Maltodextrin nanoparticles, preparation method and application thereof

Abstract

The application discloses malt dextrin nanoparticles and a preparation method and application thereof, and the malt dextrin nanoparticles are prepared by self-assembly of a malt dextrin active oxygen responsive carrier material; the malt dextrin active oxygen responsive carrier material is a carrier material with active oxygen response prepared by linking an oxidation-sensitive group phenylboronic acid pinacol ester to malt dextrin through a covalent bond. The application provides a new drug effect of malt dextrin nanoparticles, provides a potential combined drug target for patients with antibiotic-resistant bacterial infections, and develops a potential new treatment strategy for malt dextrin. The malt dextrin plays a role in improving the effect of antibiotics on bacterial removal under the condition of a small amount, and has very small toxic and side effects, and is expected to be further researched and applied to the clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a maltodextrin nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Recurrent and chronic infections caused by intracellular bacteria (ICB) have long been a major challenge in clinical practice. ICB escapes from lysosomes and resides and reproduces in the cytoplasm or intracellular vacuoles to evade immune attack, eventually leading to cell apoptosis. A large number of bacteria produced thereby can cause serious infections, including tuberculosis, endocarditis, osteomyelitis, necrotizing pneumonia, and sepsis, etc. Clinical treatment of intracellular bacterial infection often requires long-term and large-dose antibiotics for treatment. However, due to the body's own cell barrier, water-soluble antibiotics cannot effectively enter the cells, and the distribution of fat-soluble antibiotics to the cells is limited, making it difficult to achieve an effective bacteriostatic concentration, and cannot effectively target intracellular bacteria.

[0003] After intracellular infection of bacteria, ICB is prone to enter a persistent state due to the microenvironmental stress of intracellular active oxygen, acidic environment, nutrient deficiency, and antibiotic exposure. Even if the intracellular antibiotics reach the bacteriostatic concentration, they cannot effectively eliminate the intracellular bacteria. Therefore, the treatment of clinical ICB infection often faces failure and recurrence.

[0004] Recent studies have shown that providing carbon sources to persistent cells can activate the ability of the metabolic system of bacteria, restore the metabolic activity of persistent bacteria by restoring the activity of aconitase and increasing the ATP level, and thus reverse the drug resistance of persistent cells. However, how to effectively deliver carbon sources to the intracellular and be effectively utilized by ICB still lacks research.

[0005] The patent "Active oxygen radical sensitive cyclodextrin material as a drug delivery carrier and its preparation method" (publication number: CN103755839A) provides that cyclodextrin is reacted with imidazole carbonyl oxy-benzoic acid pinacol to prepare an active oxygen radical sensitive cyclodextrin material, which provides a drug delivery carrier. The patent "Active oxygen responsive moxifloxacin nano preparation and its preparation method" (publication number: CN109010311A) loads moxifloxacin onto a cyclodextrin drug carrier modified based on 4-hydroxybenzoic acid pinacol ester, and the outer layer is covered with lecithin and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 nanoparticles to prepare an active oxygen responsive moxifloxacin nano preparation, which has high dispersibility and long circulation, and can effectively improve the effect of moxifloxacin on inhibiting bacteria. The inventors tried to use cyclodextrin with different sugar units in combination with rifampicin to reverse the drug resistance of Staphylococcus aureus persistent cells, but the results failed to effectively eliminate the number of intracellular bacteria.

[0006] Current researches only target single sugar glucose to restore the killing effect of aminoglycosides on some bacteria, but these researches only target extracellular persistent cells, and there is currently no solution to the problem of intracellular persistent cell drug resistance. SUMMARY

[0007] To solve the problem of persistent cell drug resistance in the prior art, the present application provides a maltodextrin nanoparticle and a preparation method and application thereof, which uses maltodextrin as a carbon source for bacteria to reverse the drug resistance of persistent Staphylococcus aureus. The present application also develops a maltodextrin active oxygen-responsive carrier material, and provides a new treatment strategy for the combination of the maltodextrin nanoparticle prepared by self-assembly and rifampicin. The technical solutions of the present application are as follows:

[0008] As a first aspect of the present application, a maltodextrin nanoparticle is provided, which is prepared by self-assembly of a maltodextrin active oxygen-responsive carrier material; the maltodextrin active oxygen-responsive carrier material is prepared by covalently linking an oxidation-sensitive group phenylboronic acid pinacol ester to maltodextrin.

[0009] Preferably, the particle size of the maltodextrin nanoparticle is in the range of 88.8±8 nm.

[0010] As a second aspect of the present application, a preparation method of the maltodextrin nanoparticle is provided, which comprises the following steps: reacting 4-hydroxyphenylboronic acid pinacol ester (PBAP) with N,N'-carbonyldiimidazole (CDI), and then reacting the obtained product with maltodextrin to obtain a maltodextrin active oxygen-responsive carrier material (OXMD), and forming the maltodextrin active oxygen-responsive carrier material (OXMD) by coating the outside of the maltodextrin active oxygen-responsive carrier material (OXMD) with lecithin and phospholipid polyethylene glycol copolymer (DSPE-PEG).

[0011] Specifically, the preparation method comprises the following steps:

[0012] Step 1: 4-hydroxyphenylboronic acid pinacol ester and N,N'-carbonyldiimidazole are dissolved in an organic solvent, and then magnetically stirred for reaction; an organic solvent is added to the reaction solution, and then washed with water; the organic solvent layer solution is taken, washed again, and then taken; the organic solvent is removed by rotary evaporation, and then vacuum dried to obtain activated hydroxyphenylboronic acid pinacol ester;

[0013] Step 2: 4-dimethylaminopyridine, maltodextrin, and the activated hydroxyphenylboronic acid pinacol ester obtained in step 1 are dissolved in an organic solvent, and then magnetically stirred overnight in anhydrous and anaerobic conditions; the reaction solution is precipitated with water, centrifuged, the upper liquid is discarded, washed, and then freeze-dried to obtain a white solid powder of the maltodextrin active oxygen-responsive carrier material;

[0014] Step 3, lecithin and phospholipid polyethylene glycol copolymer are dissolved in organic solvent respectively and added to heated water, then stirred by magnetic force and cooled to room temperature; the maltodextrin active oxygen responsive carrier material obtained in step 2 is weighed, dissolved in organic solvent, and slowly added dropwise to the above-mentioned room temperature solution under high stirring speed, after the reaction is completed, rotary evaporation is performed, and then ultrafiltration centrifugation is performed to remove the maltodextrin active oxygen responsive carrier material that is not loaded, thereby obtaining maltodextrin nanoparticles.

[0015] Preferably, the organic solvent is selected from dichloromethane, dimethyl sulfoxide, ethanol and methanol.

[0016] Preferably, the preparation method comprises the following steps:

[0017] After 1.22 g of 4-hydroxyphenylboronic acid pinacol ester (PBAP) and 1.68 g of N,N'-carbonyldiimidazole (CDI) are dissolved in 10 mL of anhydrous dichloromethane and magnetically stirred at 40°C for 1 hour, 20 mL of anhydrous dichloromethane is added to the reaction solution, which is then washed with 15 mL of ultrapure water three times, and the lower layer solution is taken out, and then 10 mL of saturated sodium chloride is used to wash twice, and the lower layer solution is taken out, and then anhydrous sodium sulfate is added to remove the residual aqueous solution, and after standing, centrifugation is performed to take the lower layer solution, rotary evaporation is performed to remove the solvent, and then vacuum drying is performed, thereby obtaining 1.09 g of activated PBAP (CDI-PBAP) white solid powder. 0.27 g of 4-dimethylaminopyridine (DMAP), 0.19 g of maltodextrin (Maltodextrin), and the above-mentioned 1.09 g of CDI-PBAP are weighed and dissolved in 5 mL of anhydrous dimethyl sulfoxide (DMSO) respectively, and then magnetically stirred under anhydrous and anaerobic conditions at 40°C overnight. The reaction solution is precipitated with ultrapure water, centrifuged at 6000xg, the upper layer liquid is discarded, washed with ultrapure water three times, and then freeze-dried to obtain 0.84 g of maltodextrin active oxygen responsive carrier material (OXMD) white solid powder.

[0018] 15 mL of ultrapure water is heated to 65°C for standby use. 6 mg of lecithin and 12 mg of DSPE-PEG are dissolved in 1 mL of anhydrous ethanol respectively and added to the 65°C ultrapure water solution, and then magnetically stirred at 65°C for 2 hours, and then cooled to room temperature. 60 mg of OXMD is dissolved in methanol, slowly added dropwise to the above-mentioned room temperature solution under high stirring speed, and then reacted for 2 hours. After the reaction is completed, rotary evaporation is performed to 5 mL, and then ultrafiltration centrifugation is performed at 6000xg for 10 minutes to remove the OXMD that is not loaded, thereby obtaining maltodextrin nanoparticles (MDNP).

[0019] As a third aspect of the present application, it is to provide the use of the maltodextrin nanoparticles in the preparation of a drug for treating bacterial infection.

[0020] As an optional solution, the therapeutic bacterial infection drug is selected from a drug combined with an antibiotic, or is a composition of maltodextrin and an antibiotic.

[0021] Maltodextrin is a common polysaccharide food material, mainly used as a drying aid for food. It is also widely used as a pharmaceutical excipient, as a filler, sweetener, embedding agent, etc. In the present application, maltodextrin is used as a carbon source for bacteria, and combined with an antibiotic, especially rifampicin, to significantly inhibit the growth of persistent Staphylococcus aureus. The principle is that the carbon source for bacteria can restore the sensitivity of persistent cells to antibiotics.

[0022] As a fourth aspect of the present application, a pharmaceutical composition comprising the maltodextrin nanoparticles is provided.

[0023] Further, the pharmaceutical composition further comprises an antibiotic selected from vancomycin, ciprofloxacin, oxacillin, chloramphenicol, rifampicin, levofloxacin and linezolid.

[0024] Further, the pharmaceutical composition further comprises at least one pharmaceutical inactive ingredient, which can be a carrier, excipient and diluent commonly used in pharmacy.

[0025] Further, the pharmaceutical composition is in the form of a powder, granules, tablets, capsules, suspensions, emulsions, syrups, oral or topical sprays, suppositories and sterile injection solutions. Preferably, it is an injection, which can be administered intravenously or locally.

[0026] Since previous studies only targeted extracellular persistent cells, the present application particularly explores the application of polysaccharide maltodextrin in enhancing the sensitivity of rifampicin to treat intracellular bacteria. The present application is achieved by providing a nanoparticle that can provide a carbon source for bacteria intracellularly. The nanoparticle, combined with an antibiotic, can efficiently penetrate into cells; target intracellular Staphylococcus aureus and restore its sensitivity to rifampicin, enhancing the bactericidal effect of rifampicin; in drug-resistant persistent Staphylococcus aureus induced by oxidative stress, the combination of maltodextrin and rifampicin significantly inhibits the growth of Staphylococcus aureus, and the combination of maltodextrin nanoparticles and rifampicin can significantly eliminate intracellular Staphylococcus aureus in the liver and kidney.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] 1. The present application provides a new drug effect of maltodextrin nanoparticles, which can enhance the sensitivity of antibiotics to treat intracellular bacteria and enhance the bactericidal effect of antibiotics; and can solve the problem of drug resistance to intracellular persistent cells.

[0029] 2, The application provides a potential combined drug target for patients with rifampicin-resistant bacterial infection, and develops a potential treatment new strategy for maltodextrin.

[0030] 3, The product provided by the application enables maltodextrin to reverse the drug resistance of persistent Staphylococcus aureus as a carbon source of bacteria, and the application provides a maltodextrin active oxygen-responsive carrier material and a treatment new strategy of using the maltodextrin active oxygen-responsive carrier material and rifampicin in combination after self-assembly into nanoparticles.

[0031] 4, In the technical solution of the application, maltodextrin plays a role in improving the effect of rifampicin on clearing bacteria in a small amount, and has little toxic and side effects, and is expected to be further researched and applied in clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0032] The drawings accompanying the specification of the application form part of the application and serve to further understand the application, the illustrative embodiments of the application and the description thereof serve to explain the application and do not constitute an improper limitation of the application.

[0033] Figure 1 Maltodextrin reverses the drug resistance of Staphylococcus aureus to rifampicin under oxidative stress;

[0034] Figure 1 In the figure, A is a result of drug resistance reversal determination; B is a result of ATP determination; and C is a result of aconitase determination.

[0035] Figure 2 Maltodextrin active oxygen-responsive carrier material and preparation of maltodextrin nanoparticles;

[0036] Figure 2 In the figure, A is a process mode diagram for preparation of the maltodextrin-responsive carrier material; B is a process mode diagram for preparation of the maltodextrin nanoparticles; C is a result of particle size determination of the maltodextrin; D is a result of potential determination of the maltodextrin nanoparticles; and E is a result of active oxygen responsiveness determination of the maltodextrin nanoparticles.

[0037] Figure 3 Maltodextrin nanoparticles reverse the drug resistance of intracellular Staphylococcus aureus to rifampicin.

[0038] Figure 3 In the figure, A is a result of cell viability determination; B is a result of flow cytometry uptake determination; C is a result of confocal cell uptake determination; D is a result of intracellular degradation determination; E is a result of intracellular and bacterial co-localization determination; and F is a result of intracellular drug resistance reversal determination.

[0039] Figure 4 Maltodextrin nanoparticles and rifampicin are used in combination to inhibit the growth of intracellular bacteria.

[0040] Figure 4In the figure, A is the modeling figure of the intracellular bacteria transfer infection of mice using maltodextrin nanoparticles and rifampicin; B is the flow cytometry co-localization determination result of the cells infected with Staphylococcus aureus and maltodextrin nanoparticles; C is the confocal co-localization determination result of the cells infected with Staphylococcus aureus and maltodextrin nanoparticles in the liver; D is the confocal co-localization determination result of the cells infected with Staphylococcus aureus and maltodextrin nanoparticles in the kidney; E is the determination result of the total bacterial amount, extracellular bacterial amount and intracellular bacterial amount of the organs liver and kidney.

[0041] Figure 5 The Staphylococcus aureus of the various oligosaccharides reversing oxidative stress is resistant to various antibiotics.

[0042] Figure 5 In the figure, A is the determination comparison result of the oligosaccharides: manno-oligosaccharide, dextran 100, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, maltodextrin reversing the rifampicin resistance of Staphylococcus aureus persistent cells; B is the determination comparison result of maltodextrin reversing the vancomycin, ciprofloxacin, oxacillin, chloramphenicol, rifampicin, levofloxacin, linezolid resistance of Staphylococcus aureus persistent cells.

[0043] Figure 6 The schematic diagram of the mechanism of maltodextrin nanoparticle combined therapy for intracellular bacterial infection. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0045] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and furthermore, it should be understood that when the terms "comprise" and / or "include" are used in the specification, there is a presence of the features, steps, operations, devices, components and / or their combinations.

[0046] Example 1, maltodextrin restores the sensitivity of persistent Staphylococcus aureus to rifampicin

[0047] 1. Bacterial culture and persistent bacteria treatment

[0048] Staphylococcus aureus was purchased from the Institute of Agricultural Resources Planning, Chinese Academy of Agricultural Sciences, and cultured in LB liquid culture at 37°C, 180 rpm in a shaking incubator to OD600=0.3. 1x10 9Staphylococcus aureus CFU / mL was incubated with 200 μg / mL FITC in LB medium for 1.5 hours at 37℃, 180 rpm to obtain FITC-labeled Staphylococcus aureus for standby.

[0049] Maltodextrin was dissolved in sterile PBS buffer solution to prepare 2 mmol / L, 6 mmol / L, 10 mmol / L for standby.

[0050] Rifampicin was dissolved in dimethyl sulfoxide (DMSO) to prepare 0.1 μm / L solution for standby.

[0051] 1x10 8 After Staphylococcus aureus CFU / mL was treated with 120 mM H2O2 for 20 minutes and washed with sterile PBS buffer solution for 3 times, the oxidative stress-induced persistent Staphylococcus aureus was obtained, and resuspended in a mixed solution containing different concentrations of maltodextrin and rifampicin, and treated at 37℃, 180 rpm for 24 hours, then diluted and plated on solid medium with sterile PBS for counting. As shown in FIG. 1A, rifampicin alone cannot effectively eliminate persistent Staphylococcus aureus, while maltodextrin can restore the sensitivity of persistent Staphylococcus aureus to rifampicin, and has a concentration-dependent effect, which increases with the increase of its concentration. Figure 1 As shown in FIG. 1A, rifampicin alone cannot effectively eliminate persistent Staphylococcus aureus, while maltodextrin can restore the sensitivity of persistent Staphylococcus aureus to rifampicin, and has a concentration-dependent effect, which increases with the increase of its concentration.

[0052] 2, Mechanism of maltodextrin restoring the sensitivity of persistent bacteria to rifampicin

[0053] Persistent Staphylococcus aureus was treated with 1 mM maltodextrin, the control group was treated with PBS, and the normally metabolizing Staphylococcus aureus was used as a control. After 1 hour of treatment, the aconitase activity and ATP level were determined according to the aconitase detection kit (purchased from Solabio) and the ATP detection kit (BacTiter-GloTM Microbial Cell Viability Assay, Promega) instructions. As shown in FIG. 2A and FIG. 2B, Figure 1 B, Figure 1 As shown in FIG. 2A and FIG. 2B, the aconitase activity and ATP level of persistent cells are lower than those of normally metabolizing Staphylococcus aureus, while the addition of maltodextrin can enhance the aconitase activity of persistent cells and increase the ATP level.

[0054] Example 2, Verification test of maltodextrin nanoparticles restoring the sensitivity of intracellular persistent Staphylococcus aureus to rifampicin

[0055] 1, Preparation of reactive oxygen species-responsive maltodextrin

[0056] As shown in FIG. 3A and FIG. 3B, Figure 2A. 1.22 g of 4-hydroxyphenylboronic acid pinacol ester (PBAP) and 1.68 g of N,N'-carbonyldiimidazole (CDI) were dissolved in 10 mL of anhydrous dichloromethane and stirred magnetically at 40°C for 1 hour. 20 mL of anhydrous dichloromethane was added to the reaction solution, which was then washed three times with 15 mL of ultrapure water. The lower layer was removed, and the solution was washed twice with 10 mL of saturated sodium chloride. The lower layer was removed, and the residual aqueous solution was removed with anhydrous sodium sulfate. After standing, the lower layer was removed by centrifugation. The solvent was removed by rotary evaporation, and the product was dried in a vacuum oven to obtain 1.09 g of activated PBAP (CDI-PBAP) as a white solid powder. 0.27 g of 4-dimethylaminopyridine (DMAP), 0.19 g of maltodextrin, and 1.09 g of CDI-PBAP were dissolved in 5 mL of anhydrous dimethyl sulfoxide (DMSO), respectively, and then mixed. The reaction solution was precipitated with ultrapure water, centrifuged at 6000xg, and the supernatant was discarded. The precipitate was washed three times with ultrapure water and freeze-dried to obtain 0.84 g of maltodextrin active oxygen-responsive carrier material (OXMD) as a white solid powder.

[0057] 2. Preparation and characterization of maltodextrin nanoparticles

[0058] 15 mL of ultrapure water was heated to 65°C. 6 mg of lecithin and 12 mg of DSPE-PEG were dissolved in 1 mL of anhydrous ethanol and added to the 65°C ultrapure water solution. After stirring magnetically at 65°C for 2 hours, the solution was cooled to room temperature. 60 mg of OXMD was dissolved in methanol and slowly added to the above-mentioned room temperature solution under high stirring speed. The reaction was carried out for 2 hours. After the reaction was completed, the solution was rotary evaporated to 5 mL, and the un-encapsulated OXMD was removed by ultrafiltration centrifugation at 6000xg for 10 minutes to obtain maltodextrin nanoparticles (MDNP), as shown in Figure 2 B. The preparation method of maltodextrin nanoparticles labeled with fluorescent dye Cy5 was similar, except that 2% of Cy5 / DiO-DiI was added when OXMD was added. The particle size was measured by a Malvern particle size analyzer, and the morphology was characterized by scanning electron microscopy after freeze-drying, as shown in Figure 2 C. The MDNP was a uniformly distributed and regularly shaped spherical nanoparticle with an average particle size of 88±8 nm. MDNP was incubated with different concentrations of H2O2, and the transmittance of the solution was measured at OD550 at different time points. The degree of hydrolysis of the nanoparticles was calculated to indicate the active oxygen responsiveness of the maltodextrin nanoparticles. The test results are shown in Figure 2 D. The MDNP could be hydrolyzed in different concentrations of H2O2, and had a concentration-dependent effect. The higher the concentration of H2O2, the stronger the degree of hydrolysis.

[0059] 3. Cell culture and cell viability determination

[0060] Raw264.7 mouse macrophages were obtained from the Resource Center of Shanghai Institutes for Biological Sciences. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and in high-glucose DMEM supplemented with 10% FBS. Raw264.7 mouse macrophages were cultured in 96-well plates at a density of 1.0 × 10⁶ cells per well. 4 Cells were cultured at a density of [number] cells per cell line, with 100 µL of complete culture medium (DMEM containing 10% wtFBS). Prior to other experiments, cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours, cells were treated with different concentrations of MDNP for 24 hours. Cell viability was quantitatively determined using the CCK8 assay, and the results are shown below. Figure 3 As shown in Figure A, MDNP exhibits good cell compatibility and low toxicity.

[0061] 4. Cell uptake assay

[0062] 1X10 5 Raw 264.7 cells were seeded into sterile 24-well plates and cultured overnight in 500 μL of high-glucose medium (DMEM, 10% wtFBS) to allow cell adhesion. After 12 hours, the medium was replaced with 1 mM, 3 mM, and 5 mM MDNP diluted with complete medium, and the cells were incubated for another 2 hours. The medium was then aspirated, and the cells were washed three times with sterile PBS. 500 μL of sterile PBS was added to remove the adherent cells, which were then collected in 1.5 mL EP tubes. After centrifugation at 3000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL of sterile PBS. Fluorescence intensity was detected by flow cytometry. Data were processed using FlowJo 7.6.1 to obtain the dose-dependent curve of macrophage nanoparticle uptake, as shown below. Figure 3 As shown in B, the cells take up more MDNP as the MDNP concentration increases.

[0063] Inoculate 1 x 102 cells onto a sterile confocal dish. 5 Raw 264.7 cells were cultured overnight in high-glucose medium (DMEM, 10% wtFBS) to allow cell adhesion. After 12 hours, the medium was replaced with 1 mM MDNP, and the cells were co-incubated for 2 hours. The medium was then aspirated, and the cells were washed three times with sterile PBS. 75 nM Lyso-Trackergreen lysosomal fluorescent probe was added, and the cells were incubated for another 1 hour for lysosomal staining. The medium was then aspirated, and the cells were washed three times with sterile PBS. 1 mL of Hoechst 33342 nuclear dye was added, and the cells were incubated for another 30 minutes for nuclear staining. The cells were protected from light and stored in a humidified chamber for confocal microscopy imaging. Results... Figure 3 As shown in C, MDNPs exhibit high co-localization with lysosomes after being taken up by Raw264.7 cells.

[0064] 5. Intracellular degradation assay

[0065] Raw264.7 cells were seeded in 24-well plates at a concentration of 1 x 10 5 / well and incubated at 37 °C in a 5% CO2 incubator until confluence. Then 500 ng / mL LPS and 20 ng / mL IFN-γ were added to each well to stimulate macrophages for 20 hours to induce the production of a large number of reactive oxygen species. The medium was replaced with 1 mM DiO-DiI-MDNP and incubated with cells for 0h, 4h, 8h. The corresponding FRET spectra were recorded by CLSM λ scan mode with an excitation wavelength of 488 nm, scanning detection of 495-605 nm every 5 nm step, and the FRET ratio (DiO, donor) and the fluorescence intensity at 570 nm (DiI, acceptor) in the spectrum were calculated according to the fluorescence intensity at 505 nm. The detection and calculation results are shown in Figure 3 As shown in D, with the extension of the incubation time of MDNP with cells to 8 hours, the fluorescence intensity of DiO is stronger and stronger, and the FRET ratio R decreases from 0.75 to 0.58, indicating that MDNP can release maltodextrin in cells.

[0066] 6. Intracellular co-localization assay with bacteria

[0067] Raw264.7 cells were seeded in 24-well plates at a concentration of 1 x 10 5 / well and incubated at 37 °C in a 5% CO2 incubator until confluence. Then 500 ng / mL LPS and 20 ng / mL IFN-γ were added to each well to stimulate macrophages for 20 hours to induce the production of a large number of reactive oxygen species. FITC-S. aureus was used to infect the above macrophages at a concentration of MIO = 10, and was incubated at 37 °C in a 5% CO2 incubator for 2 hours. Then the original culture medium was removed, washed with sterile PBS for three times, and 50 μg / mL gentamicin was added for incubation for 30 minutes to remove extracellular bacteria. Then sterile PBS was washed for three times, and 1 mM Cy5-MDNP was added for co-incubation for 2 hours, and then the co-localization was photographed by confocal. Results Figure 3 As shown in E, the red fluorescence of MDNP and the green fluorescence of S. aureus in cells have obvious overlap, indicating that MDNP can be used by intracellular bacteria.

[0068] 7. Intracellular recovery of antibiotic sensitivity assay

[0069] Raw264.7 cells were seeded in 24-well plates at a concentration of 1 x 10 5Concentration of wells were planted in 24-well plates, and cultured to adhere to the wall in 37℃, 5% CO2 incubator. After adding 500 ng / mL LPS and 20 ng / mL IFN-γ to each well to stimulate for 20 hours, macrophages were induced to produce a large number of active oxygen. The above macrophages were infected with S. aureus at a concentration of MIO=10, and incubated at 37℃, 5% CO2 for 2 hours. After removing the original culture medium, the cells were washed with sterile PBS three times, and 50 μg / mL gentamicin was added for 30 minutes to remove extracellular bacteria. After washing with sterile PBS three times, different concentrations of materials (1 mM MDNP, 3 mM MDNP, 5 mM MDNP, 10 μg / ml Rif, 1 mM MDNP+10 μg / ml Rif, 3 mM MDNP+10 μg / ml Rif, 5 mM MDNP+10 μg / ml Rif, wherein the MDNP and rifampicin combination group, MDNP was added 2 hours before rifampicin treatment for 24 hours) were added and incubated at 37℃, 5% CO2 for 24 hours. After adding 0.5% Triton X-100 to lyse the cells for 30 minutes, the cells were diluted and plated. The results were counted as follows Figure 3 F, rifampicin cannot remove intracellular S. aureus, but the removal effect of rifampicin can be restored after the introduction of MDNP, and when the concentration of MDNP is 5 mM, the number of bacteria is reduced by an order of magnitude, indicating that MDNP can restore the sensitivity of intracellular bacteria to rifampicin.

[0070] Example 3, Maltodextrin Nanoparticles and Rifampicin Combination to Inhibit the Growth of Intracellular Bacteria in Mice

[0071] 1. Animal model and treatment

[0072] 4-6 week old BALB / c mice (female, 18±4 g) were used for animal experiments. The mice were purchased from Chongqing Enbi Biological Technology Co., Ltd. For the mouse intracellular bacterial transfer infection model, the donor group of mice was 9, and 5x10 7 CFU / mL of S. aureus, 24 hours after infection, the mice were euthanized, 8 mL of pre-cooled sterile PBS was injected into the abdominal cavity of the mice, and the peritoneal lavage fluid was collected and centrifuged at 4500 rpm for 5 minutes at 4℃. After adding 50 μg / mL gentamicin solution to incubate for 30 minutes to remove extracellular bacteria, the cells were washed with sterile PBS three times and resuspended in DMEM medium for counting and dilution to 1x10 7 / mL for standby. See the schematic Figure 4 A.

[0073] 2. Co-localization assay of S. aureus infected cells and maltodextrin nanoparticles

[0074] The above cells were co-incubated with 5 μΜ DiI fluorescent dye for 30 minutes to label the cell membrane or co-incubated with DMEM medium without fluorescent dye as a blank control group, and then washed with sterile PBS three times. The recipient mice (6) were randomly divided into two groups, including the DiI labeling group and the blank group, and each group of mice was given 1x10 6 cells. After 2 hours, the mice in the DiI labeling group were given 30 mg / kg Cy5-MDNP, and the mice in the blank control group were given the same volume of sterile PBS. The mice were euthanized after 2 hours, and the organs (liver and kidney) were cut into two parts with scissors, one part was cut into pieces and added to a RIPM1640 solution containing 5 U / mL Dispase II, 2 mg / kg Collagenase V, and 5 U / mL DNase at 37°C for 30 minutes. The cells were collected by passing through a 70-mesh cell sieve, and the co-localization of DiI-labeled cells and maltodextrin nanoparticles was detected by flow cytometry. The data were processed by Flowjo, and the results are shown in Figure 4 B, 86.6% of the MDNP entering the liver was taken up by DiI-labeled cells; 95.7% was taken up in the kidney. The remaining half of the organs were paraffin-embedded, sectioned, fixed with 4% paraformaldehyde, then incubated with F480 antibody at 4°C overnight, then combined with fluorescent secondary antibody, and photographed under confocal microscope. The results are shown in Figure 4 C, D, MDNP and DiI-labeled macrophages have obvious co-localization in the liver, and no obvious co-localization is observed in the kidney.

[0075] 3. Determination of the inhibition of the growth of intracellular bacteria by maltodextrin nanoparticles and rifampicin combination

[0076] A total of 20 recipient mice were randomly divided into four groups, including the control group (PBS), the rifampicin (Rif) group, the 30 mg / kg maltodextrin nanoparticle + rifampicin group (a+Rif), and the 100 mg / kg maltodextrin nanoparticle + rifampicin group (b+Rif). Each mouse was given 1x10 6One, 24 hours after infection, different concentrations of maltodextrin nanoparticles were given through the tail vein, and 2 hours later, rifampicin was given (or the control group / rifampicin group was given alone) for 24 hours. After 24 hours, the mice were euthanized, and the organs liver and kidney were taken, cut into small pieces, and added to a solution of RIPM1640 containing 5 U / mL Dispase II, 2 mg / kg Collagenase V, and 5 U / mL DNase at 37°C for 30 minutes. The cells were collected by passing through a 70-mesh cell sieve and divided into three equal parts: one part was the total bacterial count, the cells were lysed by adding 0.5% Triton-X 100 solution for 30 minutes, and then diluted and plated for counting; one part was the extracellular bacterial count, after centrifugation at 3000 rpm for 3 minutes, the supernatant was taken and diluted and plated for counting; one part was the intracellular bacterial count, after centrifugation at 3000 rpm for 3 minutes, the upper solution was discarded, 50 μg / mL gentamicin was added for 30 minutes to remove extracellular bacteria, and then washed with sterile PBS three times, then lysed the cells by adding 0.5% Triton-X 100 solution for 30 minutes, and then diluted and plated for counting. The counting results are shown in Figure 4 E, 100 mg / kg MDNP can significantly enhance the rifampicin clearance of intracellular bacteria in the liver and kidney of mice.

[0077] Comparative Example, Various oligosaccharides reverse the antibiotic resistance of Staphylococcus aureus under oxidative stress

[0078] Using the method provided in Example 1, bacterial culture, persistent bacterial treatment, and antibiotic treatment were carried out to investigate the effect of various oligosaccharides on restoring the sensitivity of persistent Staphylococcus aureus to rifampicin, and the results are shown in Figure 5 The linear oligosaccharide maltodextrin restores the sensitivity of persistent bacteria to the antibiotic rifampicin better than the cyclic oligosaccharides α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and mannose oligosaccharide, as shown in Figure 5 A. Through comparative experiments of various lipid-soluble antibiotics, the clearance effect of rifampicin is the best and most stable, as shown in Figure 5 B.

[0079] The mechanism of maltodextrin nanoparticle combination therapy for intracellular bacterial infection is as shown in Figure 6 After the maltodextrin nanoparticles are taken up by macrophages, they enter the lysosomes, escape from the lysosomes, and target the intracellular persistent bacteria, releasing maltodextrin in the intracellular high reactive oxygen microenvironment, restoring the metabolic activity of intracellular persistent Staphylococcus aureus, and thus enhancing the clearance effect of the antibiotic rifampicin.

[0080] Example 4, Pharmaceutical composition containing maltodextrin nanoparticles

[0081] The pharmaceutical composition contains the maltodextrin nanoparticles provided in Examples 1-3, and is a drug for treating bacterial infections.

[0082] As an embodiment, the pharmaceutical composition comprises the maltodextrin nanoparticle and a pharmaceutically inactive ingredient, which can be a carrier, an excipient and a diluent commonly used in pharmacy, and can be lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc.

[0083] The dosage form thereof is a powder, granule, tablet, capsule, suspension, emulsion, syrup, oral or external agent in the form of spray, suppository and sterile injection solution, preferably an injection, which can be administered by intravenous or local injection.

[0084] As another embodiment, the pharmaceutical composition further comprises an antibiotic selected from vancomycin, ciprofloxacin, oxacillin, chloramphenicol, rifampicin.

[0085] The pharmaceutical composition can further comprise a pharmaceutically inactive ingredient, which can be a carrier, an excipient and a diluent commonly used in pharmacy, and can adopt a dosage form commonly used in pharmacy.

[0086] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a maltodextrin nanoparticle in the manufacture of a medicament for the treatment of a bacterial infection, characterized in that, The maltodextrin nanoparticle is prepared by self-assembly of a maltodextrin active oxygen responsive carrier material; the maltodextrin active oxygen responsive carrier material is a carrier material with active oxygen response prepared by covalently linking an oxidation-sensitive group phenylboronic acid pinacol ester to maltodextrin; the maltodextrin nanoparticle is prepared by the following method: 4-hydroxyphenylboronic acid pinacol ester is reacted with N,N'-carbonyldiimidazole, and the obtained product is then reacted with maltodextrin to obtain the maltodextrin active oxygen responsive carrier material; the maltodextrin active oxygen responsive carrier material is externally coated with lecithin and phospholipid polyethylene glycol copolymer to form; The drug for treating bacterial infection is a composition of the maltodextrin nanoparticle and an antibiotic; The antibiotic is selected from vancomycin, ciprofloxacin, oxacillin, rifampicin, and levofloxacin.

2. Use of the maltodextrin nanoparticle according to claim 1 for the manufacture of a medicament for the treatment of a bacterial infection, characterized in that, The maltodextrin nanoparticle has a particle size range of 88.8±8 nm.

3. Use of the maltodextrin nanoparticle according to claim 1 for the manufacture of a medicament for the treatment of a bacterial infection, characterized in that, The method comprises the following steps: In step 1, the organic solvent is anhydrous dichloromethane; in step 2, the organic solvent is anhydrous dimethyl sulfoxide; in step 3, the organic solvent for dissolving lecithin and phospholipid polyethylene glycol copolymer is anhydrous ethanol, and the organic solvent for dissolving the maltodextrin active oxygen responsive carrier material obtained in step 2 is methanol. The maltodextrin nanoparticle is prepared by self-assembly of a maltodextrin active oxygen responsive carrier material; the maltodextrin active oxygen responsive carrier material is a carrier material with active oxygen response prepared by covalently linking an oxidation-sensitive group phenylboronic acid pinacol ester to maltodextrin; the maltodextrin nanoparticle is prepared by the following method: 4-hydroxyphenylboronic acid pinacol ester is reacted with N,N'-carbonyldiimidazole, and the obtained product is then reacted with maltodextrin to obtain the maltodextrin active oxygen responsive carrier material; the maltodextrin active oxygen responsive carrier material is externally coated with lecithin and phospholipid polyethylene glycol copolymer to form; ​ 4. Use of the maltodextrin nanoparticle according to claim 3 for the manufacture of a medicament for the treatment of a bacterial infection, characterized in that, ​ 5. A pharmaceutical composition, characterized by, ​ The pharmaceutical composition further comprises an antibiotic selected from the group consisting of vancomycin, ciprofloxacin, oxacillin, rifampicin, levofloxacin. The pharmaceutical composition further comprises an antibiotic selected from the group consisting of vancomycin, ciprofloxacin, oxacillin, rifampicin, levofloxacin.

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