Ultrasound-sensitized nanoparticles and their application, and a method for killing Helicobacter pylori

Abstract

The present invention provides an ultrasound-sensitized nanoparticle and its application as well as a method for killing Helicobacter pylori. The ultrasound-sensitized nanoparticle includes a carrier and an ultrasound sensitizer loaded on the carrier. In the present invention, the carrier includes a polymer and / or a phospholipid membrane. The present invention obtains ultrasound-sensitized nanoparticles by loading the carrier with an ultrasound sensitizer, and ultrasonically stimulates the treatment site with ultrasound waves that have a strong penetrating ability to biological tissues, thereby making it have the activity of effectively killing Helicobacter pylori. Since the ultrasound-sensitized nanoparticles do not require the addition of antibiotics, the problem of antibiotic resistance will not be caused. At the same time, the ultrasound-sensitized nanoparticles have an excellent effect of adsorbing Helicobacter pylori vacuolating toxin (VacA).

Description

Technical Field

[0001] The invention relates to the field of antibacterial, and in particular to an ultrasound-sensitized nanoparticle and an application thereof, and a method for killing Helicobacter pylori. Background Art

[0002] At present, bacterial infections have become a serious threat to public health, especially Helicobacter pylori. As one of the most common human-transmitted pathogens, Helicobacter pylori has infected at least half of the world's population, and the infection rate is often higher in countries and regions with lower economic development levels.

[0003] Helicobacter pylori infection can cause a series of chronic gastric inflammations, which continue to develop and lead to serious gastrointestinal diseases such as peptic ulcers, mucosa-associated lymphoid tissue lymphoma, and may even develop into gastric cancer. Studies have shown that for every 10% increase in the infection rate of Helicobacter pylori, the incidence of gastric cancer will increase by 25%, and the mortality rate will further increase by 18%. In 1994, the International Agency for Research on Cancer (IARC) identified Helicobacter pylori as a Class I human carcinogen, which is currently the only officially confirmed human carcinogenic bacterium. Therefore, antibacterial research on Helicobacter pylori is very necessary.

[0004] Helicobacter pylori vacuolating cytotoxins (VacA, 88kDa) are soluble proteins secreted into the extracellular space by Helicobacter pylori. VacA regulates mammalian cells in a variety of ways. It can act as an adhesin to mediate the binding of bacteria to gastric epithelial cells; VacA may also change the surface properties of gastric epithelial cells or change the secretion of various factors by these cells; VacA also destroys the integrity of the gastric epithelial barrier, thereby releasing nutrients required for the growth of Helicobacter pylori in the gastric mucus layer, etc., all of which will promote the colonization and infection of Helicobacter pylori in the stomach. In addition, studies have shown that VacA may be related to the pathogenesis of Helicobacter pylori-related peptic ulcer disease and gastric cancer.

[0005] At present, the clinical treatment of Helicobacter pylori is mainly triple therapy (a combination of proton pump inhibitors plus two antibiotics) or quadruple therapy (a combination of proton pump inhibitors plus bismuth plus two antibiotics). However, both methods inevitably use antibiotics and there is a problem of bacterial resistance. It is reported that the resistance of three antibiotics commonly used in the treatment of Helicobacter pylori, such as clarithromycin, metronidazole and levofloxacin, has reached 20-45%, 40-70% and 20-45% respectively, while some other therapeutic antibiotics such as amoxicillin, tetracycline and furazolidone have also begun to show resistance. Comprehensive experimental analysis studies on persistent Helicobacter pylori infection have shown that due to the increasing bacterial resistance year by year, the clinical treatment failure rate is close to 25%, resulting in a very high recurrence rate of Helicobacter pylori infection after antibiotic treatment.

[0006] At present, some alternative methods for treating Helicobacter pylori infection have also been developed, mainly through the development of new antibacterial substances, such as small molecule antibacterial substances from animals and plants, aromatic fatty acids, peptides, polysaccharides, etc. In addition, it is also possible to use active oxygen therapy, such as photodynamic therapy and antibacterial therapy based on nanomaterials.

[0007] However, the development of new antibacterial therapies takes a long time, and there are still many problems to be studied before the newly developed antibacterial compounds can be used in clinical treatment. In addition, most researchers still focus on the elimination of Helicobacter pylori, and often ignore the elimination of Helicobacter pylori vacuolating toxin (VacA). That is, it is not known whether the antibacterial substances such as small molecule compounds, polysaccharides, peptides and nanomaterials mentioned above can effectively eliminate the toxins released by bacteria during treatment. Therefore, these methods are obviously inappropriate for the clinical treatment of Helicobacter pylori. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide an ultrasound-sensitized nanoparticle and its application and a method for killing Helicobacter pylori. The present invention adopts ultrasound-sensitized nanoparticle-mediated ultrasound dynamic therapy, which can not only solve the severe antibiotic resistance problem in the treatment of Helicobacter pylori, but also remove the toxins released by the bacteria, thereby better protecting gastric mucosal cells.

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides an ultrasound-sensitized nanoparticle, comprising a carrier and an ultrasound sensitizer loaded on the carrier;

[0011] The ultrasonic sensitizer includes an organic small molecule ultrasonic sensitizer and / or an inorganic ultrasonic sensitizer;

[0012] The organic small molecule ultrasound sensitizer includes any one or more of porphyrin, hematoporphyrin, verteporfin, hematoporphyrin monomethyl ether, protoporphyrin IX, galloporphyrin, chlorophyll, chlorophyll-a, pheophorbide A, chlorin e6, pyropheophorbide a hexyl ether, mono-asparagine chlorin e6, m-tetrahydroxychlorophenol, indocyanine green, IR-780 iodide, curcumin or hypocrellin B;

[0013] The inorganic ultrasound sensitizer includes titanium dioxide nanoparticles and / or ferrosoferric oxide nanoparticles;

[0014] The carrier comprises a polymer and / or a phospholipid membrane;

[0015] The polymer is selected from any one or more of polylactic acid, polycaprolactone, polyglycolic acid, polylysine, polyglutamic acid, polylactic acid-glycolic acid copolymer or polylactic acid-glycolic acid-polyethylene glycol copolymer.

[0016] Preferably, the components of the phospholipid membrane include but are not limited to any one or more of phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphoinositide, phosphatidylserine, phosphatidic acid, diphosphatidylglycerol or sphingomyelin.

[0017] More preferably, the carrier comprises a polymer and a phospholipid membrane.

[0018] Preferably, the mass ratio of the polymer to the phospholipid membrane is (2-5):(8-5).

[0019] More preferably, the polymer is selected from poly(lactic-co-glycolic acid).

[0020] More preferably, the component of the phospholipid membrane is lecithin.

[0021] Preferably, the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:25 or 50:50.

[0022] Preferably, the mass ratio of the ultrasound sensitizer to the carrier is (2-10):(90-98).

[0023] Preferably, the diameter of the ultrasound sensitizing nanoparticles is 10 nm to 10 μm.

[0024] Preferably, the ultrasound-sensitized nanoparticles are prepared by nano-coprecipitation method, coprecipitation-gel method, emulsification method, sol-gel method or thermal decomposition method.

[0025] In a second aspect, the present invention provides a drug for ultrasonic dynamic treatment of Helicobacter pylori infection, comprising the ultrasound-sensitized nanoparticles involved in the above technical solution and pharmaceutically acceptable excipients.

[0026] In a third aspect, the present invention provides a method for killing Helicobacter pylori, comprising the following steps:

[0027] The ultrasound-sensitized nanoparticles or ultrasound-dynamic drugs for treating Helicobacter pylori infection involved in the above technical solution are used to kill Helicobacter pylori under ultrasound stimulation to obtain the product.

[0028] Preferably, the duration of the ultrasonic stimulation is 2 to 30 minutes, and the intensity of the ultrasonic stimulation is 0.1 to 10 W / cm 2 .

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides an ultrasound-sensitized nanoparticle with simple ingredients, low cost and biological safety, which includes a carrier and an ultrasound sensitizer loaded on the carrier. In the present invention, the carrier includes a polymer and / or a phospholipid membrane, and the ultrasound sensitizer is a sensitizer well known to those skilled in the art. The present invention obtains ultrasound-sensitized nanoparticles by loading the carrier with an ultrasound sensitizer, and ultrasonically stimulates the treatment site with the help of ultrasound waves that have a strong penetrating ability to biological tissues, thereby enabling it to have the ability to efficiently kill Helicobacter pylori. Since the ultrasound-sensitized nanoparticles do not require the addition of antibiotics, the problem of antibiotic resistance of Helicobacter pylori will not be caused. At the same time, the ultrasound-sensitized nanoparticles have an excellent effect of adsorbing Helicobacter pylori vacuolating toxin (VacA).

[0031] According to research, the method of using the ultrasound-sensitized nanoparticle-mediated ultrasound power to kill Helicobacter pylori will not interfere with the gastric intestinal flora when killing Helicobacter pylori. This method can protect gastric mucosal cells and has efficient spatiotemporal controllability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The hydrodynamic diameter diagram of the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane obtained in Example 1;

[0033] Figure 2 This is a graph showing the results of the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin obtained in Example 1 killing Helicobacter pylori;

[0034] Figure 3 This is a result diagram of the Helicobacter pylori load in the stomach of mice after the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane obtained in Example 1 were treated with ultrasound dynamic therapy mediated by the lecithin membrane;

[0035] Figure 4This is a diagram showing the adsorption effect of the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane obtained in Example 1 on vacuolating toxin (VacA) of Helicobacter pylori;

[0036] Figure 5 The hydrodynamic diameter diagram of ultrasound-sensitized nanoparticles Ce6-PLGA@Lecithin coated with lecithin membrane obtained in Example 2;

[0037] Figure 6 This is a graph showing the results of the ultrasound-sensitized nanoparticles Ce6-PLGA@Lecithin coated with lecithin membrane obtained in Example 2 killing Helicobacter pylori;

[0038] Figure 7 This is a graph showing the adsorption of Helicobacter pylori vacuolating toxin (VacA) by the ultrasound-sensitized nanoparticles Ver-PLGA@PEG obtained in Example 3. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In view of the problem of antibiotic resistance and the unpredictable problem of whether the therapeutic drugs can remove the toxins released by bacteria in the clinical treatment methods of Helicobacter pylori in the prior art, the present invention provides an ultrasound-sensitized nanoparticle, which includes a carrier and an ultrasound sensitizer loaded on the carrier. It should be noted that the position where the ultrasound sensitizer is loaded on the carrier can be any one or more positions within the carrier, on the carrier, or between the carriers. In the present invention, the mass ratio of the ultrasound sensitizer to the carrier is preferably (2-10): (90-98), more preferably (5-10): (90-95), and most preferably 9:91. Dynamic light scattering (DLS) test was performed on the ultrasound-sensitized nanoparticles, and it was found that their diameter was 10nm to 10μm.

[0041] In the present invention, the carrier includes a polymer and / or a phospholipid membrane. Wherein, the polymer includes a material formed by copolymerization of one or more of the following polymers: for example, polylactic acid (PLA), polycaprolactone (PCL), polyglycolic acid (PGA), polylysine or polyglutamic acid, polylactic acid-glycolic acid copolymer (PLGA) or PLGA and polyethylene glycol (PEG) copolymer (PLGA-PEG), preferably including PLGA and / or PLGA-PEG. The average molecular weight of the PLGA is generally selected from 2,000 to 40,000 Da, preferably 20,000 to 40,000 Da; the average molecular weight of the PEG in the PLGA-PEG is generally selected from 1,000 to 10,000 Da, preferably 2,000-4,000 Da.

[0042] The phospholipid membrane is composed of any of a number of lipids containing diglycerides, phosphate groups, and simple organic molecules (such as choline). Specifically, the components of the phospholipid membrane include, but are not limited to, phosphatidylcholine (2-diacyl-sn-glycero-3-phosphocholine, PC, i.e., phosphatidylcholine in a narrow sense, choline molecules), phosphatidylethanolamine (Phosphatidylethanolamine, PE, also known as cephalin, ethanolamine molecules), phosphoinositol (Inositol phosphate, IP, inositol molecules), phosphatidylserine (Phosphatidylserine, PS, serine molecules), phosphatidic acid (Phosphatidic acid, PA), diphosphatidylglycerol (DPG, cardiolipin) or sphingomyelin (SM), etc. In some embodiments of the present invention, the phospholipid membrane is preferably a phosphatidylcholine membrane.

[0043] In the present invention, the ultrasound sensitizer includes an organic small molecule ultrasound sensitizer and / or an inorganic ultrasound sensitizer. Wherein, the organic small molecule ultrasound sensitizer comprises one or more of the following composite substances: porphyrin and its derivatives such as hematoporphyrin (Hp), verteporfin (Ver), hematoporphyrin monomethyl ether (HMME), protoporphyrin IX (PpIX), gallium porphyrin (ATX-70, ATX-S10); chlorophyll and its derivatives such as chlorophyll-a (Chl-a), pheophorbide A, chlorin e6 (Ce6), pyropheophorbide a hexyl ether (HPPH), mono-asparagine chlorin-e6 (NPe6), m-tetrahydroxychlorophenol (mTHPC); indocyanine compounds such as indocyanine green (Indocyanine green); green, ICG); small molecule dyes such as IR-780 iodide (IR-780iodide) and other organic small molecule ultrasonic sensitizers such as curcumin (Curcumin) or hypocrellin B (Hypocrellin B, referred to as HB); the inorganic ultrasonic sensitizer comprises one or more of the following composite substances: titanium dioxide (TiO2) nanoparticles, ferrous oxide (Fe3O4) nanoparticles. In the present invention, the ultrasonic sensitizer can also be a substance obtained by any combination of the above-mentioned organic ultrasonic sensitizers and / or inorganic ultrasonic sensitizers.

[0044] The present invention loads an ultrasound sensitizer on a carrier to obtain ultrasound sensitized nanoparticles, and ultrasound stimulation is performed on the treatment site by means of ultrasound waves that have strong penetration ability on biological tissues, so that the ultrasound sensitized nanoparticles have the activity of effectively killing Helicobacter pylori. Since the ultrasound sensitized nanoparticles do not require the addition of antibiotics, the problem of antibiotic resistance will not be caused. At the same time, the ultrasound sensitized nanoparticles have an excellent effect of adsorbing Helicobacter pylori vacuolating toxin (VacA).

[0045] In some preferred embodiments of the present invention, the carrier comprises a polymer and a phospholipid membrane, and the mass ratio of the polymer to the phospholipid membrane is preferably (2-5):(8-5). Wherein, the polymer is selected from poly(lactic acid-co-glycolic acid), and the molar ratio of lactic acid to glycolic acid in the poly(lactic acid-co-glycolic acid) is preferably 75:25 or 50:50, more preferably 75:25; and the phospholipid membrane is preferably a lecithin membrane.

[0046] When the above technical scheme is followed, the obtained ultrasound-sensitized nanoparticles are generally referred to as ultrasound-sensitized nanoparticles coated with phospholipid membranes, that is, ultrasound-sensitizer-polymer@phospholipid membrane nanoparticles. According to tests, ultrasound-sensitized nanoparticles coated with phospholipid membranes have a better effect of adsorbing Helicobacter pylori vacuolating toxin (VacA), because the phospholipid membrane on the surface of the nanoparticles has a stronger binding force to Helicobacter pylori vacuolating toxin.

[0047] In the present invention, the ultrasound-sensitized nanoparticles can be prepared by nano-coprecipitation method, coprecipitation-gel method, emulsification method, sol-gel method or thermal decomposition method well known to those skilled in the art. In some embodiments of the present invention, the nano-coprecipitation method is preferably used for preparation, which specifically comprises the following steps:

[0048] (1) adding 2-10 wt % of an ultrasonic sensitizer and 90-98 wt % of a carrier into an appropriate amount of an organic solvent to obtain a dispersion of the ultrasonic sensitizer and the carrier;

[0049] (2) adding the obtained dispersion dropwise into distilled water, stirring to evaporate the organic solvent, obtaining a suspension of the ultrasound sensitizer and the carrier, i.e., ultrasound-sensitized nanoparticle suspension, and then centrifuging to obtain ultrasound-sensitized nanoparticles.

[0050] According to the present invention, first, 2 to 10 wt% of an ultrasonic sensitizer and 90 to 98 wt% of a carrier are added to an appropriate amount of an organic solvent to obtain a dispersion of the ultrasonic sensitizer and the carrier. Wherein, the ultrasonic sensitizer and the carrier are as described in the relevant content of the above technical solution, and will not be repeated here. The organic solvent is mainly used to provide a liquid environment, and can be any one or more of acetone, ether, methanol, ethanol, dichloromethane, chloroform or butanone. The mass ratio of the ultrasonic sensitizer and the carrier as a solute to the organic solvent is preferably 1: (20 to 200), and more preferably 1: (80 to 150). The present invention has no particular restrictions on the manner in which the ultrasonic sensitizer and the carrier are added to the organic solvent. The ultrasonic sensitizer can be added after the carrier is added to the organic solvent; the ultrasonic sensitizer can also be added after the carrier is added to the organic solvent; or the ultrasonic sensitizer and the carrier can be added to the organic solvent at the same time.

[0051] Then, the obtained dispersion is added dropwise into distilled water, the dropping speed is preferably 0.02-20 mL / s, the distilled water is preferably heated to 30-85°C, more preferably heated to 35-50°C, and most preferably heated to 37°C, the volume ratio of the dispersion to distilled water is preferably 1:(3-100), more preferably 1:(5-50), and stirred to evaporate the organic solvent, the stirring rate is preferably 50-1000 rpm, more preferably 200-600 rpm, and most preferably 300 rpm, the stirring time is preferably 8-48 hours, more preferably 16-24 hours, after the stirring is completed, the suspension after the ultrasonic sensitizer and the carrier are combined, that is, the ultrasonic sensitized nanoparticle suspension. According to the present invention, the obtained ultrasonic sensitized nanoparticle suspension is centrifuged to obtain ultrasonic sensitized nanoparticles. The centrifugal speed can be 800-15,000 g, and the centrifugal time can be 5-30 minutes. In some embodiments of the present invention, the centrifugation is preferably gradient centrifugation, specifically: first set low-speed centrifugation (800-1000g, 5-20 minutes) to remove large-sized nanoparticles, and then set high-speed centrifugation (10,000-15,000g, 5-20 minutes) to collect ultrasound-sensitized nanoparticles.

[0052] It should be noted that when the carrier includes both a polymer and a phospholipid membrane, in some embodiments of the present invention, the ultrasound sensitizer-polymer@phospholipid membrane nanoparticles are preferably prepared according to the following method:

[0053] (1) adding 2-10 wt % of an ultrasonic sensitizer and 90-98 wt % of a carrier into an appropriate amount of an organic solvent to obtain a dispersion of the ultrasonic sensitizer and the carrier;

[0054] (2) adding the obtained dispersion dropwise into distilled water, stirring to evaporate the organic solvent, obtaining a suspension of the ultrasound sensitizer and the carrier, i.e., ultrasound-sensitized nanoparticle suspension, and then centrifuging to obtain ultrasound-sensitized nanoparticles;

[0055] (3) After the ultrasound-sensitized nanoparticles are mixed with the phospholipid membrane, water bath sonication is performed, and then centrifugation is performed to obtain ultrasound-sensitizer-polymer@phospholipid membrane nanoparticles.

[0056] The steps (1) and (2) are as described in the relevant contents of the above technical solution and will not be repeated here. In the step (3), the dosage ratio of the nanoparticles to the phospholipid membrane is (2-5):(8-5), which can be 2:6, 3:6, 3:7 or 4:6, etc. The temperature of the water bath ultrasound is 15-60°C, preferably 20-30°C; the time is 5-15 minutes, preferably 5-10 minutes; the ultrasonic frequency is 30-70kHz, preferably 50-60kHz. After the water bath ultrasound is completed, it is preferably centrifuged at 5000-12,000g for 5-20 minutes, and more preferably at 10,000g for 10 minutes. After the centrifugation is completed, the ultrasound sensitizer-polymer@phospholipid membrane nanoparticles can be obtained.

[0057] It should be noted that in the above-mentioned usage ratio of nanoparticles to phospholipid membrane, the usage of phospholipid membrane is not less than the usage of nanoparticles. The reason is that an excess of phospholipid membrane can make all the surfaces of nanoparticles be covered by phospholipid membrane as much as possible, thereby improving the membrane encapsulation rate and the degree of membrane coverage of nanoparticles.

[0058] The preparation method of the ultrasound-sensitized nanoparticles provided by the present invention is simple, does not require expensive instruments, has low cost, and is easy to achieve large-scale production.

[0059] In the present invention, the point values ​​listed above are merely for enumeration and are not limited thereto. Other point values ​​within the numerical range are applicable and will not be described in detail here.

[0060] The ultrasound-sensitized nanoparticles provided by the present invention can be directly orally administered. For oral administration, the ultrasound-sensitized nanoparticles can be formulated into solutions, emulsions, suspensions, dispersions, such as oral liquids, syrups; or formulated with solid carriers into tablets, capsules, pills, granules or powders, etc. Based on this, the present invention also provides a drug for ultrasound-dynamic treatment of Helicobacter pylori infection, which includes the ultrasound-sensitized nanoparticles and pharmaceutically acceptable excipients.

[0061] The ultrasound-sensitized nanoparticles or ultrasound-dynamic drugs for treating Helicobacter pylori infection provided by the present invention can be used to kill Helicobacter pylori. Therefore, the present invention also provides a method for killing Helicobacter pylori, comprising the following steps:

[0062] The ultrasound-sensitized nanoparticles or the ultrasound-dynamic drug for treating Helicobacter pylori infection is used to kill Helicobacter pylori under ultrasound stimulation to obtain the product.

[0063] In the present invention, the duration of the ultrasonic stimulation is preferably 2 to 30 minutes, more preferably 10 to 20 minutes; the intensity of the ultrasonic stimulation is 0.1 to 10 W / cm 2 , more preferably 0.5-4W / cm 2 .

[0064] In some embodiments of the present invention, ultrasound sensitized nanoparticles are dispersed in phosphate buffered saline and mixed evenly to prepare antibacterial solutions with final concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL (calculated based on the dosage of ultrasound sensitizer); the above antibacterial solutions are mixed with 1.5×10 6 CFU / mL of Helicobacter pylori were mixed evenly; the mixed solution was subjected to light-proof ultrasound (0.5W / cm 2 , 10 minutes), and then the Helicobacter pylori in the mixed solution was counted by plate colony counting method. The results show that the ultrasound sensitized nanoparticles have the activity of effectively killing Helicobacter pylori under ultrasound stimulation.

[0065] In some embodiments of the present invention, ultrasound-sensitized nanoparticles are dispersed in a phosphate buffered saline solution and mixed evenly to prepare an antibacterial solution with a final concentration of 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL (calculated based on the dosage of the ultrasound sensitizer); the antibacterial solution is orally administered to a mouse model of gastric Helicobacter pylori infection, and ultrasound (0.5 W / cm 2 , 10 minutes); 48 hours after the treatment, the stomach of the above mice was taken out and homogenized after adding phosphate buffered saline; the Helicobacter pylori in the homogenate was counted by plate colony counting method to evaluate the load of Helicobacter pylori in the stomach of mice. The results showed that the ultrasound sensitized nanoparticles can significantly reduce the load of Helicobacter pylori in the stomach of mice under ultrasound stimulation.

[0066] In some embodiments of the present invention, ultrasound-sensitized nanoparticles are incubated with Helicobacter pylori culture supernatant for a period of time and then centrifuged to obtain nanoparticles (i.e., precipitates) and supernatants; the content of Helicobacter pylori vacuolating toxin (VacA) adsorbed on the surface of the nanoparticles (i.e., precipitates) and the content of Helicobacter pylori vacuolating toxin (VacA) remaining in the supernatant are determined by enzyme-linked immunosorbent assay. The results show that ultrasound-sensitized nanoparticles have a certain toxin adsorption capacity and a detoxification effect, and when the ultrasound-sensitized nanoparticles are ultrasound-sensitized nanoparticles coated with phospholipid membranes, they show a more excellent toxin adsorption capacity.

[0067] In order to further illustrate the present invention, the following examples are used to explain it in detail. The experimental raw materials used in the following examples of the present invention are all common commercial products. The following wt% represents the mass percentage; CFU represents the colony forming unit; CFU / mL represents the total number of colonies per milliliter of solution.

[0068] Example 1

[0069] This embodiment provides an ultrasound-sensitized nanoparticle Ver-PLGA@Lecithin coated with a lecithin membrane, and the preparation method thereof is as follows:

[0070] (1) Selecting poly(lactic-co-glycolic acid) (PLGA) as a carrier. The molar ratio of lactic acid to glycolic acid in the poly(lactic-co-glycolic acid) (PLGA) is 75:25; the average molecular weight of the poly(lactic-co-glycolicacid) (PLGA) is 40,000 Da;

[0071] (2) Verteporfin (Ver) was selected as ultrasound sensitizer;

[0072] (3) 1 mg (9 wt%) of ultrasound sensitizer Verteporfin and 10 mg (91 wt%) of carrier PLGA were added to 1.25 mL of acetone to obtain a dispersion, which was then added dropwise into 10 mL of distilled water, heated to 37° C., and stirred in the open air at 300 rpm for 14 hours to evaporate the acetone, and then the obtained suspension was subjected to gradient centrifugation. The gradient centrifugation was first set to low speed centrifugation (800 g, 10 minutes) and then set to high speed centrifugation (10,000 g, 10 minutes) to obtain ultrasound sensitized nanoparticles Ver-PLGA;

[0073] (4) The prepared ultrasound-sensitized nanoparticles Ver-PLGA were mixed with lecithin membrane (Lecithin) (the mass ratio of PLGA to lecithin membrane was kept at 1:2) and then subjected to water bath ultrasound (temperature of 25°C, time of 5 min, ultrasonic frequency of 53 kHz), followed by centrifugation (10,000 g, 10 min) to obtain ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane.

[0074] Dynamic light scattering (DLS) was used to test the hydrodynamic diameter of Ver-PLGA@Lecithin. Figure 1 The results show that the average particle size of ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin is 196.2nm.

[0075] Sterilization test

[0076] The present invention disperses the prepared Ver-PLGA@Lecithin nanoparticles in a phosphate buffered saline solution and mixes them evenly to prepare a Ver-PLGA@Lecithin antibacterial solution; the antibacterial solution is mixed with 1.5×10 6 CFU / mL of Helicobacter pylori were mixed evenly as the experimental group; then the mixed solution was subjected to light-proof ultrasound (0.5 W / cm 2 , 10 minutes), and then the Helicobacter pylori in the mixed solution was counted by plate colony counting method. The control group was not subjected to light-proof ultrasound.

[0077] Among them, the experimental group included 5 groups, and the final concentrations of the Ver-PLGA@Lecithin antibacterial solution after mixing with Helicobacter pylori in the 5 groups were 0μg / mL, 25μg / mL, 50μg / mL, 75μg / mL, and 100μg / mL (calculated based on the dose of verteporfin), respectively.

[0078] The culture conditions of Helicobacter pylori are 37° C. and a microaerobic atmosphere (10% CO 2 + 5% O 2 + 85% N 2 ).

[0079] Figure 2 The figure is a result of the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane killing Helicobacter pylori in Example 1 (Experiment 1 and Experiment 2 refer to two experiments conducted under the same experimental conditions). The results show that under ultrasonic stimulation, as the concentration of ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane increases, its bactericidal activity increases, and 100μg / mL of Ver-PLGA@Lecithin nanoparticles can reduce the bacterial concentration by 2.94 logCFU / mL, and have good activity in killing Helicobacter pylori.

[0080] Mouse model testing

[0081] The prepared ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane were dispersed in phosphate buffer solution (PBS) and mixed evenly to prepare Ver-PLGA@Lecithin antibacterial solution (i.e., experimental group); the antibacterial solution was orally administered to a mouse model infected with Helicobacter pylori in the stomach, and ultrasound (0.5 W / cm 2 , 10 minutes), 48 hours after the end of treatment, the stomach of the above mice was taken out and homogenized with phosphate buffered saline, and the Helicobacter pylori in the homogenate was counted by plate colony counting method to evaluate the load of Helicobacter pylori in the stomach of mice. The control group was a bacterial infection but no treatment group (i.e., no medication).

[0082] Among them, the number of bacterial colonies carried in the gastric Helicobacter pylori mouse model was 1.0×10 8 CFU / mL. The culture conditions of Helicobacter pylori are 37°C and a microaerobic atmosphere (10% CO2+5% O2+85% N2).

[0083] Figure 3 The results of the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin prepared in Example 1 and treated with ultrasound dynamic therapy mediated by the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin can significantly reduce the load of Helicobacter pylori in the stomach of mice under ultrasound stimulation, which is about 50 times lower than the bacterial load of the control group.

[0084] Toxin adsorption capacity test

[0085] The present invention mixes the obtained ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane with the culture supernatant of Helicobacter pylori (i.e., the experimental group), incubates for a period of time and then centrifuges to obtain nanoparticles (i.e., precipitates) and supernatants; and determines the content of Helicobacter pylori vacuolating toxin (VacA) adsorbed on the surface of the nanoparticles (i.e., precipitates) and the content of Helicobacter pylori vacuolating toxin (VacA) remaining in the supernatant by enzyme-linked immunosorbent assay. The control group is the culture supernatant of Helicobacter pylori without any nanoparticles added.

[0086] Figure 4 The figure is the result of the adsorption of Helicobacter pylori vacuolating toxin (VacA) by the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane obtained in Example 1 (Experiments 1 and 2 refer to two experiments conducted under the same experimental conditions). The results show that compared with the control group, the residual amount of Helicobacter pylori vacuolating toxin (VacA) in the supernatant was reduced by 76%, indicating that the ultrasound-sensitized nanoparticles Ver-PLGA@Lecithin coated with lecithin membrane have a strong toxin adsorption capacity and show an excellent detoxification effect.

[0087] Example 2

[0088] This embodiment provides an ultrasound-sensitized nanoparticle Ce6-PLGA@Lecithin coated with a lecithin membrane, and the preparation method thereof is as follows:

[0089] (1) Selecting poly(lactic-co-glycolic acid) (PLGA) as a carrier. The molar ratio of lactic acid to glycolic acid in the poly(lactic-co-glycolic acid) (PLGA) is 75:25; the average molecular weight of the poly(lactic-co-glycolicacid) (PLGA) is 30,000 Da;

[0090] (2) Chlorin e6 (Ce6) was selected as the ultrasound sensitizer;

[0091] (3) 1 mg (10 wt%) of ultrasound sensitizer dihydrochlorin e6 and 9 mg (90 wt%) of carrier PLGA were added to 1.2 mL of acetone to obtain a dispersion, which was then added dropwise to 8 mL of distilled water and heated to 35° C. The dispersion was stirred in the open air at 400 rpm for 16 hours to evaporate the acetone, and then the obtained suspension was subjected to gradient centrifugation. The gradient centrifugation was first set to low speed centrifugation (900 g, 8 minutes) and then to high speed centrifugation (12,000 g, 8 minutes) to obtain ultrasound sensitized nanoparticles Ce6-PLGA;

[0092] (4) The prepared ultrasound-sensitized nanoparticles Ce6-PLGA were mixed with lecithin membrane (Lecithin) (the mass ratio of PLGA to lecithin membrane was kept at 1:3) and then subjected to water bath ultrasound (temperature of 20°C, time of 8 min, ultrasonic frequency of 60 kHz), followed by centrifugation (12,000 g, 8 min) to obtain ultrasound-sensitized nanoparticles Ce6-PLGA@Lecithin coated with lecithin membrane.

[0093] Dynamic light scattering (DLS) was used to test the hydrodynamic diameter of Ce6-PLGA@Lecithin. Figure 2 The results show that the average particle size of ultrasound-sensitized nanoparticles Ce6-PLGA@Lecithin is 191.7 nm.

[0094] Referring to the method of Example 1, an experimental group and a control group were set up, wherein the experimental group included 5 groups, and the final concentrations of Ce6-PLGA@Lecithin antibacterial solution after mixing with Helicobacter pylori in the 5 groups were 0 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL (calculated based on the dosage of dihydrochlorin Ce6). Then, the test was performed according to the bactericidal test method of Example 1.

[0095] Figure 6The results of the ultrasound-sensitized nanoparticles Ce6-PLGA@Lecithin coated with lecithin membrane in killing Helicobacter pylori in Example 2 (Experiment 1 and Experiment 2 refer to two experiments conducted under the same experimental conditions). The results show that under ultrasonic stimulation, as the concentration of ultrasound-sensitized nanoparticles Ce6-PLGA@Lecithin coated with lecithin membrane increases, its bactericidal activity increases, and 100 μg / mL of Ce6-PLGA@Lecithin nanoparticle composition can reduce the bacterial concentration by 3.26 log CFU / mL, and has excellent activity in killing Helicobacter pylori.

[0096] Example 3

[0097] This embodiment provides an ultrasound-sensitized nanoparticle Ver-PLGA@PEG, and the preparation method thereof is as follows:

[0098] (1) PLGA-PEG copolymer (PEG: polyethylene glycol) is selected as the carrier. The molar ratio of lactic acid to glycolic acid in the poly(lactic-co-glycolic acid, PLGA) is 75:25; the average molecular weight of the poly(lactic-co-glycolic acid, PLGA) is 36,000 Da; the average molecular weight of the polyethylene glycol (PEG) is 2,000 Da;

[0099] (2) Verteporfin (Ver) was selected as ultrasound sensitizer;

[0100] (3) 1 mg (8 wt %) of ultrasound sensitizer Verteporfin and 11.5 mg (92 wt %) of carrier PLGA-PEG were added to 1.5 mL of acetone to obtain a dispersion, which was then added dropwise into 12 mL of distilled water and heated to 40° C. The dispersion was stirred in the open air at 450 rpm for 12 hours to evaporate the acetone, and then the obtained suspension was subjected to gradient centrifugation. The gradient centrifugation was first set to low speed centrifugation (1000 g, 6 minutes) and then to high speed centrifugation (15,000 g, 6 minutes) to obtain ultrasound sensitized nanoparticles Ver-PLGA@PEG.

[0101] The present invention mixes the obtained ultrasound-sensitized nanoparticles Ver-PLGA@PEG with the culture supernatant of Helicobacter pylori (i.e., the experimental group), incubates for a period of time and then centrifuges to obtain nanoparticles (i.e., precipitates) and supernatants; and determines the content of Helicobacter pylori vacuolating toxin (VacA) adsorbed on the surface of the nanoparticles (i.e., precipitates) and the content of Helicobacter pylori vacuolating toxin (VacA) remaining in the supernatant by enzyme-linked immunosorbent assay. The control group is the culture supernatant of Helicobacter pylori without any nanoparticles added.

[0102] Figure 7 The figure is the result of the adsorption of Helicobacter pylori vacuolating toxin (VacA) by the ultrasound-sensitized nanoparticle Ver-PLGA@PEG obtained in Example 3 (Experiment 1 and Experiment 2 refer to two experiments conducted under the same experimental conditions). The results show that compared with the control group, the residual amount of Helicobacter pylori vacuolating toxin (VacA) in the supernatant was reduced by 28%, indicating that the ultrasound-sensitized nanoparticle Ver-PLGA@PEG has a certain toxin adsorption capacity and shows a certain detoxification effect.

[0103] Comparative Example 1

[0104] This comparative example provides a standard clinical treatment method for gastric Helicobacter pylori infection, namely triple therapy (a regimen comprising two antibiotics and a proton pump inhibitor (PPI)), and the specific steps are as follows:

[0105] (1) Experimental group: Omeprazole (proton pump inhibitor, 400 μmol / kg mouse), amoxicillin (antibiotic, 28.5 mg / kg mouse) and clarithromycin (antibiotic, 14.3 mg / kg mouse) were orally administered to a mouse model with gastric Helicobacter pylori infection. After 48 hours of treatment, the stomach of the mouse was removed and homogenized with phosphate buffered saline. The Helicobacter pylori in the homogenate was counted by plate colony counting method to evaluate the load of Helicobacter pylori in the mouse stomach. The control group was a bacterial infection but no treatment group (i.e., no medication).

[0106] Among them, the number of bacterial colonies carried in the gastric Helicobacter pylori mouse model was 1.0×10 8 CFU / mL. The culture conditions of Helicobacter pylori are 37°C and a microaerobic atmosphere (10% CO2+5% O2+85% N2).

[0107] Figure 3 The data of the "Triple therapy" group correspond to the load of Helicobacter pylori in the stomach of mice after triple therapy treatment in the comparative example. The results show that triple therapy can effectively reduce the load of Helicobacter pylori in the stomach of mice.

[0108] It is illustrated in combination with Example 1 and Comparative Example 1 that the ultrasound-sensitized nanoparticle-mediated sonodynamic therapy provided by the present invention has an effect of killing Helicobacter pylori comparable to that of the triple therapy, but the sonodynamic therapy described in the present invention does not induce antibiotic resistance, which is significantly better than the triple therapy commonly used in the prior art.

[0109] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of ultrasound-sensitized nanoparticles in the preparation of drugs for ultrasound-dynamic treatment of Helicobacter pylori infection. It is characterized in that It comprises a carrier and an ultrasound sensitizer loaded on the carrier; The ultrasonic sensitizer includes an organic small molecule ultrasonic sensitizer; The organic small molecule ultrasound sensitizer includes verteporfin and / or dihydrochlorin e6; The carrier includes a polymer and a phospholipid membrane; The polymer is selected from any one or more of polylactic acid-co-glycolic acid or polylactic acid-co-glycolic acid-polyethylene glycol.

2. The use according to claim 1, It is characterized in that The components of the phospholipid membrane include but are not limited to any one or more of phosphatidylcholine, phosphatidylethanolamine, phosphoinositide, phosphatidylserine, phosphatidic acid, diphosphatidylglycerol or sphingomyelin.

3. The use according to claim 1, It is characterized in that The mass ratio of the polymer to the phospholipid membrane is (2-5):(8-5).

4. The use according to claim 1, It is characterized in that The polymer is selected from poly(lactic acid-co-glycolic acid); The component of the phospholipid membrane is lecithin.

5. The use according to claim 1, It is characterized in that The molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:25 or 50:

50.

6. The use according to claim 1, It is characterized in that The mass ratio of the ultrasonic sensitizer to the carrier is (2-10):(90-98); The diameter of the ultrasound-sensitized nanoparticles is 10 nm to 10 μm.

7. The use according to claim 1, It is characterized in that The ultrasound sensitized nanoparticles are prepared by nanometer coprecipitation method, coprecipitation-gel method, emulsification method, sol-gel method or thermal decomposition method.

8. The use according to claim 1, It is characterized in that The application is performed under ultrasound stimulation conditions; The duration of the ultrasound stimulation is 2 to 30 min, and the intensity of the ultrasound stimulation is 0.1 to 10 W / cm 2 .

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