucus layer, and also has the disadvantage of low passive diffusion efficiency. In view of this, the development of a nanoformulation that can efficiently cross the gastric mucus layer without damaging the inherent functions of the mucus layer will help to achieve safe and effective drug delivery.

[0006] Enzyme-driven nanocarriers or robots have been used in the field of drug delivery in recent years (e.g., Chinese Patents 2023105002163, 2023104386823, and 2021116525349). These inventions exhibit excellent biocompatibility and self-propulsion, but their application in gastric and related diseases has been rarely reported. This is primarily because enzymes, as proteins, can be affected by gastric acid, leading to conformational changes, protonation of functional groups, and denaturation and inactivation.

[0007] Intracellular infection caused by Helicobacter pylori immune escape is currently not addressed in the treatment of Helicobacter pylori infection, and it is also an important factor that makes it impossible to eradicate Helicobacter pylori. Inventions related to nanoformulations for the treatment of intracellular infections of other bacteria (such as Mycobacterium tuberculosis, Salmonella and Staphylococcus aureus, etc.) have been confirmed (e.g., Chinese patents 2022101452049 and 2021106974178). By giving nanomaterials the ability to enter cells and / or increasing the retention time of nanoformulations in cells, efficient clearance of intracellular bacteria can be achieved; however, bacterial intracellular infections are not only present in the cytoplasm, but also have the characteristics of hiding in autophagosomes. Although the ability of nanoformulations to enter cells is enhanced or the intracellular retention time is prolonged, nanoformulations cannot restore the level of cellular autophagy and cannot eradicate bacteria hiding in autophagosomes, resulting in repeated bacterial infections.

[0008] Studies on the pharmacological effects of autophagy agonists have shown that restoring or enhancing host cell autophagy is beneficial for clearing intracellular bacteria. Furthermore, cyclodextrins, as a class of polymer compounds, have also been reported to have pharmacological effects that activate autophagy. Studies have shown that autophagy agonists and related nanoformulations have therapeutic potential for treating intracellular bacterial infections. By modulating the autophagy pathway, new therapeutic strategies can be developed to combat infections caused by intracellular bacteria, particularly for Helicobacter pylori intracellular infection, a relatively underappreciated area.

[0009] Helicobacter pylori is classified as a Class I carcinogen by the World Health Organization and the International Agency for Research on Cancer, and its eradication is attracting widespread attention. Existing technologies focus on addressing how to more effectively deliver antimicrobial drugs to the site of infection, but often overlook the intracellular infection of Helicobacter pylori, which makes eradication difficult. Summary of the Invention

[0010] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a driven oral nanoformulation for intracellular Helicobacter pylori, which can eliminate Helicobacter pylori infection from the root by improving stability in the stomach, enhancing permeability of the gastric mucus layer, giving bacteria targeting and eliminating intracellular infectious bacteria.

[0011] Another object of the present invention is to provide an application of the driven oral nanoformulation targeting intracellular Helicobacter pylori.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] In a first aspect, a driven oral nanoformulation for intracellular Helicobacter pylori is provided, wherein the nanoformulation is composed of 10 to 30 parts by weight of a loaded drug, 80 to 95 parts by weight of a phospholipid, 5 to 20 parts by weight of a bacteria-targeting functional polymer A-PEG-B, and 10 to 30 parts by weight of a mucus layer penetrating functional inclusion compound X@CD-TPGS; in the bacteria-targeting functional polymer A-PEG-B, A is a bacteria-targeting peptide; PEG is polyethylene glycol 2000; and B is a phospholipid; the mucus layer penetrating functional inclusion compound X@CD-TPGS is an inclusion compound formed by X and CD-TPGS, wherein X is a catalytic enzyme, TPGS is vitamin E polyethylene glycol 1000 succinate, and CD is cyclodextrin.

[0014] Furthermore, the nanoformulation is composed of 15 to 25 parts by weight of loaded drug, 85 to 90 parts by weight of phospholipid, 8 to 13 parts by weight of bacteria-targeting functional polymer A-PEG-B, and 15 to 22 parts by weight of mucus layer penetrating functional inclusion compound X@CD-TPGS.

[0015] Furthermore, the nanoformulation is composed of 20 parts by weight of loaded drug, 90 parts by weight of phospholipid, 10 parts by weight of bacteria-targeting functional polymer A-PEG-B, and 20 parts by weight of mucus layer penetrating functional inclusion compound X@CD-TPGS.

[0016] Furthermore, the nano-carrier of the nano-preparation is a micelle, a liposome, or a nanoparticle.

[0017] Furthermore, the phospholipid is any one of 1,2-distearoylphosphatidylcholine, polylactic acid-glycolic acid 1000, polyglycolic acid 2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, hydrogenated soybean phosphatidylcholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0018] Furthermore, the loaded drug is an autophagy stimulating drug and / or an antibiotic.

[0019] Furthermore, the autophagy agonist drug is any one of rapamycin, metformin, resveratrol, atorvastatin, and artemisinin, and the antibiotic is any one of metronidazole, levofloxacin, amoxicillin, clarithromycin, tetracycline, and furazolidone; the present invention achieves the effect of clearing intracellular Helicobacter pylori infection by activating host cell autophagy through the rational combination of autophagy agonist and cyclodextrin.

[0020] Furthermore, in the bacteria-targeting functional polymer A-PEG-B, A is a bacteria-targeting peptide, PEG is polyethylene glycol, and B is a phospholipid. The molar ratio of the bacteria-targeting peptide, polyethylene glycol 2000, and phospholipid in the bacteria-targeting functional polymer A-PEG-B is 1:1:1.

[0021] The sequence of the bacteria targeting peptide is any one of WKVRKSFFKLQGK, FHRNHRSPVTLL, RKRWWVVKRKWWV, KSKVGFLQLLFHKK, KGKGVFLQIQLFHK, and YCALQKRFILKMC.

[0022] Furthermore, the bacteria-targeting functional polymer A-PEG-B was prepared by the following method: 100 mg of B-PEG2000-NHS was weighed and dissolved in 5 mL of DMF, and a resin to which 1.1 eq of the bacteria-targeting peptide with undeprotected side chain amino groups was added. 3 eq of triethylamine was then added, and the mixture was reacted at room temperature for 12 hours to cleave the bacteria-targeting peptide from the resin. The solution was then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed for 24 hours using pure water as the dialysis medium. The solution in the dialysis bag was collected and freeze-dried to obtain the bacteria-targeting functional polymer A-PEG-B.

[0023] The molecular weight of polyethylene glycol is 2000.

[0024] The phospholipid is one of 1,2-distearoylphosphatidylcholine, polylactic acid-glycolic acid 1000, polyglycolic acid 2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, hydrogenated soybean phosphatidylcholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0025] Furthermore, the mucus-penetrating inclusion complex X@CD-TPGS is an inclusion complex formed by X and CD-TPGS, wherein X is a catalytic enzyme, TPGS is vitamin E polyethylene glycol succinate, and CD is cyclodextrin. The present invention utilizes the propelling properties of the catalytic enzyme as a propellant for the nanoformulation, promoting its rapid penetration through the gastric mucus layer. By selecting the appropriate catalytic enzyme species and techniques for maintaining enzyme stability, the present invention enables the application of the catalytic enzyme as an auxiliary driving agent in the acidic environment of the stomach.

[0026] The molar ratio of catalytic enzyme, vitamin E polyethylene glycol 1000 succinate and cyclodextrin in X@CD-TPGS is 1:1:1.

[0027] The cyclodextrin is any one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, methyl-β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin.

[0028] The catalytic enzyme is one of lipase, catalase, urease, amino acid decarboxylase, superoxide dismutase, and glutaminase.

[0029] Furthermore, the mucus layer penetrating functional inclusion complex X@CD-TPGS was prepared by the following method: 0.2 mmol glutaric acid, 0.1 mmol 4-dimethylaminopyridine (DMAP) and 0.24 mmol EDC were added to each 1 mL of anhydrous dimethylsulfoxide (DMSO) for dissolution, magnetically stirred under nitrogen protection at room temperature for 2 hours, and then 0.04 mmol TPGS was added. The reaction was continued under nitrogen protection at room temperature with magnetic stirring for 24 hours. The crude product was then transferred to a dialysis bag (molecular weight cutoff 1000 Da) and dialyzed in deionized water for 48 hours to remove DMSO, catalyst and unreacted raw materials. Finally, the carboxylation product of TPGS, TPGS-COOH, was obtained by lyophilization.

[0030] 10 μmol TPGS-COOH, 40 μmol EDC, and 70 μmol NHS were added to each 1 mL of anhydrous dimethylsulfoxide (DMSO) to dissolve and activate with stirring for 30 min. Then, 30 μmol cyclodextrin was added and stirred for reaction for 24 h. The crude product was dialyzed against deionized water in a dialysis bag (molecular weight cutoff 2000 Da) for 48 h to remove cyclodextrin-NH2, EDC, NHS, and DMSO, and then lyophilized to obtain the product TPGS-βCD.

[0031] In a second aspect, the present invention provides a method for preparing a nanoformulation, comprising the following steps:

[0032] (1) The loaded drug and A-PEG-B were weighed in proportion and dissolved in 2 mL of chloroform. The dissolved material was added to a round-bottom flask and subjected to reduced pressure rotary evaporation in a 60°C water bath for 1 hour to remove the organic solvent and form a homogeneous film at the bottom of the round-bottom flask. 2 mL of pure water was added to the round-bottom flask and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the film at the bottom of the round-bottom flask. The resulting solution was then broken up by ultrasonication at 100 W for 5 minutes.

[0033] (2) The solution obtained in step (1) and X@CD-TPGS were mixed with the nanocarrier, and incubated at 37° C. and 120 rpm for 2 to 6 hours to obtain the nanoformulation.

[0034] In a third aspect, the present invention further provides the use of the above-mentioned nanoformulation in treating Helicobacter pylori infection.

[0035] The nanoformulation showed good stability under acidic conditions in the stomach, mucus layer penetration efficiency, bacterial targeting ability and therapeutic effect of eliminating intracellular Helicobacter pylori.

[0036] Beneficial effects:

[0037] Compared to existing technologies, this invention innovatively proposes a nanoformulation design strategy that achieves precise drug delivery to Helicobacter pylori by synergizing bacterial-targeting peptides with a safe mucus-penetrating strategy. This synergistic autophagy agonist and cyclodextrin activate autophagy to eliminate intracellular Helicobacter pylori infection, eradicating the infection at its source. This invention offers oral stability, efficient penetration of the gastric mucus layer, targeted bacteria, and intracellular bacterial clearance, providing a new perspective and possibility for the treatment of Helicobacter pylori, with practical significance and promising application prospects.

[0038] Compared with the existing technology, the nanoformulation for treating Helicobacter pylori infection provided by the present invention has the following advantages: it can well realize drug loading, the mucus layer penetrating functional inclusion compound on the carrier can respond to the gastric mucus layer environment, provide power for the carrier to penetrate the mucus layer and maintain the stability of the carrier under gastric acid conditions, the bacteria targeting functional polymer on the carrier can enable the carrier to target bacteria, the carrier can improve the clearance rate of intracellular infected bacteria, eliminate Helicobacter pylori infection from the root, and improve the efficiency and bactericidal effect of the nanoformulation in reaching the Helicobacter pylori infection site. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without inventiveness and labor. Among them:

[0040] Figure 1 The particle size diagram and potential diagram of the nanoformulation prepared in the example are shown.

[0041] Figure 2 The results show the stability of the nanoformulations prepared in the examples in solutions with different pH values.

[0042] Figure 3 The mucus layer penetration ability of the nanoformulations prepared in the examples is shown, where # represents p≤0.05 vs. Comparative Example 1, * represents p≤0.05 vs. Comparative Example 2, and $ represents p≤0.05 vs. Comparative Example 3.

[0043] Figure 4 The results show that the nanoparticles prepared in the examples have good targeting performance on bacteria.

[0044] Figure 5 The antibacterial effects of the nanoformulations prepared in the examples on extracellular and intracellular bacteria in in vitro experiments are shown, where # represents p≤0.05 vs. comparative example 1, * represents p≤0.05 vs. comparative example 2, and $ represents p≤0.05 vs. comparative example 3.

[0045] Figure 6 The data show that the nanoformulation prepared in the examples improves the autophagy level of infected cells, where # represents p≤0.05 vs. Comparative Example 1, * represents p≤0.05 vs. Comparative Example 2, and $ represents p≤0.05 vs. Comparative Example 3.

[0046] Figure 7 The results show that the nanoformulation prepared in the example has an efficacy against Helicobacter pylori infection in vivo, with &p≤0.05 vs. Triple therapy. DETAILED DESCRIPTION

[0047] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Note: In the examples, R represents arginine, W represents tryptophan, V represents valine, K represents lysine, S represents serine, G represents glycine, F represents phenylalanine, L represents leucine, Q represents glutamine, and A represents threonine.

[0049] Example 1

[0050] Clarithromycin: 1 mg

[0051] Rapamycin: 1 mg 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine: 9 mg

[0052] YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1 mg

[0053] Catalase@Hydroxypropyl-β-cyclodextrin-TPGS: 2 mg

[0054] The bacteria-targeting functional polymer YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine is prepared by the following steps:

[0055] The bacterial targeting peptide YCALQKRFILKMC was synthesized by solid-phase synthesis. 100 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000-NHS was weighed and dissolved in 5 mL of DMF. A resin to which 1.1 eq of the bacterial targeting peptide YCALQKRFILKMC with undeprotected side chain amino groups was added was added. 3 eq of triethylamine was then added and reacted at room temperature for 12 h. The bacterial targeting peptide YCALQKRFILKMC was cleaved from the resin. The solution was then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed for 24 h using pure water as the dialysis medium. The solution in the dialysis bag was collected and freeze-dried to obtain a bacterial targeting functional polymer.

[0056] YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0057] The preparation method of the mucus layer penetrating functional inclusion compound catalase@hydroxypropyl-β-cyclodextrin-TPGS comprises the following steps:

[0058] (1) 0.2 mmol glutaric acid, 0.1 mmol 4-dimethylaminopyridine (DMAP) and 0.24 mmol EDC were added to each 1 mL of anhydrous dimethylsulfoxide (DMSO) to dissolve the mixture. The mixture was magnetically stirred for 2 h under nitrogen protection at room temperature. Then 0.04 mmol TPGS was added and the reaction was continued under nitrogen protection at room temperature with magnetic stirring for 24 h. The crude product was then transferred to a dialysis bag (molecular weight cutoff 1000 Da) and dialyzed in deionized water for 48 h to remove DMSO, catalyst and unreacted raw materials. Finally, the carboxylation product of TPGS, TPGS-COOH, was obtained by lyophilization.

[0059] (2) 10 μmol TPGS-COOH, 40 μmol EDC and 70 μmol NHS were added to each 1 mL of anhydrous dimethylsulfoxide (DMSO) to dissolve, stirred and activated for 30 min, and then 30 μmol hydroxypropyl-β-cyclodextrin was added and stirred for reaction for 24 h. The crude product was dialyzed with deionized water in a dialysis bag (molecular weight cutoff 2000 Da) for 48 h to remove cyclodextrin-NH2, EDC, NHS and DMSO, and then lyophilized to obtain the product hydroxypropyl-β-cyclodextrin-TPGS.

[0060] (3) Dissolve catalase powder in deionized water at a ratio of 0.005:1 (mol:L) to deionized water, and shake evenly. Dissolve hydroxypropyl-β-cyclodextrin-TPGS in deionized water at a ratio of 0.01:1 (mol:L) to TPGS-hydroxypropyl-β-cyclodextrin and deionized water, and shake evenly. Mix the catalase solution and the hydroxypropyl-β-cyclodextrin-TPGS solution at a volume ratio of 1:1, and stir magnetically at room temperature for 24 hours to obtain catalase@hydroxypropyl-β-cyclodextrin-TPGS.

[0061] A nanoformulation preparation method comprises the following steps: 1 mg of clarithromycin, 1 mg of rapamycin, 9 mg of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1 mg of YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine are weighed and dissolved in 2 mL of chloroform. The dissolved materials are added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent and form a homogeneous film on the bottom of the round-bottom flask. 2 mL of pure water is added to the round-bottom flask, and the round-bottom flask is placed in a rotary evaporator in a 60°C water bath for 2 hours to allow the film on the bottom of the round-bottom flask to completely hydrate and fall off. The resulting solution is then disrupted by ultrasonication at 100 W for 5 minutes. The resulting solution is then mixed with 2 mg of catalase@hydroxypropyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0062] Example 2

[0063] Amoxicillin: 1 mg

[0064] Rapamycin: 1 mg 1,2-Distearoylphosphatidylcholine: 9 mg

[0065] WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1 mg

[0066] Urease@Hydroxypropyl-β-cyclodextrin-TPGS: 2 mg

[0067] Referring to Example 1, WKVRKSFFKLQG was used to replace YCALQKRFILKMC to prepare WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0068] Referring to Example 1, urease was used instead of catalase to prepare urease@hydroxypropyl-β-cyclodextrin-TPGS.

[0069] A nano preparation, the preparation method comprises the following steps:

[0070] 1 mg of amoxicillin, 1 mg of rapamycin, 9 mg of 1,2-distearoylphosphatidylcholine, and 1 mg of WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine were weighed and dissolved in 2 mL of chloroform. The dissolved materials were added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent and form a homogeneous film at the bottom of the round-bottom flask. 2 mL of pure water was added to the round-bottom flask, and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the film at the bottom of the round-bottom flask. The resulting solution was then ultrasonicated at 100 W for 5 minutes to disrupt the solution. The resulting solution was mixed with 2 mg of urease@hydroxypropyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0071] Example 3

[0072] Clarithromycin: 1 mg

[0073] Rapamycin: 1 mg 1,2-Distearoylphosphatidylcholine: 9 mg

[0074] WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1 mg

[0075] Catalase@Methyl-β-cyclodextrin-TPGS: 2 mg

[0076] Referring to Example 1, WKVRKSFFKLQG was used to replace YCALQKRFILKMC to prepare WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0077] Referring to Example 1, methyl-β-cyclodextrin was used instead of hydroxypropyl-β-cyclodextrin to prepare catalase@methyl-β-cyclodextrin-TPGS.

[0078] A nano preparation, the preparation method comprises the following steps:

[0079] 1 mg of clarithromycin, 1 mg of rapamycin, 9 mg of 1,2-distearoylphosphatidylcholine, and 1 mg of WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine were weighed and dissolved in 2 mL of chloroform. The dissolved materials were added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent, forming a homogeneous film at the bottom of the round-bottom flask. 2 mL of pure water was added to the round-bottom flask, and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the film at the bottom of the round-bottom flask. The resulting solution was then disrupted by sonication at 100 W for 5 minutes. The resulting solution was mixed with catalase@methyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0080] Example 4

[0081] Clarithromycin: 0.72 mg

[0082] Rapamycin: 0.72 mg 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine: 8.4 mg

[0083] YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1.02 mg

[0084] Catalase@Hydroxypropyl-β-cyclodextrin-TPGS: 1.86 mg

[0085] According to the preparation method of Example 1, a driven oral nanoformulation targeting intracellular Helicobacter pylori was prepared.

[0086] Example 5

[0087] Amoxicillin: 1 mg

[0088] Rapamycin: 1 mg 1,2-Distearoylphosphatidylcholine: 9.1 mg

[0089] WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1.05 mg

[0090] Urease@Hydroxypropyl-β-cyclodextrin-TPGS: 2.1 mg

[0091] According to the preparation method of Example 2, a driven oral nanoformulation targeting intracellular Helicobacter pylori was prepared.

[0092] Example 6

[0093] Clarithromycin: 0.6 mg

[0094] Rapamycin: 0.9 mg 1,2-Distearoylphosphatidylcholine: 8.4 mg

[0095] WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1.08 mg

[0096] Catalase@Methyl-β-cyclodextrin-TPGS: 1.56 mg

[0097] According to the preparation method of Example 3, a driven oral nanoformulation targeting intracellular Helicobacter pylori was prepared.

[0098] Comparative Example 1

[0099] Levofloxacin: 2 mg

[0100] Hydrogenated soy phosphatidylcholine: 9 mg 1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1 mg

[0101] Hydroxypropyl-β-cyclodextrin-TPGS: 1 mg

[0102] Referring to Example 1, hydroxypropyl-β-cyclodextrin-TPGS was prepared.

[0103] A nano preparation, the preparation method comprises the following steps:

[0104] 2 mg of levofloxacin, 9 mg of hydrogenated soy phosphatidylcholine, and 1 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine were weighed and dissolved in 2 mL of chloroform. The dissolved materials were added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent and form a homogeneous film at the bottom of the round-bottom flask. 2 mL of pure water was added to the round-bottom flask, and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the thin film at the bottom of the round-bottom flask. The resulting solution was then ultrasonicated at 100 W for 5 minutes to disrupt the solution. The resulting solution was mixed with 1 mg of hydroxypropyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0105] Comparative Example 2

[0106] Levofloxacin: 1 mg

[0107] Rapamycin: 1 mg 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine: 9 mg

[0108] YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1 mg

[0109] Hydroxypropyl-β-cyclodextrin-TPGS: 1 mg

[0110] With reference to Example 1, YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine was prepared, where YCALQKRFILKMC is a peptide sequence that does not have bacterial targeting function.

[0111] Referring to Example 1, hydroxypropyl-β-cyclodextrin-TPGS was prepared.

[0112] A nano preparation, the preparation method comprises the following steps:

[0113] 1 mg of levofloxacin, 1 mg of rapamycin, 9 mg of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1 mg of YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine were weighed and dissolved in 2 mL of chloroform. The dissolved materials were added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent and form a homogeneous film on the bottom of the round-bottom flask. 2 mL of pure water was added to the round-bottom flask, and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the film on the bottom of the round-bottom flask. The resulting solution was then ultrasonicated at 100 W for 5 minutes to disrupt the solution. The resulting solution was mixed with 1 mg of hydroxypropyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0114] Comparative Example 3

[0115] Levofloxacin: 1 mg

[0116] Scoparone: 1mg 1,2-Distearoylphosphatidylcholine: 9mg

[0117] IKISGKWKAQKRFLK-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1 mg

[0118] Hydroxypropyl-β-cyclodextrin-TPGS: 1 mg

[0119] With reference to Example 1, IKISGKWKAQKRFLK-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine was prepared, where IKISGKWKAQKRFLK is a peptide sequence that does not have bacterial targeting function.

[0120] Referring to Example 1, hydroxypropyl-β-cyclodextrin-TPGS was prepared.

[0121] A nano preparation, the preparation method comprises the following steps:

[0122] 1 mg of levofloxacin, 1 mg of scoparia lactone, 9 mg of 1,2-distearoyl phosphatidylcholine, and 1 mg of IKISGKWKAQKRFLK-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine were weighed and dissolved in 2 mL of chloroform. The dissolved materials were added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent and form a homogeneous film on the bottom of the round-bottom flask. 2 mL of pure water was added to the round-bottom flask, and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the film on the bottom of the round-bottom flask. The resulting solution was then disrupted by ultrasonication at 100 W for 5 minutes. The resulting solution was mixed with hydroxypropyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0123] Comparative Example 4

[0124] Clarithromycin: 1 mg

[0125] Rapamycin: 1 mg 1,2-Distearoylphosphatidylcholine: 5 mg

[0126] WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 5 mg

[0127] Urease@Methyl-β-cyclodextrin-TPGS: 2 mg

[0128] Referring to Example 1, WKVRKSFFKLQG was used to replace YCALQKRFILKMC to prepare WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0129] Referring to Example 1, methyl-β-cyclodextrin was used to replace hydroxypropyl-β-cyclodextrin, and urease was used to replace catalase to prepare urease@methyl-β-cyclodextrin-TPGS.

[0130] A nano preparation, the preparation method comprises the following steps:

[0131] 1 mg of clarithromycin, 1 mg of rapamycin, 5 mg of 1,2-distearoylphosphatidylcholine, and 5 mg of WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine were weighed and dissolved in 2 mL of chloroform. The dissolved materials were added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent and form a homogeneous film on the bottom of the round-bottom flask. 2 mL of pure water was added to the round-bottom flask, and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the film on the bottom of the round-bottom flask. The resulting solution was then disrupted by sonication at 100 W for 5 minutes. The resulting solution was mixed with urease@methyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0132] Comparative Example 5

[0133] Amoxicillin: 2 mg

[0134] Rapamycin: 2 mg 1,2-Distearoylphosphatidylcholine: 9 mg

[0135] FHRNHRSPVTLL-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1 mg

[0136] Catalase@Methyl-β-cyclodextrin-TPGS: 5 mg

[0137] Referring to Example 1, FHRNHRSPVTLL was used to replace YCALQKRFILKMC to prepare FHRNHRSPVTLL-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0138] Referring to Example 1, methyl-β-cyclodextrin was used instead of hydroxypropyl-β-cyclodextrin to prepare catalase@methyl-β-cyclodextrin-TPGS.

[0139] A nano preparation, the preparation method comprises the following steps:

[0140] 2 mg of amoxicillin, 2 mg of rapamycin, 9 mg of 1,2-distearoylphosphatidylcholine, and 1 mg of FHRNHRSPVTLL-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine were weighed and dissolved in 2 mL of chloroform. The dissolved materials were added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent, forming a homogeneous film at the bottom of the round-bottom flask. 2 mL of pure water was added to the round-bottom flask, and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the film at the bottom of the round-bottom flask. The resulting solution was then disrupted by sonication at 100 W for 5 minutes. The resulting solution was mixed with catalase@methyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0141] Comparative Example 6

[0142] Amoxicillin: 2 mg

[0143] Rapamycin: 2 mg

[0144] Hydrogenated soy phosphatidylcholine: 9mg

[0145] WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine: 1 mg

[0146] Urease@Methyl-β-cyclodextrin-TPGS: 2 mg

[0147] Referring to Example 1, WKVRKSFFKLQG was used to replace YCALQKRFILKMC to prepare WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

[0148] Referring to Example 1, methyl-β-cyclodextrin was used to replace hydroxypropyl-β-cyclodextrin, and urease was used to replace catalase to prepare urease@methyl-β-cyclodextrin-TPGS.

[0149] A nano preparation, the preparation method comprises the following steps:

[0150] 2mg of amoxicillin, 2mg of rapamycin, 9mg of hydrogenated soy phosphatidylcholine, and 1mg of WKVRKSFFKLQG-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine were weighed and dissolved in 2mL of chloroform. The dissolved materials were added to a round-bottom flask and subjected to thin film evaporation under reduced pressure in a 60°C water bath for 1 hour to remove the organic solvent and form a homogeneous film at the bottom of the round-bottom flask. 2mL of pure water was added to the round-bottom flask, and the round-bottom flask was placed in a rotary evaporator in a 60°C water bath for 2 hours to completely hydrate and remove the thin film at the bottom of the round-bottom flask. The resulting solution was then disrupted by ultrasonication at 100W for 5 minutes. The resulting solution was mixed with urease@methyl-β-cyclodextrin-TPGS and incubated at 37°C and 120 rpm for 6 hours to obtain the nanoformulation.

[0151] Test Example 1

[0152] Particle size distribution and zeta potential determination of nanoformulations

[0153] 1 mL of the nanoformulations obtained in Comparative Examples 1 to 6 and Examples 1 to 3 was accurately measured and placed in a 10 mL volumetric flask, and diluted to the mark with ultrapure water. The particle size and zeta potential of the nanoformulations were measured.

[0154] Depend on Figure 1 It can be seen that the particle size distribution of the nanoformulations obtained in Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 1, Example 2, and Example 3 is between 180-280 nm, and the Zeta potential is between -30 and 0 mV. However, the particle size of the preparation prepared in Comparative Example 4 was measured to be 501.51±18.37 nm, and the Zeta potential was -38.18±0.40 mV. The resulting particle size was too large, which was not conducive to the penetration of the preparation in the mucus layer (a particle size of about 200 nm is the preferred particle size for penetrating the mucus layer). Therefore, this formulation was not used for the functional evaluation of the subsequent preparation.

[0155] In addition, sample instability was observed during the preparation process of Comparative Examples 5 and 6. For example, precipitation occurred after the resulting preparations were left to stand overnight, indicating that the stability of the preparations was poor. Therefore, the formulations were not used for functional evaluation of subsequent preparations.

[0156] Test Example 2

[0157] Stability of nanoformulations in artificial gastric fluids with different pH values

[0158] Weigh 5.14 mg of KCl, 1.22 mg of KH2PO4, 21 mg of NaHCO3, 27.58 mg of NaCl, 0.20 mg of MgCl2·6(H2O), 0.53 mg of NH4Cl, 0.17 mg of CaCl2, and appropriate amounts of pepsin and mucin. Pour these materials into an appropriate amount of PBS solution, add a stir bar, and stir on a magnetic stirrer overnight until the pepsin and mucin are completely dissolved in the mother solution. Adjust the pH of the solution to 2.0 with 1.0 mol / L HCl to prepare artificial gastric juice.

[0159] The pH of artificial gastric fluid was adjusted to 2.0 (simulating gastric acid), 5.5, 6.8, and 7.4 (simulating gastric mucus layer, pH 5.5-7.4) using HCl or NaOH. 0.2 mL of the nanoformulation solution was added to 0.8 mL of artificial gastric fluid at different pH values, mixed thoroughly, and the particle size of the mixture was measured at 0, 1, 2, 4, and 8 hours.

[0160] Depend on Figure 2 It can be seen that the nanoformulations obtained in Example 1, Example 2 and Example 3 can be stably present for 8 hours in a simulated gastric acid environment with a pH of 2.0 and a simulated gastric mucus layer environment with a pH of 5.5-7.4, which can meet the requirements of drug administration. However, Comparative Example 1, Comparative Example 2 and Comparative Example 3 cannot maintain stability in a low pH (pH 2.0 and 5.5) environment, and the particle size increases from 220nm (the particle size when initially prepared) to 600-2000nm, indicating that its structure is destroyed in a low pH environment. It can be concluded that the selection of a suitable phospholipid composition and / or catalytic enzyme reaction protection is conducive to maintaining the stability of the nanoformulation in the acidic environment of the stomach.

[0161] Test Example 3

[0162] Mucus layer penetration performance of nanoformulations

[0163] Mucin binding rate: A certain amount of mucin was dissolved in 0.300 mL of phosphate buffered saline (PBS, pH 6.8, the pH of the gastric mucus layer is 6.8) to prepare a suspension with a concentration of 1.0% (w / v), and stirred overnight to ensure full suspension. 0.100 mL of the nanoformulation, from which free drug had been removed, was dispersed in a mucin solution. After incubation at 37°C and 100 rpm for 2 h, the solution was centrifuged at 5000 rpm for 10 min. 0.100 mL of the supernatant was removed and 2.400 mL of methanol was added to disrupt the nanoformulation (this portion of the nanoformulation was not bound to mucin). The drug concentration (C0) of the disrupted nanoformulation was measured using high-performance liquid chromatography. For comparison, an equal amount (0.100 mL) of the nanoformulation was dispersed in 0.3 mL of phosphate buffered saline (PBS, pH 6.8). After incubation at 37°C and 100 rpm for 2 h, the solution was centrifuged at 5000 rpm for 10 min. 0.100 mL of the supernatant was removed and 2.400 mL of methanol was added to disrupt the nanoformulation. The drug concentration (C1) was measured. The percentage of the nanoformulation bound to mucin was calculated.

[0164]

[0165] Wherein: C0 is the drug concentration in the supernatant of the group co-incubated with mucin solution, and C1 is the drug concentration in the supernatant of the control group.

[0166] Mucus layer penetration ability:

[0167] Mucus obtained from porcine stomachs was added to 5 mL of 0.1 M PBS (Ph 7.4) at a ratio of 1 g mucus to 4°C (4°C) and stirred for 1 hour. The mixture was then centrifuged at <100 rpm, 10400 g, and 10°C for 2 hours. The supernatant was discarded and the centrifugation was repeated several times until the supernatant was colorless. The supernatant was removed after centrifugation, and the precipitate was the desired purified gastric mucus.

[0168] 100 μL of gastric mucus (thickness approximately 1.2 mm, gastric luminal mucosal mucus thickness 0.3-3 mm) was spread onto the donor chamber filter of a Transwell plate. 500 μL of PBS buffer (pH 7.4) was added to the receptor chamber and equilibrated at 37°C for 15 min. 100 μL of the nanoformulation, from which free drug had been removed, was added to the donor chamber and incubated at 37°C, 100 rpm (in a small shaker air bath to prevent water from entering the apparatus). At 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, and 4 h, 50 μL of the receptor chamber was sampled and the same volume of PBS buffer was added. The sample was then demulsified with 450 μL of methanol and analyzed by HPLC. The drug concentration was recorded as Cn.

[0169] The penetration results are expressed as the apparent permeability coefficient (Papp, cm / S), which is calculated as follows:

[0170]

[0171] Where dQ is the amount of drug detected in the receptor compartment (μg), dt is the experimental time (s), dQ / dt is the amount of drug transported per unit time, dQ / dt is the slope of the linear regression of the cumulative drug release amount Q versus time t, and A is the surface area of ​​the Transwell membrane (cm 2 ), C0 is the initial concentration of fluorescently labeled nanocarriers added to the upper chamber (μg / mL = μg / cm 3 ).

[0172] The mucin binding rate of a drug can be used as one of the criteria for evaluating its ability to penetrate the gastric mucus layer. Lower mucin binding rates indicate a lower likelihood of retention in the gastric mucus layer. Penetration is expressed as Papp; higher Papp values ​​indicate a greater ability of the drug to penetrate the mucosa and a faster penetration rate.

[0173] like Figure 3 As shown, the nanoformulations obtained in Examples 1, 2, and 3 have low mucin binding rates and good mucus layer penetration, indicating that the nanoformulations obtained in Examples 1, 2, and 3 are not easily retained in the gastric mucus layer. In contrast, Comparative Examples 1, 2, and 3 have higher mucin binding rates, but do not contain mucus layer-penetrating inclusion compounds and have the lowest mucus layer penetration. This leads to the conclusion that the catalytic enzyme in the mucus layer-penetrating inclusion compound provides the driving force for the nanoformulation to penetrate the mucus layer.

[0174] Test Example 4

[0175] Targeting properties of nanoparticles to bacteria

[0176] Microthermophoresis (MST) refers to the directional motion of molecules in a microscopic temperature gradient field, and is widely used in verifying whether there is binding between molecules, quantitative determination of intermolecular interaction affinity, etc. LPS, as the protein in the outer layer of the cell wall of Gram-negative bacteria, is used as a monomer substitute for Helicobacter pylori in this experiment to verify the targeting of nanoformulations to bacteria. Comparative Example 1, Comparison 5, Example 6 obtained nanoformulations and 0.15mol / L carbonate buffer solution of pH 9.0 were mixed according to a volume ratio of 9: 1 (making solution pH> 8.5), 0.2mg of FITC powder was accurately weighed and added to the above solution, reacted in the dark at room temperature, and stirred for 12h. Subsequently, the solution was transferred to a dialysis bag with a molecular weight cut-off of 8k-14k, dialyzed with water as a dialysis medium to remove free FITC, and the solution in the bag was collected to obtain FITC-labeled nanocarriers. Prepare 16 LPS aqueous solutions in 16 microcentrifuge tubes using a 1 mg / mL LPS solution (50 μM) as the stock solution using serial dilutions. Each tube contains 10 μL of LPS solution. Add 10 μL of FITC-labeled carrier sequentially and mix thoroughly using a pipette. Insert 16 MST standard capillaries into each microcentrifuge tube, using the capillary siphon to load the sample to at least two-thirds of its height.

[0177] Place the sample-loading capillaries on the instrument's sample loading platform according to the serial number, select the blue light channel, set the exciter power to 20%, and the MST power to medium for testing.

[0178] like Figure 4 As shown, the Kd of the nanoformulation obtained in Example 1 with LPS is 218nM, the Kd of the nanoformulation obtained in Example 2 with LPS is 404nM, and the Kd of the nanoformulation obtained in Example 3 with LPS is 990nM. The nanoformulations obtained in Example 1, Example 2 and Example 3 have the ability to bind to LPS and can target bacteria. In contrast, the Kd of the nanoformulations obtained in Comparative Examples 1, 2 and 3 with LPS was not measured, indicating that they do not have the ability to target bacteria, which is related to their lack of bacteria-targeting functional polymers (the peptide sequences YCALQKRFILKMC and IKISGKWKAQKRFLK selected in Comparative Examples 2 and 3 do not have bacteria-targeting function). It can be concluded that in the present invention, bacteria-targeting functional polymers are necessary for nanoformulations to target bacteria.

[0179] Test Example 5

[0180] Antibacterial effects of nanoparticles against extracellular and intracellular bacteria in vitro

[0181] RAW2647 cells were seeded into cell culture dishes at a density of 2*10^5 cells / mL, with 1 mL per dish. The cells were cultured in a 37°C 5% CO2 incubator for 12 h, allowing the cells to adhere to the wall and reach a density of approximately 50%.

[0182] Discard the culture medium and replace it with a bacterial solution diluted in complete culture medium to an MOI of 1:50 per well. Simultaneously, add the preparation or drug diluted in sterile PBS to a final concentration of 3.90 μg / mL. A positive control group containing no bacterial solution and a negative control group without the preparation were established. Place the plate in a cell culture incubator and incubate at 37°C for 24 hours.

[0183] Extracellular bacteria count: Collect 0.5 mL of the supernatant (extracellular culture medium) from each well, add 0.5 mL of PBS to the wells to wash the plate, repeat this operation twice, and collect the PBS and extracellular culture medium together in the same EP tube. Remove 50 μL of the extracellular bacterial suspension and evenly spread it on the pre-prepared solid culture medium plate using a spreader rod. After 36 hours of incubation, photograph the plate and record the growth of extracellular bacteria on the plate.

[0184]

[0185] Intracellular bacterial count: Add 0.5 mL of 0.4% Triton X-100 to the culture dish and incubate in a cell incubator for 30 min. Collect the cell lysate. Add 0.5 mL of PBS to each well to rinse the plate. Repeat this process twice. Collect the PBS and cell lysate together in the same EP tube. Remove 50 μL of the cell lysate and spread it evenly onto the pre-prepared solid culture medium plate using a spreader. After 36 h of incubation, photograph the plate and record the growth of extracellular bacteria.

[0186]

[0187] like Figure 5 As shown, the nanoformulations obtained in Example 1, Example 2 and Example 3 have an inhibition rate of more than 80% on extracellular bacteria and an inhibition rate of more than 75% on intracellular bacteria, showing good extracellular and intracellular bacteria clearance effects, and Examples 4 to 6 have similar antibacterial efficacy. However, the nanoformulations obtained in Comparative Examples 1, 2 and 3 only show an intracellular bacteria clearance effect but not an extracellular bacteria clearance effect, which is related to the fact that the nanoformulations do not have a bacteria-targeting functional polymer component. It can be concluded that targeting extracellular bacteria helps to increase the contact between the preparation and bacteria and release the drug through contact release. The clearance of intracellular bacteria depends on the combined action of the antibacterial components and autophagy activation components of the nanoformulation.

[0188] Test Example 6

[0189] Effects of nanoparticles on autophagy levels in infected cells in vitro

[0190] RAW 264.7 macrophages were diluted with complete culture medium and cultured at a concentration of 1 × 10 5 200 μL of cells / mL were inoculated into a 96-well plate. The edges of the 96-well plate were filled with sterile PBS. A set of zeroing wells was set up with only culture medium but no cells. The 96-well plate with cells was placed in a 5% carbon dioxide constant temperature incubator at 37°C and cultured for 12 hours until the cell density in each well of the 96-well plate was approximately 70%. The culture medium in the cell culture dish was discarded, and the cells were washed twice with sterile PBS. 90 μL of bacterial culture medium (MOl=0 (negative control) 50) was added to each well. The nanoformulations obtained from Comparative Example 1, Comparative Example 5, and Example 6 diluted with PBS were added and incubated in a 37°C 5% CO2 incubator for 24 hours.

[0191] Aspirate the culture medium from the treated cells. Add 0.1 mL of MDC staining solution to each well of the 96-well plate and incubate in a cell culture incubator at 37°C in the dark for 30 min. Aspirate the MDC staining solution and wash three times with 0.1 mL of Assay Buffer (provided in the kit) each time. Aspirate the Assay Buffer and add 0.1 mL of Assay Buffer. Fluorescence detection was performed using a fluorescence microplate reader with an excitation wavelength of 335 nm and an emission wavelength of 512 nm.

[0192] like Figure 6 As shown, the nanoformulations obtained in Example 1, Example 2 and Example 3 are beneficial to improving the autophagy level of infected cells. This test example shows that the autophagy level of cells infected with Helicobacter pylori (PBS group) is inhibited, and the increase in autophagy level is related to the intracellular bacteria (such as Figure 5 The clearance effect was positively correlated.

[0193] Test Example 7

[0194] Efficacy of nanoparticles against Helicobacter pylori infection in vivo

[0195] Healthy female C57 mice (6-8 weeks) were used to establish the HP infection model. The mice were fasted for 6 hours and deprived of water for 2 hours before each administration. 5% NaHCO₃ 0.2 mL / mouse was administered orally to neutralize gastric acid. Thirty minutes later, 0.3 mL of 1*10 9 CFU / mL suspension was added, and food and water were given 4 hours after the bacteria were added, once every 2 days for 2 weeks.

[0196] Two weeks after the administration of the bacteria, the mice were fed normally for one week to allow HP to colonize in the stomach. The mice were randomly divided into three treatment groups (n=8), and received treatment with PBS, triple therapy (omeprazole + amoxicillin + clarithromycin), comparative example 1, comparative example 2, comparative example 3, and the nanoformulations obtained in Example 1, Example 2 and Example 3, respectively. They were fasted for 6 hours before each administration and deprived of water for the first 2 hours. Oral administration of 5% NaHCO3 0.3 mL / mouse was used to neutralize the gastric acid of the mice. After 30 minutes, the mice were given treatment with different preparations, and food and water were given 2 hours after administration. The different preparations were administered by oral gavage, 0.6 mL / mouse, once a day, for 1 week. 48 hours after the last administration, the mice were killed and the stomach was removed from the abdominal cavity (fasted 48 hours before sacrifice). The stomach was cut open along the greater curvature of the stomach, the gastric contents were removed and rinsed with PBS. The gastric tissue was homogenized using a high-speed shearing instrument. The gastric tissue of each group of animals was homogenized and diluted, and then spread on a plate. The colonies were counted after incubation at 37°C for 24 to 48 hours.

[0197] like Figure 7 As shown, in the in vivo animal infection model, the nanoformulations obtained in Example 1, Example 2 and Example 3 all have the best antibacterial properties for extracellular bacteria, intracellular bacteria and total bacterial count (the sum of the number of extracellular bacteria and intracellular bacteria). Compared with the current clinical conventional therapy (triple therapy), the nanoformulations obtained in Example 1, Example 2 and Example 3 showed outstanding antibacterial efficacy against intracellular bacteria. The nanoformulations obtained in Example 1, Example 2 and Example 3 reflect the necessity of eliminating intracellular bacteria in the treatment of Helicobacter pylori infection in vivo, as well as the importance of protecting the stability of the formulation structure and the design of bacterial targeting.

[0198] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be encompassed by the claims of the present invention. Experimental methods and reagents without specific conditions in the examples are all based on conventional conditions in the art.

Claims

1. A driven oral nanoformulation for intracellular Helicobacter pylori, characterized in that: The nanoformulation is composed of 10 to 30 parts by weight of a loaded drug, 80 to 95 parts by weight of a phospholipid, 5 to 20 parts by weight of a bacteria-targeting functional polymer A-PEG-B, and 10 to 30 parts by weight of a mucus layer penetrating functional inclusion compound X@CD-TPGS; the loaded drug is selected from any one or two of clarithromycin, rapamycin, and amoxicillin; the phospholipid is any one of 1,2-distearoylphosphatidylcholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; in the bacteria-targeting functional polymer A-PEG-B, A is a bacteria-targeting peptide, PEG is polyethylene glycol 2000, B is a phospholipid, and A-PEG-B is selected from YCALQKRFILKMC-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, WKVRKSFFKLQG -PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine; the mucus layer penetrating functional inclusion compound X@CD-TPGS is an inclusion compound formed by X and CD-TPGS, wherein X is a catalytic enzyme, TPGS is vitamin E polyethylene glycol 1000 succinate, CD is a cyclodextrin, and the X@CD-TPGS is selected from catalase@hydroxypropyl-β-cyclodextrin-TPGS, urease@hydroxypropyl-β-cyclodextrin-TPGS, and catalase@methyl-β-cyclodextrin-TPGS; the driven oral nanoformulation is prepared by the following method: (1): Weigh the loaded drug, phospholipid, and bacteria-targeting functional polymer A-PEG-B in proportion and dissolve them in chloroform or methanol. Add the dissolved materials to a round-bottom flask and evaporate them into a thin film under reduced pressure in a water bath at 45-60 °C for 0.5-1 hour to remove the organic solvent and form a homogeneous film at the bottom of the round-bottom flask. Add pure water to the round-bottom flask and place the round-bottom flask in a rotary evaporator in a water bath at 45-60 °C for 0.5-2 hours to allow all the thin films at the bottom of the round-bottom flask to hydrate and fall off. Use an ultrasonic crusher to ultrasonicate at 100W for 5 minutes to crush the resulting solution. The ratio of loaded drug to chloroform or ethanol is 1g:1L, and the ratio of loaded drug to pure water is 1g:1L. (2): The solution obtained in step (1) and the mucus layer penetrating functional inclusion complex are mixed, and incubated at 35-38°C and a rotation speed of 100-150 rpm for 5-8 hours to obtain the nanoformulation.

2. The nanoformulation according to claim 1, characterized in that The nanoformulation consists of 15-25 parts by weight of loaded drug, 85-90 parts by weight of phospholipid, 8-13 parts by weight of bacteria-targeting functional polymer A-PEG-B, and 15-22 parts by weight of mucus layer penetrating functional inclusion compound X@CD-TPGS.

3. The nanoformulation according to claim 1, characterized in that The molar ratio of the bacteria targeting peptide, polyethylene glycol 2000 and phospholipid in the bacteria targeting functional polymer A-PEG-B is 1:1:1; the bacteria targeting functional polymer A-PEG-B is prepared by the following method: taking 100 mg of B-PEG2000-NHS as an example, 100 mg of B-PEG2000-NHS is weighed and dissolved in 5 mL of DMF, a resin connected with 1.1 eq of a bacteria targeting peptide whose side chain amino group is not deprotected by Boc is added, and then 3 eq of triethylamine is added. The reaction is carried out at room temperature for 12 hours to cut the bacteria targeting peptide from the resin. The solution is then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed for 24 hours using pure water as the dialysis medium, and the solution in the dialysis bag is collected and freeze-dried to obtain the bacteria targeting functional polymer A-PEG-B.

4. The nanoformulation according to claim 1, characterized in that The molar ratio of the catalytic enzyme, vitamin E polyethylene glycol 1000 succinate and cyclodextrin in the mucus layer penetrating functional inclusion compound X@CD-TPGS is 1:1:1; the mucus layer penetrating functional inclusion compound X@CD-TPGS is prepared by the following method: (1) 0.2 mmol glutaric acid, 0.1 mmol DMAP and 0.24 mmol EDC are added to each 1 mL DMSO to dissolve, and magnetic stirring is carried out for 1.5-3 h under nitrogen protection at room temperature, and then 0.04 mmol TPGS is added, and the reaction is continued under nitrogen protection at room temperature by magnetic stirring for 20-24 h, and then the crude product is transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed in deionized water for 48 h to remove DMSO, catalyst and unreacted raw materials, etc., and finally lyophilized to obtain the carboxylation product of TPGS TPGS-COOH; (2) 10 μmol TPGS-COOH, 40 μmol EDC and 70 μmol NHS are added to each 1 mL DMSO to dissolve, and stirred for activation for 30 min, then add 30 μmol cyclodextrin, stir and react for 20-24 h, dialyze the crude product with deionized water in a dialysis bag with a molecular weight cutoff of 2000 Da for 45-50 h to remove cyclodextrin-NH2, EDC, NHS and DMSO, and freeze-dry to obtain the product TPGS-βCD; (3) dissolve the catalytic enzyme powder in deionized water at a ratio of 0.005 mol:1 L of catalytic enzyme to deionized water, shake evenly, dissolve TPGS-cyclodextrin in deionized water at a ratio of 0.01 mol:1 L of TPGS-cyclodextrin to deionized water, shake evenly, mix the catalytic enzyme solution with the TPGS-cyclodextrin solution at a volume ratio of 1:1, and stir magnetically at room temperature for 24 h to obtain X@CD-TPGS.

5. The nanoformulation according to claim 1, characterized in that The nanoparticle preparation is micelle, liposome, or nanoparticle.

6. A method for preparing the nanoformulation according to any one of claims 1 to 5, characterized in that: Prepared by the following method: (1): Weigh the loaded drug, phospholipid, and bacteria-targeting functional polymer A-PEG-B in proportion and dissolve them in chloroform or methanol. Add the dissolved materials to a round-bottom flask and evaporate them into a thin film under reduced pressure in a water bath at 45-60 °C for 0.5-1 hour to remove the organic solvent and form a homogeneous film at the bottom of the round-bottom flask. Add pure water to the round-bottom flask and place the round-bottom flask in a rotary evaporator in a water bath at 45-60 °C for 0.5-2 hours to allow all the thin films at the bottom of the round-bottom flask to hydrate and fall off. Use an ultrasonic crusher to ultrasonicate at 100W for 5 minutes to crush the resulting solution. The ratio of loaded drug to chloroform or ethanol is 1g:1L, and the ratio of loaded drug to pure water is 1g:1L. (2): The solution obtained in step (1) and the mucus layer penetrating functional inclusion complex are mixed, and incubated at 35-38°C and a rotation speed of 100-150 rpm for 5-8 hours to obtain the nanoformulation.

7. Use of the nanoformulation according to any one of claims 1 to 5 in the preparation of a medicament for treating Helicobacter pylori infection.

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