Neuraminidase-targeted drug-loaded nanoparticles and their preparation method and application

Abstract

The present invention discloses drug-loaded nanoparticles targeting neuraminidase, as well as a preparation method and application thereof, belonging to the technical field of pharmaceutical preparations. The present invention connects polysialic acid and polylactic acid glycolic acid via an ester bond to obtain drug-loaded nanoparticles with stable structure and performance. The drug-loaded nanoparticles have the function of targeting neuraminidase and have relatively high efficiency in entering cells. Furthermore, antibiotics are dispersed in the drug-loaded nanoparticles, and an inflammatory modulator is adsorbed on the surface of the drug-loaded nanoparticles. The drug-loaded nanoparticles can deliver the antibiotics and the inflammatory modulator to Grasseria suis in pigs, releasing the antibiotics and the inflammatory modulator in the slightly acidic environment of the infected site in the pig, thereby efficiently killing Grasseria suis and reducing inflammatory damage caused by bacterial infection and toxic damage of the antibiotics. Therefore, the present invention has good application prospects in the preparation of drugs for preventing and / or treating Grasseria suis infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to drug-loaded nanoparticles targeting neuraminidase, and a preparation method and application thereof. Background Art

[0002] Under large-scale farming conditions, bacterial infection is one of the main causes of morbidity and mortality in livestock and poultry, resulting in severe economic losses. The widespread use of antibiotics has significantly reduced infection and mortality rates in livestock and poultry, but long-term overuse has led to bacterial resistance and residues in livestock products, limiting their potential application.

[0003] Glaesserella parasuis (Gps) is a common respiratory bacteria species in pigs, present in all stages of pig life. Swine Glaesser's disease typically develops after the introduction of new pigs or infection with other pathogens. Piglets can be infected with Gps through contact with sows. Different serotypes of Gps have been isolated from pigs, with the predominant serotypes of Gps prevalent in my country being highly virulent strains 4 and 5. The morbidity and mortality rates range from 5% to 10%, with the incidence in latently infected pigs reaching as high as 75%.

[0004] Nitric oxide, an effector molecule of the immune system, is primarily derived from the L-arginine metabolic pathway mediated by nitric oxide synthase (NOS). The key rate-limiting step is the competitive binding of arginase (ARG) with NOS for arginine. In pathological responses, the produced nitric oxide has certain negative effects on the host, including immune rejection, neurodegeneration, and septic shock. For example, infection with Gps significantly induces NO production in porcine alveolar macrophages. Furthermore, bacterial activation of NF-κB signaling mediates the upregulation of NOS2, promoting NO production and playing a key role in amplifying the inflammatory response. For example, the NOS2 / NO ratio is highly correlated with the development of lung inflammation. Repeated stimulation of airway epithelial cells by external pathogens prompts them to secrete large amounts of proinflammatory cytokines. High levels of inflammatory cytokines exacerbate chronic damage and malignant regeneration of other lung cells, leading to the exudation of large amounts of fibrinogen from the alveoli and the development of lung pathologies such as pulmonary fibrosis.

[0005] Responsive nanoparticle drug delivery systems are a type of nanoparticle that achieves intelligent controlled release based on drastically changing external physical and chemical factors. They typically offer precise drug delivery and high therapeutic compliance. These delivery platforms increase drug concentration only upon reaching the lesion, avoiding the overtoxicity and premature failure associated with premature release. Treatment strategies based on responsive nanocarriers have been widely applied to bacterial infections, including pH-sensitive, redox-sensitive, enzyme-responsive, temperature-responsive, and responsive to strong external stimuli.

[0006] However, existing drug delivery systems have problems such as poor targeting, poor stability and low efficiency in entering cells when delivering antibiotics and inflammatory regulators used to kill GPS. Summary of the Invention

[0007] The present invention aims to provide drug-loaded nanoparticles targeting neuraminidase, as well as their preparation method and application. This approach aims to address the problems of poor targeting, poor stability, and low cell entry efficiency in existing drug delivery systems for delivering antibiotics and inflammatory modulators used to kill GPS.

[0008] In a first aspect, the present invention provides a drug-loaded nanoparticle targeting neuraminidase, the drug-loaded nanoparticle comprising polysialic acid and polylactic-co-glycolic acid; wherein the carboxyl group on the side chain of polysialic acid is connected to the hydroxyl group in polylactic-co-glycolic acid via an ester bond.

[0009] In the drug-loaded nanoparticles provided by the present invention, polysialic acid and polylactic acid glycolic acid are connected through an ester bond, thereby making the structure and performance of the drug-loaded nanoparticles stable. The drug-loaded nanoparticles have the function of targeting neuraminidase and have a faster efficiency of entering cells.

[0010] In some embodiments, the drug-loaded nanoparticles further include antibiotics, which are dispersed in the drug-loaded nanoparticles; the mass ratio of the antibiotics to the drug-loaded nanoparticles is (0.3-0.6):1, and the antibiotics include at least one of tedilosin, tilmicosin, clarithromycin, roxithromycin, and tylosin.

[0011] In this invention, antibiotics are dispersed within drug-loaded nanoparticles to prevent drug loss during delivery. Since neuraminidase, a potential bacterial virulence factor, has been shown to be expressed in Glaseria suis and involved in the breakdown of sialic acid, these drug-loaded nanoparticles can deliver antibiotics to Glaseria suis in pigs (targeting neuraminidase). The antibiotics are then released in the slightly acidic environment of the infected area, effectively killing Glaseria suis.

[0012] In some embodiments, the drug-loaded nanoparticles further include an inflammatory regulator, which is adsorbed on the surface of the drug-loaded nanoparticles by electrostatic adsorption; the mass ratio of the inflammatory regulator to the drug-loaded nanoparticles is (0.05~0.15):1, and the inflammatory regulator includes at least one of S-methylisothiourea, 1400W, L-NMMA, ginsenoside and isocysteine.

[0013] In the present invention, an inflammatory regulator is further adsorbed on the surface of the drug-loaded nanoparticles. The inflammatory regulator is delivered to the site of infection of Glaseria suis along with the drug-loaded nanoparticles and released, thereby reducing the inflammatory damage caused by bacterial infection and the toxic damage of antibiotics.

[0014] In a second aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned drug-loaded nanoparticles targeting neuraminidase.

[0015] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0016] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Exemplarily, pharmaceutically acceptable carriers include buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with drug administration. Each carrier must be "acceptable," meaning compatible with the other ingredients of the composition and not harmful to the patient.

[0017] In a third aspect, the present invention provides a method for preparing drug-loaded nanoparticles targeting neuraminidase as described above, comprising the following steps: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine to a polysialic acid aqueous solution, respectively, to fully dissolve them, then adding a polylactic acid hydroxy organic solution for condensation reaction, and purifying the resulting product to obtain a polysialic acid-polylactic acid glycolic acid intermediate compound; dissolving the polysialic acid-polylactic acid glycolic acid intermediate compound and an inflammatory regulator in water to obtain an aqueous solution; dissolving an antibiotic drug and a cationic surfactant in an organic solvent to obtain an organic solution; mixing the aqueous solution and the organic solution and ultrasonically oscillating them, then stirring them, and centrifuging, washing and freeze-drying the resulting product to obtain drug-loaded nanoparticles.

[0018] In the preparation method provided by the present invention, antibiotic drugs are loaded into a polysialic acid-polylactic acid-glycolic acid intermediate compound by an emulsification co-solvent volatilization method, and then, under the action of a cationic surfactant, an inflammation regulator is adsorbed on the surface of the polysialic acid-polylactic acid-glycolic acid intermediate compound by electrostatic adsorption, thereby obtaining spherical drug-loaded nanoparticles with uniform particle size distribution and stable structural properties.

[0019] In some preferred embodiments, during the preparation of the polysialic acid-polylactic acid-glycolic acid intermediate compound, the purification step of the obtained product specifically includes: adding an organic solvent to the product to obtain a flocculent precipitate, centrifuging the flocculent precipitate, dissolving the obtained precipitate in phosphate buffer and then dialyzing to obtain drug-loaded nanoparticles.

[0020] In some more preferred embodiments, the organic solvent includes chloroform and petroleum ether; the centrifugation includes: centrifugation at a temperature of 0-4°C and a rotation speed of 12,000×g for 15-25 minutes; and the dialysis time is 1-3 days.

[0021] In the present invention, the amounts of chloroform and petroleum ether can be conventionally adjusted according to actual use needs. The amount of chloroform used can be, for example, 2 times the volume of the product, and the amount of petroleum ether used can be, for example, 1 times the volume of the product. The pH value of the phosphate buffer is 8, and it includes the following components: 5.59 g of dipotassium hydrogen phosphate, 0.41 g of potassium dihydrogen phosphate, and the volume is adjusted to 1 L with deionized water.

[0022] In some embodiments, during the preparation of the polysialic acid-polylactic acid glycolic acid intermediate compound, the concentration of polysialic acid is 8-12 mM, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 9-13 mM, and the concentration of 4-dimethylaminopyridine is 9-13 mM; the concentration of polylactic acid hydroxyl is 8-12 mM; and the condensation reaction step specifically includes: reacting at a temperature of 35-45°C for 15-20 hours.

[0023] In some embodiments, the preparation process of the aqueous phase solution further includes the step of adding 0.5~2wt% polyvinyl alcohol (PVA) to water; in the preparation process of the organic phase solution, the mass ratio of the antibiotic drug to the cationic surfactant is 1:(0.05~0.15), and the cationic surfactant includes hexadecyltrimethylammonium bromide (CTAB), and the organic solvent includes chloroform.

[0024] In some embodiments, during the preparation of drug-loaded nanoparticles, the stirring treatment step specifically includes: stirring at a temperature of -1 to 1°C for 16 to 48 hours.

[0025] In a fourth aspect, the present invention provides the use of any of the above-mentioned neuraminidase-targeted drug-loaded nanoparticles, any of the above-mentioned pharmaceutical compositions, or any of the above-mentioned preparation methods in the preparation of drugs for preventing and / or treating swine Grasseria infection.

[0026] The beneficial effects of the present invention are as follows: different from the prior art, the present invention connects polysialic acid and polylactic acid glycolic acid through ester bonds to obtain drug-loaded nanoparticles with stable structure and performance, and the drug-loaded nanoparticles have the function of targeting neuraminidase and have a faster efficiency of entering cells; further, antibiotics are dispersed in the drug-loaded nanoparticles, and inflammation regulators are adsorbed on the surface of the drug-loaded nanoparticles. The drug-loaded nanoparticles can deliver antibiotics and inflammation regulators to Grasseria suis (targeting neuraminidase) in the pig's body, and release antibiotics and inflammation regulators in the acidic environment of the infected site in the pig's body, thereby efficiently killing Grasseria suis and reducing inflammatory damage caused by bacterial infection and toxic damage of antibiotics. Therefore, the drug-loaded nanoparticles have good application prospects in the preparation of drugs for preventing and / or treating Grasseria suis infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The polysialic acid-polylactic acid glycolic acid intermediate compound (PSA@PLGA) prepared in Example 1 of the present invention 1 HNMR spectrum;

[0028] Figure 2 FT-IR spectrum of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention;

[0029] Figure 3A TEM electron micrograph and particle size distribution diagram of the polysialic acid-polylactic acid-glycolic acid intermediate compound prepared in Example 1 of the present invention;

[0030] Figure 3B TEM electron micrograph and particle size distribution diagram of drug-loaded nanoparticles (PSA@PLGA-TD) prepared in Example 1 of the present invention;

[0031] Figure 3C TEM electron micrograph and particle size distribution diagram of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention;

[0032] Figure 4 zeta potential distribution spectrum of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention;

[0033] Figure 5 This is the UV-visible spectrophotometric spectrum of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention;

[0034] Figure 6This is a graph of the responsive release of drugs from drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention;

[0035] Figure 7 This is a graph showing changes in NO content when cells were treated with drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention, n=3. ;

[0036] Figure 8 This is a graph showing the results of cell uptake of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention, n=3. ;

[0037] Figure 9 This is an in situ fluorescence image of the cell uptake of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention;

[0038] Figure 10 This is a diagram showing the extracellular antibacterial effect of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention, n=3. ;

[0039] Figure 11 This is a diagram showing the extracellular antibacterial plate coating results of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention;

[0040] Figure 12 This is a fluorescence image of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention regulating cellular inflammation. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making creative efforts are within the scope of protection of the present invention.

[0042] For experimental methods in the examples where specific conditions are not specified, generally conventional conditions and conditions described in the manual or conditions recommended by the manufacturer were followed. The general equipment, materials, reagents, etc. used were all commercially available unless otherwise specified.

[0043] The present invention provides a drug-loaded nanoparticle targeting neuraminidase, which comprises polysialic acid, polylactic-co-glycolic acid, an antibiotic drug and an inflammation regulator; wherein the carboxyl group on the side chain of the polysialic acid is connected to the hydroxyl group in the polylactic-co-glycolic acid through an ester bond; the antibiotic drug is dispersed in the drug-loaded nanoparticle, and the inflammation regulator is adsorbed on the surface of the drug-loaded nanoparticle through electrostatic adsorption.

[0044] The preparation method of the drug-loaded nanoparticles comprises the following steps: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine to a polysialic acid aqueous solution and fully dissolving them, then adding a polylactic acid hydroxy organic solution to carry out a condensation reaction, purifying the obtained product to obtain a polysialic acid-polylactic acid glycolic acid intermediate compound; dissolving the polysialic acid-polylactic acid glycolic acid intermediate compound and an inflammation regulator in water to obtain an aqueous phase solution; dissolving an antibiotic drug and a cationic surfactant in an organic solvent to obtain an organic phase solution; mixing the aqueous phase solution and the organic phase solution, performing ultrasonic oscillation, and then stirring the mixture; and centrifuging, washing, and freeze-drying the obtained product to obtain the drug-loaded nanoparticles.

[0045] For example, the drug-loaded nanoparticles were synthesized using tediluosin (TD) and S-methylisothiourea (SMT) as raw materials.

[0046] Example 1 Synthesis and structural characterization of drug-loaded nanoparticles (PSA@PLGA-TD / SMT)

[0047] A method for preparing drug-loaded nanoparticles (PSA@PLGA-TD / SMT) targeting neuraminidase comprises the following steps:

[0048] 1) Dissolve 10 mM polysialic acid and 11 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide in water and stir at room temperature for 1 h.

[0049] 2) Add 11 mM 4-dimethylaminopyridine to the solution in step 1);

[0050] 3) Dissolve 10 mM poly(lactic-co-glycolic acid) in dimethyl sulfoxide (DMSO) and slowly inject the solution obtained in step 2) with stirring;

[0051] 4) The solution in step 3) was reacted at 40°C for 20 hours;

[0052] 5) Add 2 volumes of pre-cooled chloroform (temperature 5°C) and 1 volume of pre-cooled petroleum ether (temperature 5°C) to the mixture obtained in step 4), shake thoroughly to suspend, and let stand at room temperature for 5 minutes to allow the product to form a flocculent precipitate;

[0053] 6) Place the product obtained in step 5) in a high-speed refrigerated centrifuge and centrifuge at 4°C and 12,000 × g for 20 minutes, and remove the supernatant;

[0054] 7) Dissolve the precipitate obtained in step 6) in 3-5 mL of phosphate buffer (containing 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate, diluted to 1 L with deionized water; pH 8) and dialyze the precipitate against 100-300 volumes of phosphate buffer at room temperature for 2 days. The dialyzed product is lyophilized to obtain a polysialic acid-polylactic-co-glycolic acid intermediate compound;

[0055] 8) Take 10 mg of the product obtained in step 7) and add it to an aqueous solution containing 1% polyvinyl alcohol (PVA), and at the same time add 1 mg of S-methylisothiourea;

[0056] 9) Weigh 5 mg of tediluosin and 0.5 mg of cetyltrimethylammonium bromide and dissolve them in chloroform;

[0057] 10) mixing the solutions obtained in step 9) and step 10) and performing ultrasonic vibration to make them uniform;

[0058] 11) Stirring the solution obtained in step 10) at 0°C for 20 hours;

[0059] 12) placing the product obtained in step 12) in a high-speed refrigerated centrifuge and centrifuging at 4°C and 12,000 × g for 20 minutes, removing the supernatant;

[0060] 13) The product obtained in step 13) is washed with ultrapure water and freeze-dried to obtain drug-loaded nanoparticles (PSA@PLGA-TD / SMT).

[0061] The prepared polysialic acid-polylactic acid glycolic acid intermediate compound was subjected to nuclear magnetic resonance spectroscopy, and the prepared drug-loaded nanoparticles (PSA@PLGA-TD / SMT) were tested by Fourier transform infrared, transmission electron microscopy, hydrated particle size, zeta potential and UV-visible spectrophotometry. The results were as follows: Figure 1-5 shown.

[0062] from Figure 1 It can be seen that the polysialic acid-polylactic acid glycolic acid intermediate compound has characteristic hydrogens of both polysialic acid and polylactic acid glycolic acid. The results of hydrogen nuclear magnetic resonance spectrum show that the polysialic acid-polylactic acid glycolic acid intermediate compound was successfully synthesized.

[0063] from Figure 2It can be seen that polysialic acid (PSA) and polylactic-co-glycolic acid (PLGA) are connected through ester bonds. FT-IR results show that drug-loaded nanoparticles (PSA@PLGA-TD / SMT) are successfully synthesized.

[0064] from Figures 3A-3C It can be seen that the prepared PSA@PLGA is hollow spherical with good dispersibility, and the median of its particle size distribution is slightly lower than 100 nm; the prepared PSA@PLGA-TD particle size is slightly larger and presents a spherical morphology; the prepared PSA@PLGA-TD / SMT particle size distribution is more uniform. The results show that the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) of the present invention have good structural stability.

[0065] from Figure 4 It can be seen that the surface ζ potential value of the prepared drug-loaded nanoparticles (PSA@PLGA-TD / SMT) is relatively stable. The results show that the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) are stably dispersed in water.

[0066] from Figure 5 It can be seen that the prepared PSA@PLGA-TD and PSA@PLGA-TD / SMT have ultraviolet absorption peaks at 250-325nm, and there is also an ultraviolet absorption peak similar to TD when the wavelength is less than 250nm. The results further show that the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) are successfully synthesized.

[0067] The above results show that the present invention successfully synthesized drug-loaded nanoparticles (PSA@PLGA-TD / SMT) using tediluosin (TD) and S-methylisothiourea as raw materials, and the structure and performance of the drug-loaded nanoparticles are stable.

[0068] Example 2 Responsive release of drugs from drug-loaded nanoparticles (PSA@PLGA-TD / SMT)

[0069] In this example, the release behavior of drugs in drug-loaded nanoparticles (PSA@PLGA-TD / SMT) in neuraminidase target systems with different concentrations was studied.

[0070] Specifically, appropriate amounts of TD and PSA@PLGA-TD / SMT were weighed and dissolved in PBS buffer (pH 7.4) to a final concentration of 5 mg / ml (based on the final concentration of TD). The PSA@PLGA-TD / SMT solution was divided into four parts, and Glaseria suis (Gps) in the logarithmic growth phase was added to each part to make the final bacterial concentrations of 0, 10, and 20 mg / ml, respectively. 5 (Low), 10 7 (Medium), 10 9(High) CFU / mL. Take 1 mL of each of the above solutions and add them to a dialysis bag with a molecular weight cutoff of 1000. Place the dialysis bag in a clean 50 mL centrifuge tube containing PBS solution, place it in a constant temperature water bath shaker, and shake it in a water bath at 37°C and 200 r / min. Take 1 mL of sample between the dialysis bag and the centrifuge tube for testing at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 hours respectively, and immediately add 1 mL of isothermal PBS buffer after sampling. The method for detecting the drug content in the sample refers to the tediluosin detection method in the pharmacopoeia. Among them, the cumulative drug release percentage (%) after the solution volume changes is calculated as follows:

[0071]

[0072] Where, V 0 is the total volume of the drug release medium, which is 40 mL; V i Indicates at time node t i The volume of the sample taken at the time; C i (mg / L) is at the time point t i The concentration of the sample taken at the time C n (mg / L) t n The concentration of the sample taken at the time point; m drug (μg) is the initial drug content of the sample to be tested.

[0073] The results are as follows Figure 6 shown.

[0074] from Figure 6 It can be seen that after the addition of Grasseria suis (containing neuraminidase (NA) target), the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) release the drug to a certain extent, and as the concentration of Grasseria suis increases, the drug has a higher release rate; the results show that the drug-loaded nanoparticles in the present invention have the release characteristics of targeting neuraminidase.

[0075] Example 3 Regulation of NO content in cells by drug-loaded nanoparticles (PSA@PLGA-TD / SMT)

[0076] In this example, the regulation of NO content in cells by drug-loaded nanoparticles (PSA@PLGA-TD / SMT) was studied.

[0077] Specifically, RAW264.7 cells in the logarithmic phase with stable characteristics and good culture were resuspended in DMED medium containing 10% FBS (V / V) and evenly plated in a 96-well plate to a final cell concentration of 10 5 / mL, and cultured in a 37°C, 5% CO2 incubator for 12 hours. The cells were divided into seven groups. The first group was treated with PBS. In the second to seventh groups, each group of cells was first infected with a bacterial solution with an MOI of 100. Then, the third to seventh groups of cells were treated with TD, SMT, PSA@PLGA (its concentration was consistent with the PSA@PLGA-TD shell concentration), PSA@PLGA-TD, and PSA@PLGA-TD / SMT at a concentration of 32μg / mL (calculated as the final concentration of TD or SMT), respectively. Three biological replicates were set up in the group. After culturing for 2, 4, and 8 hours, 50μL of cell supernatant was taken for use. The samples were transferred to a new 96-well plate and the corresponding reagents were added using the method specified in the nitric oxide detection kit. The NO content in the cell supernatant of each well was measured at 540nm using an enzyme reader. The results are shown as follows. Figure 7 shown.

[0078] from Figure 7 It can be seen that after the cells are treated with the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) of the present invention, the NO content is low. The results show that the PSA@PLGA-TD / SMT can target cells infected with Grasseria suis (Gps) and release TD and SMT, killing Grasseria suis on the one hand, while SMT reduces the inflammatory response.

[0079] Example 4 Cellular Uptake of Drug-Loaded Nanoparticles (PSA@PLGA-TD / SMT)

[0080] In this example, the ability of cells to take up drug-loaded nanoparticles (PSA@PLGA-TD / SMT) was tested.

[0081] Specifically, each group of RAW264.7 cells was treated with 32 μg / mL (based on the final concentration of TD) of TD, PSA@PLGA-TD, and PSA@PLGA-TD / SMT, and three biological replicates were set up in each group. The culture medium was discarded at 1, 2, 3, 4, 5, and 6 hours for each group, and the cells were washed with PBS three times. Then, pure water was added and the cells were placed at -20°C, frozen and thawed three times, centrifuged at 12000 r / min, 4°C for 10 minutes, and the supernatant was taken for testing. The content detection method was based on the tediluosin detection method in the pharmacopoeia. The results are shown in the figure. Figure 8 shown.

[0082] from Figure 8 It can be seen that compared with TD, drug-loaded nanoparticles (PSA@PLGA-TD / SMT) are more easily taken up by cells.

[0083] Example 5 Cellular Uptake and Intracellular Localization of Drug-Loaded Nanoparticles (PSA@PLGA-TD / SMT)

[0084] In this example, the intracellular localization of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) after cellular uptake was studied.

[0085] Because TD does not autofluoresce, its fluorescence quenches quickly, and its luminescence intensity is low, it is necessary to use an easily detectable analog substitute (C6) for intracellular localization detection. C6 is a fluorescent dye commonly used for in vivo tracking of active molecule delivery systems and for studying cellular uptake and transport mechanisms.

[0086] Specifically, clean cell slides were placed in 24-well plates, and each group of cells was treated with PBS, 1 mg / mL C6, PSA@PLGA-C6, and PSA@PLGA-C6 / SMT (based on the final concentration of C6). The cell culture medium was discarded at 0.5 and 2 hours, respectively, and the cells were fixed with 4% PFA, counterstained with Dil and DAPI, and then mounted with anti-quenching mounting medium. The cells were examined under a fluorescence microscope. Figure 9 shown.

[0087] from Figure 9 As can be seen, fluorescence appears in the cells, and the results further show that cells can more efficiently absorb drug-loaded nanoparticles (PSA@PLGA-TD / SMT).

[0088] Example 6 Extracellular Antibacterial Effect of Drug-Loaded Nanoparticles (PSA@PLGA-TD / SMT)

[0089] In this example, the extracellular antibacterial ability of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) was studied.

[0090] Specifically, Glaseria suis (Gps) was inoculated into a 96-well plate to a final bacterial concentration of 10 5 CFU / mL, treated with TD, PSA@PLGA-TD, PSA@PLGA-TD / SMT gradient concentrations of 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, and cultured overnight. After treatment, the corresponding bacterial solution was diluted and coated on the plate, cultured at 37℃ for 24h, the number and distribution of colonies were observed, and the inhibition rate was calculated. The results are as follows Figure 10 and 11 shown.

[0091] from Figure 10 and 11 It can be seen that compared with the original drug TD, the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) in the present invention have a better killing effect on Glaseria suis.

[0092] Example 7 Regulation of Cellular Inflammation by Drug-Loaded Nanoparticles (PSA@PLGA-TD / SMT)

[0093] In this example, the regulation of cellular inflammation by drug-loaded nanoparticles (PSA@PLGA-TD / SMT) was studied.

[0094] Specifically, a sterile, polylysine-treated, clean cell slide was placed at the bottom of a well plate. The cell slides were divided into six groups. The first group was treated with PBS. Groups 2 through 6 were first infected with a bacterial solution at an MOI of 100. Then, cells in groups 3 through 6 were treated with TD, PSA@PLGA (the concentration corresponding to the PSA@PLGA-TD shell concentration), PSA@PLGA-TD, and PSA@PLGA-TD / SMT at a concentration of 32 μg / mL (based on the final TD or SMT concentration), respectively. Following treatment, the culture medium containing the drug and strain was discarded, and each well was washed with PBS until clean. The cells were then fixed with 4% PFA for 20 minutes at room temperature, washed with PBS until clean, and then permeabilized with a cell permeabilization solution containing 0.1% Triton X-100 for 10 minutes at room temperature. After further washing with PBS, the cells were blocked in 5% BSA in TBST for two hours. After further washing with PBS, add ARG1, NOS2, and COX2 primary antibodies and incubate at 4°C overnight. After further washing with PBS, add fluorescent secondary antibodies and incubate at room temperature in the dark for 2 hours. After further washing with PBS, add anti-fluorescence quenching mounting medium to seal the slides and perform fluorescence microscopy under a fluorescence microscope. The results are as follows Figure 12 shown.

[0095] from Figure 12 It can be seen that drug-loaded nanoparticles (PSA@PLGA-TD / SMT) can downregulate the expression of NOS2 and COX2 while upregulating the level of ARG1, which helps to resolve inflammation and repair cell tissues and organs.

[0096] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For any details not described in individual embodiments, reference may be made to the description in other embodiments.

[0097] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A drug-loaded nanoparticle targeting neuraminidase, characterized in that: The drug-loaded nanoparticles include polysialic acid and polylactic-co-glycolic acid; Wherein, the carboxyl group on the side chain of the polysialic acid is connected to the hydroxyl group in the polylactic acid glycolic acid through an ester bond; The drug-loaded nanoparticles further include an antibiotic drug, which is dispersed in the drug-loaded nanoparticles; the mass ratio of the antibiotic drug to the drug-loaded nanoparticles is (0.3-0.6):1, and the antibiotic drug is at least one of tedilosin, tilmicosin, clarithromycin, roxithromycin, and tylosin; The drug-loaded nanoparticles also include an inflammation regulator, which is adsorbed on the surface of the drug-loaded nanoparticles through electrostatic adsorption; the mass ratio of the inflammation regulator to the drug-loaded nanoparticles is (0.05~0.15):1, and the inflammation regulator is at least one of S-methylisothiourea, 1400W, and L-NMMA.

2. A pharmaceutical composition, characterized in that The drug-loaded nanoparticles targeting neuraminidase according to claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that Also included are pharmaceutically acceptable carriers.

4. A method for preparing drug-loaded nanoparticles targeting neuraminidase according to claim 1, characterized in that: The steps include: 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine are added to the polysialic acid aqueous solution to fully dissolve the mixture, and then a polylactic acid hydroxy organic solution is added to carry out a condensation reaction. The resulting product is purified to obtain a polysialic acid-polylactic acid glycolic acid intermediate compound; dissolving the polysialic acid-polylactic acid-glycolic acid intermediate compound and the inflammation regulator in water to obtain an aqueous solution; Dissolving antibiotics and cationic surfactants in an organic solvent to obtain an organic phase solution; The aqueous phase solution and the organic phase solution are mixed and ultrasonically shaken, and then stirred, and the resulting product is centrifuged, washed and freeze-dried to obtain drug-loaded nanoparticles; The cationic surfactant is cetyltrimethylammonium bromide.

5. The preparation method according to claim 4, characterized in that In the preparation process of the polysialic acid-polylactic acid glycolic acid intermediate compound, the concentration of the polysialic acid is 8~12mM, the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 9~13mM, and the concentration of the 4-dimethylaminopyridine is 9~13mM; the concentration of the polylactic acid hydroxyl group is 8~12mM; and the condensation reaction step specifically includes: reacting at a temperature of 35~45°C for 15~20h.

6. The preparation method according to claim 4, characterized in that The preparation process of the aqueous phase solution also includes the step of adding 0.5~2wt% polyvinyl alcohol to water; in the preparation process of the organic phase solution, the mass ratio of the antibiotic drug to the cationic surfactant is 1:(0.05~0.15), and the organic solvent includes chloroform.

7. The preparation method according to claim 4, characterized in that In the preparation process of the drug-loaded nanoparticles, the stirring treatment step specifically includes: stirring for 16 to 48 hours at a temperature of -1 to 1°C.

8. Use of the neuraminidase-targeting drug-loaded nanoparticles according to claim 1, the pharmaceutical composition according to any one of claims 2 to 3, or the neuraminidase-targeting drug-loaded nanoparticles prepared by the preparation method according to any one of claims 4 to 7 in the preparation of a drug for preventing and / or treating Glaseria suis infection.

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