Acid-stable, pH-responsive nanoparticle targeting Helicobacter pylori, preparation method, and use thereof

Abstract

The present invention discloses a nanoparticle with acid stability, pH responsiveness and targeting Helicobacter pylori, as well as a preparation method and use. The nanoparticle has a core-shell structure; wherein the core structure is an amino-modified mesoporous iron oxide nanoparticle loaded with manganese porphyrin; and the shell structure is a polymer coating targeting Helicobacter pylori. The pH-sensitive nanoparticles prepared by the present invention have acid stability and good mucus permeability. The pH-sensitive nanoparticles can accurately target Helicobacter pylori in the stomach. Magnetic resonance imaging (MRI) based on the particles can provide real-time information on the biodistribution of the drug in the body, guide ultrasonic irradiation when the drug release is maximum, and activate the drug to produce 1 O2 can eliminate Helicobacter pylori, and its inhibitory effect on Helicobacter pylori in vivo is comparable to that of antibiotic-based triple therapy, but it has no obvious mammalian cell toxicity and interference with normal intestinal flora, and has better biosafety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nanoparticle with acid stability, pH responsiveness and targeting Helicobacter pylori, as well as a preparation method and use thereof. Background Art

[0002] Helicobacter pylori (H.pylori) infection is one of the important factors leading to gastric cancer. It is also closely related to a variety of digestive tract diseases such as chronic gastritis, peptic ulcer, and gastric mucosal atrophy. Accurate and rapid detection and killing of it are of great significance to public health. At present, the detection methods of H.Pylori are divided into two categories: invasive examinations that require endoscopy and non-invasive examinations that do not require endoscopy. Invasive examinations mainly include endoscopy, rapid urease test, histopathological examination, polymerase chain reaction, and bacterial culture. Invasive examinations often reduce patient compliance, and endoscopes that are not thoroughly disinfected in hospitals may cause cross-infection of Helicobacter pylori. Non-invasive examinations reduce patient discomfort and are more convenient and quick. Non-invasive examinations include urea breath test, 15 Currently, commonly used non-invasive tests, such as urine ammonia excretion tests, stool antigen tests, and serological tests, do not directly detect H. pylori and often have the risk of false positives. Using intact H. pylori cells as a target is the most convenient and accurate method for detecting H. pylori. Therefore, developing a painless, accurate, and direct method for detecting H. pylori in vivo is crucial.

[0003] Clinically, antibiotic-based treatments for Helicobacter pylori infection, such as triple or quadruple therapy, often result in poor therapeutic efficacy due to antibiotic degradation and insufficient residence time of antibiotics in the gastrointestinal tract. The increasing abuse of antibiotics not only leads to the development of drug resistance in Helicobacter pylori but also to the adverse clearance of intestinal commensal bacteria. Therefore, eradication of Helicobacter pylori remains a huge challenge.

[0004] SDT converts oxygen into singlet oxygen (SO) by using sonosensitizers and appropriate ultrasound (US) stimulation. 1 O2) to kill bacterial cells without the risk of antibiotic resistance. 1O2 has a short lifespan and a limited migration distance, which severely limits the efficacy of SDT. In addition, the in vivo treatment of Helicobacter pylori faces the following difficulties. The drug must first be able to withstand the extremely acidic pH environment and the pepsin environment. Gastric emptying and the mucus layer hinder the drug from reaching the infected environment, and H. pylori is very prone to drug resistance. Therefore, there is an urgent need to develop a non-antibiotic treatment method to inhibit H. pylori infection, and it should meet the following five requirements: First, overcome the harsh gastric acid environment for drug administration; second, overcome the gastric mucus barrier for drug administration; third, accurately target Helicobacter pylori to increase the concentration of the drug at the infection site; fourth, do not produce Helicobacter pylori resistance; and fifth, do not affect the normal function of intestinal probiotics. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a nanoparticle with acid stability, pH responsiveness and targeting Helicobacter pylori, as well as a preparation method and use thereof.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] A nanoparticle that is acid-stable, pH-responsive, and targets Helicobacter pylori has a core-shell structure; wherein the core structure is amino-modified mesoporous iron oxide nanoparticles loaded with manganese porphyrin; and the shell structure is a polymer coating that targets Helicobacter pylori.

[0008] Furthermore, the polymer coating is made of an enteric polymer and modified with urea-PEG.

[0009] Furthermore, the preparation method of the coating is as follows:

[0010] Urea-PEG was synthesized from ureaacetic acid and PEG2000 via amidation reaction, and then modified onto the enteric polymer side chain via esterification reaction.

[0011] Furthermore, the enteric polymer is Eudragit L100-55.

[0012] The present invention prepares nanoparticles with a core-shell structure that are acid-stable, pH-responsive, mucus-penetrating, and specifically target Helicobacter pylori, as well as possessing nuclear magnetic resonance imaging (NMR) and SDT activity. The nanoparticles are composed of two effector units: (1) a multifunctional polymer-coated ELP, which imparts acid stability, mucus-penetrating properties, and the ability to target the urea channel protein (UreI) on the Helicobacter pylori cell wall. (2) a MRET effector unit, HMIN@MnTCPP—mesoporous iron oxide nanoparticles (HMINs) loaded with manganese porphyrin (MnTCPP), which quenches the NMR signal of MnTCPP.

[0013] The present invention first synthesized a multifunctional polymer molecule ELP, and modified its side chain with urea-based PEG2000, so that it has the function of mucus penetration and specific targeting of H. pylori, and can specifically release drugs in the H. pylori infection environment. Secondly, mesoporous ferrosoferric oxide nanoparticles (HMIN) were synthesized, and HMIN was amino-treated and then loaded with manganese porphyrin to prepare the MRET effect unit HMIN@MnTCPP (HM). ELP was coated on the surface of HM to prepare the nanoparticle EHM. The urea group of ELP can target the urea channel protein (UreI) on the cell wall of Helicobacter pylori and dissolve at the site of Helicobacter pylori infection to release manganese porphyrin. Manganese porphyrin is a commonly used sonosensitizer with excellent nuclear magnetic resonance imaging performance and sonodynamic therapy efficacy. The strengthening of the nuclear magnetic resonance imaging signal of manganese porphyrin indicates the presence of Helicobacter pylori infection. At the same time, nuclear magnetic resonance imaging can monitor the biological distribution of manganese porphyrin for nuclear magnetic resonance imaging-guided sonodynamic therapy, producing 1 O2 fights Helicobacter pylori.

[0014] Furthermore, the mass ratio of the mesoporous iron oxide nanoparticles to the manganese porphyrin is 4:1 to 1:2.

[0015] A method for preparing the above-mentioned nanoparticles comprises the following steps:

[0016] After S1 and ureaacetic acid were activated in EDC and NHS for 30 minutes, PEG2000 was added and reacted for 12 hours. The reaction of PEG2000 was monitored by thin layer chromatography to determine whether it was completely reacted. After the reaction was complete, unreacted ureaacetic acid, EDC and NHS were removed by dialysis and freeze-dried to obtain a white powder.

[0017] S2 and Eudragit L100-55 were activated in EDC at 25°C for 24 hours, then the powder obtained in step S1 was added and reacted for 48 hours. The EDC and the powder obtained in step S1 were dialyzed and freeze-dried to obtain a white powder.

[0018] S3. Disperse ferric chloride, sodium citrate and urea in a 100 mL round-bottom flask containing ultrapure water, stir rapidly until completely dissolved, and then slowly add polyacrylamide in batches under continuous stirring, and continue stirring for 30 minutes; then transfer the above mixed solution to a 100 mL polytetrafluoroethylene-lined stainless steel reactor, and react at a constant temperature of 200°C for 12 hours. After the reaction is completed, naturally cool to room temperature, separate the precipitate with an external magnet, discard the supernatant, and wash the precipitate three times with ultrapure water and anhydrous ethanol alternately, and collect the final black precipitate;

[0019] S4. Take the product of S3 and add it dropwise to a PEI solution (polyethyleneimine) under rapid stirring. Stir rapidly at a rate of 400 r / min for 24 h. After the reaction is completed, separate the precipitate with an external magnet, discard the supernatant, and wash it three times with ultrapure water to wash away unreacted PEI. Collect the precipitate and freeze-dry it;

[0020] S5. The product of S4 and MnTCPP were uniformly dispersed in water, sonicated continuously for 3 h at room temperature, shaken for 21 h, magnetically separated and washed three times, and freeze-dried.

[0021] S6. Rapidly mix the product of S5 with Eudragit L100-55 methanol solution, then immediately add it to a rapidly stirring polypropylene alcohol aqueous solution (pH = 3). After rapid stirring for 20 minutes, centrifuge at 4000 rpm for 3 minutes, and wash the precipitate three times with simulated gastric acid solution.

[0022] Furthermore, the mass fraction of the polymer in the polymer coating solution is 2-5%.

[0023] A preparation for treating related diseases caused by Helicobacter pylori infection, comprising the above-mentioned nanoparticles, or the nanoparticles prepared by the above-mentioned method.

[0024] Beneficial effects of the present invention:

[0025] The pH-sensitive nanoparticles prepared by the present invention have acid stability and good mucus permeability. MRI shows that the pH-sensitive nanoparticles can accurately target Helicobacter pylori in the stomach, provide real-time information on the biodistribution of drugs in the body, and can be irradiated with ultrasound when the drug release is the most accumulated to activate the drug production. 1 O2 can eliminate H. pylori, and its in vivo therapeutic effect is comparable to that of antibiotic-based triple therapy, but with better biosafety and will not harm mammalian cells and normal intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flow chart for the preparation of pH-sensitive nanoparticles and diagram of their mechanism of action;

[0027] Figure 2 For ELP 1 H NMR spectrum;

[0028] Figure 3 The SEM and SEM images of EHM and element distribution map are shown;

[0029] Figure 4 The results of the EHM's acid stability, pH-responsive drug release, nuclear magnetic resonance imaging performance, mucus permeability, and targeting properties are shown in the figure.

[0030] Figure 5 To investigate the in vitro antibacterial activity of EHM;

[0031] Figure 6 This is a diagram showing the validation results of the Helicobacter pylori (ATCC11637) infection model in C57BL / 6 mice;

[0032] Figure 7 This is the result of magnetic resonance imaging of Helicobacter pylori (ATCC11637) infection detected in EHM.

[0033] Figure 8 This is the result diagram of the in vivo therapeutic effect of EHM on Helicobacter pylori (ATCC11637) infection;

[0034] Figure 9 Conduct biosafety inspection for EHM;

[0035] Figure 10 This is a graph showing the effects of EHM and standard triple therapy on intestinal flora;

[0036] Figure 11 This figure shows the effects of EHM and standard triple therapy on intestinal microbial abundance levels. DETAILED DESCRIPTION

[0037] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0038] Example 1 Preparation of pH-sensitive nanoparticles

[0039] 1. Preparation of urea-PEG

[0040] 5 mg / mL ureaacetic acid, 5 mg / mL EDC, and 4 mg / mL NHS were dissolved in 2 mL DMF and activated at 25°C for 30 minutes. Then, 10 mg / mL PEG2000 was added and the reaction continued for 12 hours. The complete reaction of PEG2000 was monitored by thin-layer chromatography. After the reaction was complete, unreacted ureaacetic acid, EDC, and NHS were removed by dialysis against 200D for 12 hours. The urea-PEG was then freeze-dried to obtain a white powder. H-NMR spectroscopy showed that its structure was consistent with that of the target compound.

[0041] 2. Preparation of urea-PEG-modified Eudragit L100-55

[0042] 25 mg / mL Eudragit L100-55 and 3 mg / mL EDC were dissolved in 2 mL DMF and activated at 25°C for 24 h. 20 mg / mL urea-PEG was added and the reaction continued for 48 h. Unreacted EDC and urea-PEG were removed by dialysis at 2500 D for 12 h. The ELP was obtained by freeze-drying to obtain a white powder. H NMR spectroscopy showed that its structure was consistent with that of the target compound ( Figure 2 ).

[0043] 3. Preparation of amino-modified HMIN

[0044] (1) Disperse 0.811 g of ferric chloride (13.5 g / L), 1.76 g of sodium citrate (30.0 g / L), and 0.541 g of urea (9.00 g / L) in a 100 mL round-bottom flask containing 60 mL of ultrapure water and stir rapidly until completely dissolved. Slowly add 0.30 g of polyacrylamide (PAM, 5.0 g / L) in batches while stirring continuously for 30 minutes. The mixed solution is then transferred to a 100 mL polytetrafluoroethylene-lined stainless steel reactor and reacted at 200 °C for 12 hours. After the reaction is completed, it is naturally cooled to room temperature. The precipitate is separated by an external magnet, the supernatant is discarded, and the precipitate is washed three times with ultrapure water and anhydrous ethanol alternately. The final black precipitate is collected to obtain HMIN.

[0045] (2) 40 mL of 2.5 g / L HMIN was added dropwise to 10 mL of 20 g / L PEI solution under rapid stirring at 400 rpm for 24 h. After the reaction was complete, the precipitate was separated using an external magnet, the supernatant was discarded, and the solution was washed three times with ultrapure water to remove unreacted PEI. The precipitate was collected and freeze-dried to obtain HMIN-NH2.

[0046] 4. Loading manganese porphyrin

[0047] 10 mg / mL HMIN-NH2 and 10 mg / mL MnTCPP were evenly dispersed in water, continuously ultrasonicated for 3 h at room temperature, shaken for 21 h, magnetically separated and washed three times, and freeze-dried to obtain HMIN-NH2@MnTCPP.

[0048] 5. Preparation of nanoparticles with core-shell structure

[0049] 2 mg of HMIN-NH2@MnTCPP was quickly mixed with 2.5% urea-PEG-modified Eudragit L100-55 methanol solution, and then immediately added to 5 mL of rapidly stirring 1% PVA aqueous solution (pH = 3). After rapid stirring for 20 minutes, the mixture was centrifuged at 4000 rpm for 3 minutes, and the precipitate was washed three times with simulated gastric acid solution (SGF, pH = 1.2) to obtain the nanoparticle EHM.

[0050] Example 2 Characterization of pH-sensitive nanoparticles

[0051] Scanning electron microscopy (SEM) and energy spectrum showed that the particle size of EHM increased after coating with ELP, and Mn was successfully loaded and evenly distributed in EHM, indicating that the synthesis of EHM was successful (see Figure 3 B). In addition, DLS and Zeta potential were also used to monitor the preparation process of EHM, and the results were consistent with those of SEM (see Figure 3 A). Taken together, these findings confirm the successful preparation of EHM.

[0052] Example 3 pH-sensitive nanoparticle performance detection

[0053] 1. Gastric acid stability

[0054] The probe was incubated in simulated gastric fluid (pH 1.5) at a concentration of 100 μg / mL for 2 days, and the release of MnTCPP in the magnetic separation supernatant was observed after incubation. The particle size of the probe was measured after incubation in simulated gastric fluid for 1-7 days, and its morphological stability was investigated. The results are shown in Figure 4 a.

[0055] like Figure 4 As shown in a, EHM nanoparticles can be stable in simulated gastric acid solution for 3 days, which can meet the drug delivery requirements.

[0056] 2. pH release behavior

[0057] The MnTCPP release rate of the nanoparticles in the simulated gastric acid environment (pH 2.5), gastric mucus layer (pH 4.5-7.0), and gastric epithelial environment (pH 7.4) was investigated respectively. ELP-HMIN@MnTCPP nanoprobes were added to buffers in different pH environments and incubated at 37°C. At 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 9h, and 12h of incubation, the precipitate was separated by an external magnet and the supernatant was collected. The concentration of Mn element in each supernatant sample was measured by ICP-OCE, and the cumulative release curve of MnTCPP was drawn. The experiment was repeated 3 times for each sample, and the average value was calculated. The results are shown in Figure 4 b and c.

[0058] like Figure 4 As shown in Figures b and c, MnTCPP is released in small amounts in simulated gastric acid, but is rapidly and extensively released in a pH 6.0 environment. To simulate the in vivo transport process of the nanoparticles, we also used specific incubation intervals (2 hours at pH 1.2, 2 hours at pH 6.0, and 8 hours at pH 7.4) to simulate the environmental changes from gastric fluid to the mucosal layer and then to the H. pylori colonization area (gastric mucosal epithelium) to evaluate the nanoparticle release behavior. EHM can partially block the release of Mn in gastric acid (pH 1.2), but releases it in large quantities in the H. pylori colonization area.

[0059] 3. MRI performance

[0060] The longitudinal relaxation rates (r1) of MnTCPP, HMIN@MnTCPP, and ELP-HMIN@MnTCPP before and after pH-sensitive release were investigated and compared to verify the change in magnetic relaxation rate before and after MRET response. The specific process is as follows:

[0061] MnTCPP, HMIN@MnTCPP, and ELP-HMIN@MnTCPP were diluted with SGF solution into three groups of solutions with different manganese concentrations (0.01mM, 0.02mM, 0.03mM, 0.06mM, and 0.12mM). At the same time, a group of solutions was set up to dilute ELP-HMIN@MnTCPP into a series of different manganese concentrations using PBS buffer (pH=6.0). Then, each group of solutions was placed in a constant temperature mixer and incubated at 37°C and 200r / min for 2h. After the incubation was completed, the precipitate was separated by an external magnet and the supernatant was collected. The supernatant of each group was placed in a 1.5mL EP tube, and each sample was scanned using a 0.5T magnetic resonance imaging system using a spin echo pulse sequence to obtain the longitudinal relaxation time T1. Then, a linear regression was performed between 1 / T1 and the Mn concentration in the corresponding sample. The slope of the obtained straight line was the longitudinal relaxation rate r1 of the sample to be tested. The results are shown in FIG. Figure 4 d and e.

[0062] The specific imaging parameters are as follows: repetition time (TR) = 200 ms, echo time (TE) = 20 ms; field of view = 100 × 100 mm2; matrix size = 256 × 192; number of slices = 1; slice thickness = 8 mm; flip angle = 90°; averages = 8.

[0063] like Figure 4 As shown in d and e, when the pH of the EHM solution changes from 1.2 to 6.0, ELP dissolves and MnTCPP is released, and the longitudinal relaxation rate (r1) of the EHM solution increases from 1.78 mM to 6.0. -1 s -1 becomes 11.5mM -1 s -1 , the nuclear magnetic imaging signal becomes brighter, which shows that ELP has pH responsiveness and good T1-weighted imaging performance.

[0064] 4. Mucus penetration performance

[0065] The in vitro adhesive properties of the prepared nanoparticles were evaluated using commercial porcine mucin particles. It is generally assumed that if nanoparticles exhibit adhesive properties, the surface properties of mucus, such as surface charge, may be affected by the adhering particles. This change can be determined by measuring the zeta potential using a laser particle size analyzer. Mucin particles were thoroughly suspended in deionized water at a concentration of 1% and then mixed with various concentrations of prepared particles or polymer solutions at pH 2.5 and pH 6.0 to simulate the gastric environment. Mucin solutions were prepared by dissolving mucin in distilled water and then filtering to remove insoluble solids. After incubation at 37°C for 2 hours, the mixture was magnetically separated, and the particle size and potential of the mucus-EHM conjugates were measured using a particle size analyzer. Nanoparticles synthesized using EL-HM coated with unmodified PEG served as a control.

[0066] To evaluate the mucus penetration of the prepared ELP-HMIN@MnTCPP nanoparticles, 50 mg / mL (pH 7.4) and 20 mg / mL (pH 6) porcine gastric mucin solutions were sequentially coated on transwell inserts with a thickness of 200 μm to simulate the environment of the gastric mucosal system. The prepared ELP-HMIN@MnTCPP nanoparticle solution was added on top of the mucin layer, and the drug concentration permeating into the outer chamber at different time points was measured. The apparent permeability coefficient Papp (cm / s) was calculated using the formula:

[0067]

[0068] Where dQ / dt represents the permeation amount of nanomedicine per unit time, A is the effective permeation area, and C is the initial concentration of nanomedicine. The results are shown in Figure 4 f and g.

[0069] like Figure 4 As shown in f and g, it was found that EHM has good mucus permeability. The specific recognition ability of EHM for H. pylori was then investigated using fluorescence microscopy. The results showed that EHM can specifically recognize H. pylori, providing the possibility for subsequent EHM to target H. pylori infection sites in vivo.

[0070] 5. Targeting

[0071] H. Pylori (109 cfu / mL) was washed three times with sterile PBS (pH = 6.0, 0.01M) by centrifugation and resuspended in 80 μL PBS, incubated with 80 μL DAPI stain (100 μg / mL) at 37 ° C for 30 min, and then centrifuged and washed three times. 40 μL ELP-HMIN@MnTCPP (100 μg / mL) nanoparticles were added and incubated in a cell culture incubator at 37 ° C for 1 hour. After that, the bacteria-nanoparticle complex was captured by magnetic separation and redispersed in 50 μL sterile PBS (pH 6.0). Nanoparticles EL-HM synthesized with unmodified PEG EL as the coating material were used as a control. Then the cells were observed under a fluorescence microscope. The whole process was kept away from light. The results are shown in Figure 2. Figure 4 h.

[0072] like Figure 4 As shown in h, H. pylori treated with EHM for 2 hours exhibited strong absorption after magnetic separation. However, when treated with the control EL-HM (which lacks a targeting moiety), there was almost no fluorescence after magnetic separation. These results suggest that EHM can actively target H. pylori via the urea moiety.

[0073] 6. ROS production capability

[0074] The efficiency of singlet oxygen generation after ELP-HMIN@MnTCPP release under ultrasound was detected using a 1,3-diphenylisobenzofuran (DPBF) probe, as follows:

[0075] 15 μg / mL DPBF was mixed with free MnTCPP and ELP-HMIN@MnTCPP (MnTCPP concentration: 100 μg / mL) in an ethanol-water mixed solvent (ethanol: water = 1:1), and then subjected to low-intensity focused ultrasound irradiation (1 MHz, 1.5 W cm -2At different time points, the absorption intensity of the solution at 410 nm was monitored using a multifunctional microplate reader, and the absorption decay rate was calculated to evaluate the ability to generate singlet oxygen. Free MnTCPP was used as a positive control, and ELP-HMIN@MnTCPP before pH response was used as a negative control. The results are shown in Figure 5 .

[0076] like Figure 5 As shown in Figure 2, DPBF was used as an indicator to detect the singlet oxygen produced after low-intensity focused ultrasound irradiation of nanoparticles. 1 O2 undergoes an irreversible reaction, resulting in attenuation of UV-visible absorption at 410nm. The absorption peak intensity of EHM at 410nm in the SGF+DPBF group, the ultrasound irradiation group or the non-ultrasound group showed no significant change. In contrast, the absorption peak intensity of the EHM+DPBF group solution at 410nm showed a significant downward trend with the increase of irradiation time, indicating that EHM can produce 1 O2, and the ability to produce singlet oxygen was characterized by dependence on ultrasound irradiation time.

[0077] Example 4 Ability of pH-sensitive nanoparticles to inhibit Helicobacter pylori in vitro

[0078] 1. Experimental methods

[0079] Helicobacter pylori was cultured overnight at 37°C under microaerobic conditions until the OD600 value of the bacteria reached 0.5. Next, 30 μL of the bacterial suspension was mixed with ELP-HMIN@MnTCPP (MnTCPP concentration: 100 μg / mL) to a final volume of 300 μL. After simulating the release of ELP-HMIN@MnTCPP at different concentrations in PBS buffer solution for 2 h, the cells were exposed to ultrasound irradiation (1 MHz, 1.5 W cm -2 ) for 0, 2, 4, 6, and 8 minutes. Next, 100 μL of bacterial suspension was spread on a blood plate and cultured at 37°C for 72 hours to form viable colony units. The bacterial inhibition rate was calculated. The group without adding nanomaterials and only irradiated with ultrasound was used as a control. The results are shown in Figure 2. Figure 5 B.

[0080] The sonotoxicity of EHM against bacterial cells was determined by colony counts. EHM exhibited significant antibacterial properties after six minutes of ultrasound exposure. EHM concentration affected the level of ROS generated by ultrasound, so its inhibitory effect on H. pylori was investigated. The minimum bactericidal concentration (MBC) of the nanomedicine was determined to be the lowest drug concentration required to kill 99.9% (a three-order-of-magnitude reduction) of H. pylori.

[0081] like Figure 5As shown in C, the ultrasound treatment frequency is 1 MHz and the intensity is 1.5 W cm -2 The bacterial inhibition rate reached 99% when the time was 6 minutes and the EHM (containing 6.25 μg / mL of MnTCPP) was used, indicating that the minimum bactericidal concentration (MBC) of the nanomedicine was EHM (containing 6.25 μg / mL of MnTCPP). These results indicate that EHM can be used as a promising sonosensitizer to eradicate Helicobacter pylori infection.

[0082] Example 5: In vitro nuclear magnetic resonance imaging of pH-sensitive nanoparticles for detecting Helicobacter pylori infection

[0083] 1. Establishment of a Helicobacter pylori infection model in C57 BL / 6 mice

[0084] Four-week-old C57BL / 6 male mice weighing approximately 16–20 g were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. They were housed in the college's animal room under conditions that complied with the regulations governing laboratory animals.

[0085] First, H. pylori was washed three times by centrifugation in fresh liquid culture medium and resuspended in blank liquid culture medium. The OD value was adjusted to 1.0 at a wavelength of 600 nm. C57 BL / 6 mice were deprived of water and food overnight. Each mouse was then gavaged with 0.5 mL of 5% NaHCO₃. Four hours later, 1 mL of the bacterial suspension was administered orally. Two hours after gavage, water and food were given. This gavage was repeated every other day for four days. After three weeks of acclimatization, feces were collected and tested using a Helicobacter pylori fecal antigen kit. Four mice were randomly selected and sacrificed, and gastric tissue was removed. Gastric debris was gently washed with saline to remove the remaining gastric debris. The gastric tissue was homogenized and then cultured for bacteria. The remaining gastric tissue was then fixed with 10% paraformaldehyde solution for 24 hours, paraffin-embedded sections were prepared, and stained with hematoxylin and eosin. The gastric tissue samples were then observed under a fluorescence microscope for damage.

[0086] 2. In vivo nuclear magnetic resonance imaging performance assessment

[0087] like Figure 6 As shown in A, 4-week-old C57BL / 6 male mice were selected as the model. During the modeling process, it should be noted that water and food should be withheld the night before. After 3 weeks of adaptation, one mouse in each of the model group and the control group was randomly killed, and the infection status of the mice was identified by H&E staining. Figure 6 As shown in B and C, compared with the gastric tissue sections of the control group mice, the gastric tissue specimens of the model group infected mice showed Helicobacter pylori colonization and obvious inflammatory cell infiltration, indicating that the C57BL / 6 mouse infection model was successfully established. Figure 6As shown in D, the bacterial culture results of the gastric tissues of 5 randomly sacrificed mice were all positive, confirming the successful colonization of Helicobacter pylori in the mice. Due to the rapid renewal and shedding of the gastric mucosal epithelium, the colonized Helicobacter pylori will also fall off. Therefore, fecal DNA testing can be used as a non-invasive method for the preliminary diagnosis of Helicobacter pylori infection. Figure 6 As shown in E, feces of modeling mice were collected and tested positive using a fecal antigen kit.

[0088] The model mice were weighed and anesthetized by intraperitoneal injection of 10% chloral hydrate with a sterile syringe. T1 imaging of the mice was performed using a nuclear magnetic resonance imaging analysis system. The probe injection volume was then calculated based on the mouse weight (the MnTCPP concentration in the control probe injection was 8 mg / kg). The probe was injected into the model mice via oral gavage. T1 imaging of the mice was performed at 5 minutes, 0.5 hours, 1 hour, 2 hours, 4 hours, and 12 hours after the probe injection to obtain T1 MRI images of the mice at different time points. The results are shown in Figure 2. Figure 7 .

[0089] like Figure 7 As shown in the figure, after oral administration of EHM nanoparticles to H. pylori model mice, the gastric tissue exhibited a dark signal, indicating the probe was in the "MRET ON" state. As the probe remained in the body for an extended period, the gastric tissue gradually brightened between 0.5 and 2 hours, reaching a peak (maximum brightness) 2 hours after probe injection. This is due to the probe releasing the maximum amount of MnTCPP in the infection environment 2 hours later, consistent with in vitro experimental results, indicating the probe was in the "MRET OFF" state at this time. Oral administration of EHM nanoparticles to normal mice revealed no specific release of MnTCPP in the infected area, and the gastric tissue of the mice exhibited a dark signal. In contrast, the MR signal of free MnTCPP in the control group peaked immediately after oral administration and gradually returned to the unadministered state as the stomach emptied. MnTCPP was unable to distinguish between infected and control mice. In summary, EHM nanoparticles can specifically detect H. pylori infection in vivo, achieving an "off-on" MR signal, thereby increasing imaging sensitivity and contrast. Imaging showed that the nanomedicine accumulated most at the infection site 2 hours after gavage, which can be used to guide SDT, indicating that 2 hours after gavage of the probe is the optimal SDT time for mice.

[0090] Example 6: Ability of pH-sensitive nanoparticles to treat Helicobacter pylori infection by in vivo nuclear magnetic resonance imaging

[0091] 1. Method

[0092] The 25 C57 BL / 6 model mice infected with Helicobacter pylori from the above experiment were randomly divided into 5 groups (A, B, C, D and E), with 5 mice in each group. They were marked and placed in mouse culture cages. Water and food were withheld overnight, and the mice were weighed. Group A was given SGF solution, while Group B was given SGF and ultrasound irradiation (1 MHz, 1.5 W cm -2 Group C received EHM nanoparticles at a MnTCPP concentration of 8 mg / kg, Group D received EHM nanoparticles and ultrasound irradiation at a MnTCPP concentration of 8 mg / kg, and Group E received triple therapy (amoxicillin (30.0 mg / kg), clarithromycin (15.0 mg / kg), and lansoprazole (0.9 mg / kg) suspension for 3 days). All mice were administered by gavage. 48 hours after treatment, the mice were sacrificed to collect gastric tissue. Gastric tissue from three mice in each group was randomly sampled, weighed, and homogenized in 2.5 mL of PBS. The homogenate was then plated on an agar plate to count the number of bacteria.

[0093] 2. Results

[0094] The colony forming units (CFU) of H. pylori in the stomach of mice infected with Helicobacter pylori were quantified after treatment. Ultrasound was indicated as "+" and no ultrasound was indicated as "-".

[0095] like Figure 8 As shown, the colony counts of SGF(-) and SGF(+) were 2.0×10 7 CFU / g and 1.5×10 7 CFU / g, ultrasound can inhibit it to a certain extent. Compared with EHM(-) (1.7×10 7 CFU / g), the EHM(+) group showed superior antibacterial effect, and the colony count of EHM(-) was 1.98×10 3 CFU / g, compared with antibiotic-based treatment (triple therapy group) (2.0×10 3 CFU / g), and the EHM(+) group showed similar therapeutic efficacy without developing resistance. These results suggest the great potential of EHM as an alternative to antibiotic-based therapies for the effective eradication of H. pylori.

[0096] To further evaluate the efficacy of EHM-mediated SDT, H&E staining was used to investigate the extent of gastric inflammation and bacterial infection after treatment. HE staining was used to observe the damage and inflammatory response to H. pylori-induced gastric tissue in C57 mice. Model mice treated with SGF alone still showed significant inflammatory cell infiltration and mucosal necrosis in the gastric mucosa. Inflammatory cell infiltration was also observed in the SGF(+), EHM(-), and antibiotic groups, but EHM(+) treatment resulted in virtually no inflammatory cell infiltration or mucosal necrosis in the gastric mucosa. Together, these results suggest that EHM-mediated SDT shows great potential as an alternative to antibiotics, effectively treating H. pylori infection without adverse reactions.

[0097] Example 7: In vitro biosafety assessment of pH-sensitive nanoparticles by nuclear magnetic resonance imaging

[0098] 1. Cytotoxicity assay

[0099] Human gastric epithelial cells GES-1 were cultured in RPMI-1640 complete medium (with penicillin, streptomycin and 10% FBS) at 37°C, saturated humidity and 5% CO2. Cell growth was observed under a microscope. When the cells were in good growth condition, the logarithmic phase cells were collected for further experiments. In this experiment, CCK-8 was used to study the toxicity of ELP-HMIN@MnTCPP on GES-1 cells. After the cells grew to approximately 80% of their density, they were washed twice with PBS buffer. 200 μL of complete medium containing different concentrations of ELP-HMIN@MnTCPP was added to each well of the experimental group, and the same volume of empty medium was added to the empty control group. After 24 hours, 100 μL of new CCK-8 medium was added to each well and incubated for 2 hours. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader, and the experiment was repeated three times. The cell survival rate was calculated according to the following formula:

[0100] Cell survival rate (%) = absorbance of experimental group after zero adjustment / absorbance of control group × 100%

[0101] 2. Macrobiological safety

[0102] The changes in body weight 30 days after drug injection were monitored to macroscopically evaluate the effects of ELP-HMIN@MnTCPP treatment on digestive tract function.

[0103] 3. Hematoxylin-eosin stained pathological sections

[0104] Twenty-four hours after probe injection, mice were killed by cervical dislocation, dissected, and the hearts, livers, spleens, lungs, and kidneys were collected and fixed with 4% paraformaldehyde. The tissues were then embedded in paraffin, sectioned, stained with H&E, and photographed to investigate the physiological toxicity of the probe.

[0105] 4. Metabolic kinetics in vivo

[0106] To investigate the gastrointestinal metabolic dynamics of ELP-HMIN@MnTCPP nanoparticles, we orally administered 100 μL of the probe (200 μg / mL) to C57 BL / 6 mice and collected feces within 12 hours. The feces and probe were then digested with a digestion solution at 70°C for 6 hours. Fecal Fe and Mn content were then measured by inductively coupled plasma atomic emission spectroscopy (ICP-OES). The cumulative elimination rate of MnTCPP in feces was calculated using the following formula:

[0107] Cumulative elimination rate = Cfeces / CELP-HMIN@MnTCPP×100%

[0108] C feces and CELP-HMIN@MnTCPP are the Mn element concentrations determined by ICP-OES, respectively.

[0109] like Figure 9 As shown, the gastrointestinal Fe retention time of mice was monitored by collecting feces and measuring their Fe and Mn contents normalized to fecal weight (n=5). EHM is superparamagnetic and is easily excreted from the body along with food residues. About 95% of EHM was excreted through feces after 12 hours. The effect of treatment on digestive tract function was macroscopically assessed by changes in mouse body weight. No significant changes were observed in all experimental groups. In addition, organ coefficients were observed to evaluate the long-term toxicity of nanoparticle treatment. There were almost no substantial changes in major organs. HE staining of all organs after treatment is shown in the figure. Figure 9 Figure D. Pathological analysis of the stomach using hematoxylin and eosin (H&E) staining revealed abundant gastric glands in the lamina propria, with normal morphology and structure of principal and parietal cells, and no significant inflammation. H&E staining of organotypic sections revealed no obvious lesions. Overall, this demonstrates the high biocompatibility of EHM.

[0110] Example 8 Effects of pH-sensitive nanoparticles and standard triple therapy on intestinal flora

[0111] 1. Method

[0112] Feces of treated mice were collected for quantitative analysis of intestinal flora.

[0113] 2. Results

[0114] The intestinal contents of mice in the treatment group and triple therapy treatment were analyzed for bacterial diversity. Alpha diversity is mainly used to study the diversity of communities in a certain habitat (or sample). By evaluating a series of Alpha diversity indices, information such as the richness and diversity of species in the environmental community can be obtained. The indices reflecting community richness are: sobs, chao, and ace; this article uses the sobs index, which is the actual number of species observed; through the Alpha diversity analysis of the richness of the mouse intestinal flora, it can be seen that the intestinal flora abundance level of mice in the EHM sonodynamic therapy group is similar to that of the normal group; while the flora abundance level of mice in the triple antibiotic treatment group was significantly decreased ( Figure 10 ), which is mainly due to the toxic side effects of antibiotics on intestinal symbiotic bacteria. In addition, through the heat map of the community analysis at the genus level of intestinal contents, it can be seen that the species abundance of intestinal microorganisms in the EHM sonodynamic therapy group is similar to that of the normal group at the genus level ( Figure 11 ). However, most commensal bacteria in the triple antibiotic treatment group were downregulated at the genus level, further demonstrating the significant toxic side effects of antibiotics on the intestinal flora. These results demonstrate that EHM can target specific areas of the stomach.

[0115] The sonodynamic therapy, which responds to acidic environments, kills bacteria through ultrasound irradiation, avoiding nonspecific damage to normal tissues and intestinal commensal bacteria and demonstrating good biocompatibility. The primer sequences for real-time fluorescence quantitative PCR used for microbial diversity analysis are shown in Table 1.

[0116] Table 1 Primer sequences for real-time fluorescence quantitative PCR used for bacterial diversity analysis

[0117]

[0118] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can 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 included in the scope of the claims of the present invention.

Claims

1. A nanoparticle with acid stability, pH responsiveness and targeting Helicobacter pylori, characterized in that: It has a core-shell structure; wherein the core structure is amino-modified mesoporous ferrosoferric oxide nanoparticles loaded with manganese porphyrin; the shell structure is a polymer coating targeting Helicobacter pylori; the polymer coating is made of enteric polymer Eudragit L100-55 and modified with urea-PEG; The preparation method of the coating is as follows: Urea-PEG was synthesized from ureaacetic acid and PEG2000 via amidation reaction, and then modified onto the enteric polymer side chain via esterification reaction.

2. The nanoparticles according to claim 1, characterized in that The mass ratio of the mesoporous ferrosoferric oxide nanoparticles to the manganese porphyrin is 4:1 to 1:

2.

3. A method for preparing the nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: (1) Preparation of mesoporous Fe3O4 nanoparticles; (2) The mesoporous ferroferric oxide nanoparticles and manganese porphyrin are uniformly dispersed in the solution, and the manganese porphyrin is loaded onto the mesoporous ferroferric oxide nanoparticles to obtain HMIN@MnTCPP; (3) The polymer coating solution was mixed with HMIN@MnTCPP, and then a polyvinyl alcohol aqueous solution was added to form a core-shell structure. The nanoparticles were obtained after centrifugation and washing.

4. The preparation method according to claim 3, characterized in that The mesoporous ferrosoferric oxide nanoparticles are prepared by a one-step hydrothermal synthesis method.

5. The preparation method according to claim 3, characterized in that After mixing in step (2), the mixture needs to be ultrasonicated at room temperature for 3 to 5 hours, then shaken for 18 to 25 hours, and then magnetically separated and washed 3 to 5 times.

6. The preparation method according to claim 3, characterized in that The mass fraction of the polymer in the polymer coating solution is 2-5%.

7. A preparation for treating Helicobacter pylori infection-related diseases, characterized in that: The nanoparticles include the nanoparticles according to claim 1 or 2, or the nanoparticles prepared by the method according to any one of claims 4 to 6.

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