Neuraminidase-targeted drug-loaded nanoparticles as well as preparation method and application of neuraminidase-targeted drug-loaded nanoparticles

Abstract

The invention discloses a neuraminidase-targeted drug-loaded nanoparticle as well as a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. Polysialic acid and poly (lactic-co-glycolic acid) are connected through ester bond connection to obtain the drug-loaded nanoparticles with stable structure and performance, and the drug-loaded nanoparticles have the effect of targeting neuraminidase and also have high efficiency of entering cells; the antibiotic drug is further dispersed in the drug-loaded nanoparticles, the inflammation regulating agent is adsorbed on the surface of the drug-loaded nanoparticles, and the drug-loaded nanoparticles can deliver the antibiotic drug and the inflammation regulating agent to the pig Gleisseria in the pig body. According to the present invention, the antibiotic drug and the inflammation regulating agent are released under the acidic environment at the infection site in the pig body, such that the pig Gleisseria is efficiently killed, the inflammation damage caused by bacterial infection and the toxic damage of the antibiotic drug are reduced, and the good application prospect is provided in the preparation of the drug for preventing and / or treating the pig Gleisseria infection.

Description

Technical Field

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

[0002] Under large-scale breeding conditions, bacterial infection is one of the main causes of morbidity and mortality in livestock and poultry, causing serious economic losses. The widespread use of antibiotics has significantly reduced the infection rate and mortality rate of livestock and poultry, but long-term abuse has led to bacterial resistance and residues in livestock products, limiting their application prospects.

[0003] Glaesserella parasuis (Gps) is one of the common respiratory flora in farms and is widely present in pigs at all stages. Swine Glaesser's disease usually occurs 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 can be isolated from pigs. The main serotypes of Gps prevalent in my country are type 4 and type 5 highly virulent strains. The morbidity and mortality rates of the disease are between 5% and 10%, and the morbidity rate of latently infected pigs can be as high as 75%.

[0004] As an effector molecule of the immune system, nitric oxide is mainly derived from the L-arginine metabolic pathway mediated by nitric oxide synthase (NOS), and its key rate-limiting step is the step in which arginase (ARG) competes with NOS to bind arginine. In pathological reactions, the produced nitric oxide has certain negative effects on the host, including immune rejection, neurodegenerative effects, and septic shock. Taking GPS as an example, its infection can significantly induce the production of NO by porcine alveolar macrophages. At the same time, bacterial activation of NF-κB signaling mediates the upregulation of NOS2 and promotes the production of NO, which plays an important role in amplifying the inflammatory response. For example, NOS2 / NO is highly correlated with the development of lung inflammation. When external pathogens repeatedly stimulate airway epithelial cells, they will prompt epithelial cells to secrete a large number of proinflammatory factors. High levels of inflammatory cytokines will aggravate the chronic damage and malignant regeneration of other cells in the lungs, and a large amount of fibrinogen will infiltrate from the alveoli, thereby forming lung pathological changes such as lung tissue fibrosis.

[0005] Responsive nano-drug delivery systems are a type of nanoparticles that achieve intelligent controlled release based on drastic changes in external physical and chemical factors. They usually have the characteristics of precise drug release and high treatment compliance. This type of drug delivery platform will only increase the drug concentration when it reaches the lesion site, avoiding excessive toxicity and premature failure caused by early release. Therapeutic strategies based on responsive nano-carriers have been widely used in bacterial infections, and their types include pH-sensitive, redox, enzyme-responsive, temperature-responsive, and strong external stimulus-responsive.

[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 purpose of the present invention is to provide a drug-loaded nanoparticle targeting neuraminidase and a preparation method and application thereof, so as to solve the problems of poor targeting, poor stability and low efficiency of entering cells when delivering antibiotics and inflammatory regulators used to kill GPS in existing drug delivery systems.

[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 the polysialic acid is connected to the hydroxyl group in the 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, so that the structure and performance of the drug-loaded nanoparticles are stable, and 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 antibiotic drugs, which are dispersed in the drug-loaded nanoparticles; the mass ratio of the antibiotic drugs to the drug-loaded nanoparticles is (0.3~0.6):1, and the antibiotic drugs include at least one of tedilosin, tilmicosin, clarithromycin, roxithromycin and tylosin.

[0011] In the present invention, antibiotic drugs are dispersed in drug-loaded nanoparticles to avoid drug loss during delivery. Since neuraminidase is a potential bacterial virulence factor, it has been confirmed to be expressed in Glaseria suis and participate in the decomposition of sialic acid. Therefore, the drug-loaded nanoparticles can deliver antibiotic drugs to Glaseria suis in the pig body (targeting neuraminidase), release antibiotic drugs in the acidic environment of the infected part of the pig body, and effectively kill Glaseria suis.

[0012] In some embodiments, the drug-loaded nanoparticles also 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 isothiocarboxylate.

[0013] In the present invention, the 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 antibiotic drugs.

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

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

[0016] In the present invention, 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 agents and absorption delaying agents that are compatible with drug administration. Each carrier must be "acceptable", meaning compatible with the other ingredients of the composition and harmless 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 to fully dissolve them, and then adding a polylactic acid hydroxy organic solution for condensation reaction. After purifying the obtained product, a polysialic acid-polylactic acid glycolic acid intermediate compound is obtained; the polysialic acid-polylactic acid glycolic acid intermediate compound and an inflammatory regulator are dissolved in water to obtain an aqueous solution; an antibiotic drug and a cationic surfactant are dissolved in an organic solvent to obtain an organic solution; the aqueous solution and the organic solution are mixed and ultrasonically vibrated, and then stirred, and the obtained product is centrifuged, washed and freeze-dried 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 emulsified co-solvent volatilization method, and then, under the action of a cationic surfactant, an inflammation regulator is adsorbed onto the surface of the polysialic acid-polylactic acid-glycolic acid intermediate compound by electrostatic adsorption to obtain spherical drug-loaded nanoparticles with uniform particle size distribution and stable structural properties.

[0019] In some preferred embodiments, in the preparation process 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 a phosphate buffer solution 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 for 15 to 25 minutes at a temperature of 0 to 4°C and a rotation speed of 12,000×g; and the dialysis time is 1 to 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 also 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 for 16 to 48 hours at a temperature of -1 to 1°C.

[0025] In a fourth aspect, the present invention provides the use of any of the above-mentioned drug-loaded nanoparticles targeting neuraminidase, any of the above-mentioned pharmaceutical compositions, or drug-loaded nanoparticles targeting neuraminidase prepared by any of the above-mentioned preparation methods in the preparation of drugs for preventing and / or treating swine Glaseria 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 inflammatory regulators are adsorbed on the surface of the drug-loaded nanoparticles, and the drug-loaded nanoparticles can deliver antibiotics and inflammatory regulators to Glaseria suis (targeting neuraminidase) in pigs, and release antibiotics and inflammatory regulators in the acidic environment of the infected part of the pig, thereby efficiently killing Glaseria suis and reducing the inflammatory damage caused by bacterial infection and the toxic damage of antibiotics. Therefore, the drug-loaded nanoparticles have good application prospects in the preparation of drugs for preventing and / or treating Glaseria 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; Figure 2 FT-IR spectrum of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention; Figure 3A TEM electron microscope and particle size distribution diagram of the polysialic acid-polylactic acid-glycolic acid intermediate compound prepared in Example 1 of the present invention; Figure 3B TEM electron microscope and particle size distribution diagram of the drug-loaded nanoparticles (PSA@PLGA-TD) prepared in Example 1 of the present invention; Figure 3C TEM electron microscope and particle size distribution diagram of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention; Figure 4 This is the zeta potential distribution spectrum of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention; 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; Figure 6 This is a responsive release diagram of the drug in the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention; Figure 7This is a graph showing the change in NO content when the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention treat cells, n=3, ; 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, ; Fig. 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; Fig.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, ; Fig.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; Fig.12 This is a fluorescence image of the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) prepared in Example 1 of the present invention regulating cell inflammation. DETAILED DESCRIPTION

[0028] 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 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.

[0029] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0030] The present invention provides a drug-loaded nanoparticle targeting neuraminidase, wherein the drug-loaded nanoparticle comprises polysialic acid, polylactic acid 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 acid 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.

[0031] 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 to fully dissolve 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 performing a stirring treatment, and the obtained product is centrifuged, washed and freeze-dried to obtain the drug-loaded nanoparticles.

[0032] By way of example, the drug-loaded nanoparticles are synthesized using tediloxine (TD) and S-methylisothiourea (SMT) as raw materials.

[0033] Example 1 Synthesis and structural characterization of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) A method for preparing drug-loaded nanoparticles (PSA@PLGA-TD / SMT) targeting neuraminidase comprises the following steps: 1) Dissolve 10 mM polysialic acid and 11 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide in water and stir at room temperature for 1 h; 2) adding 11 mM 4-dimethylaminopyridine to the solution in step 1); 3) Dissolve 10 mM poly(lactic-co-glycolic acid) in dimethyl sulfoxide (DMSO), slowly inject the solution obtained in step 2), and stir; 4) reacting the solution in step 3) at 40°C for 20 hours; 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 well to suspend, and let stand at room temperature for 5 minutes to allow the product to produce flocculent precipitation; 6) placing the product obtained in step 5) in a high-speed refrigerated centrifuge, centrifuging at 4°C and 12,000×g for 20 minutes, and removing the supernatant; 7) Take 3-5 mL of phosphate buffer (containing 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate, and dilute to 1 L with deionized water; pH is 8) to dissolve the precipitate obtained in step 6), and dialyze for 2 days at room temperature using 100-300 times the volume of phosphate buffer. After the dialyzed product is freeze-dried, a polysialic acid-polylactic acid glycolic acid intermediate compound is obtained; 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; 9) Weigh 5 mg of tediluosin and 0.5 mg of cetyltrimethylammonium bromide respectively and dissolve them in chloroform; 10) mixing the solutions obtained in step 9) and step 10), and performing ultrasonic vibration to make them uniform; 11) Stirring the solution obtained in step 10) at 0°C for 20 hours; 12) placing the product obtained in step 12) in a high-speed refrigerated centrifuge, centrifuging at 4°C and 12,000×g for 20 minutes, and removing the supernatant; 13) The product obtained in step 13) is washed with ultrapure water and freeze-dried to obtain drug-loaded nanoparticles (PSA@PLGA-TD / SMT).

[0034] The prepared polysialic acid-polylactic acid-glycolic acid intermediate compound was subjected to nuclear magnetic resonance hydrogen spectrum, 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.

[0035] 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.

[0036] from Figure 2 It can be seen that polysialic acid (PSA) and polylactic-co-glycolic acid (PLGA) are connected by ester bonds, and FT-IR results show that drug-loaded nanoparticles (PSA@PLGA-TD / SMT) are successfully synthesized.

[0037] 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 100nm; 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.

[0038] from Figure 4It 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.

[0039] from Figure 5 It can be seen that the prepared PSA@PLGA-TD and PSA@PLGA-TD / SMT show 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.

[0040] 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.

[0041] Example 2 Responsive release of drugs from drug-loaded nanoparticles (PSA@PLGA-TD / SMT) In this example, the release behavior of the drug in the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) in the neuraminidase target system with different concentrations was studied.

[0042] 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 portions, and Glasella suis (Gps) in the logarithmic growth phase was added to each portion to a final concentration of 0, 10, and 20 mg / ml, respectively. 5 (Low), 10 7 (Medium), 10 9 (High) CFU / mL. Take 1mL 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 50mL 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 200r / min. Take 1mL of samples between the dialysis bag and the centrifuge tube for testing at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24h, respectively, and immediately add 1mL of isothermal PBS buffer after sampling. The method for detecting the drug content in the sample refers to the detection method of tediluosin in the pharmacopoeia. Among them, the cumulative drug release percentage (%) after the solution volume changes is calculated as follows:

[0043] In the formula, V 0 is the total volume of the drug release medium, and its value is 40 mL; Vi Indicates at time node t i The volume of the sample taken at the time; C i (mg / L) is 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.

[0044] The results are as follows Figure 6 shown.

[0045] from Figure 6 It can be seen that after adding 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.

[0046] Example 3 Regulation of NO content in cells by drug-loaded nanoparticles (PSA@PLGA-TD / SMT) In this example, the regulation of NO content in cells by drug-loaded nanoparticles (PSA@PLGA-TD / SMT) was studied.

[0047] 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 then 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 12h. 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, and 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 (based on the final concentration of TD or SMT). Three biological replicates were set up in the group, and 50μL of cell supernatant was taken after culturing for 2, 4, and 8h, respectively. 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.

[0048] from Figure 7 It can be seen that after the drug-loaded nanoparticles (PSA@PLGA-TD / SMT) in the present invention are used to treat cells, the NO content is low. The results show that PSA@PLGA-TD / SMT can target cells infected with Glaseria suis (Gps) and release TD and SMT, which can kill Glaseria suis while SMT reduces the inflammatory response.

[0049] Example 4 Cellular uptake of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) In this example, the ability of cells to take up drug-loaded nanoparticles (PSA@PLGA-TD / SMT) was tested.

[0050] 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 h in each group, and the cells were washed with PBS three times, then added with pure water and placed at -20°C, and repeatedly frozen and thawed three times. The cells were centrifuged at 12000 r / min and 4°C for 10 min, and the supernatant was taken for testing. The content detection method was based on the detection method of tediluosin in the pharmacopoeia. The results are shown in the figure. Figure 8 shown.

[0051] 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.

[0052] Example 5 Intracellular localization of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) after cellular uptake In this example, the intracellular localization of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) after cellular uptake was studied.

[0053] Since TD does not spontaneously fluoresce, has a fast fluorescence quenching, and has a low luminescence intensity, it is necessary to use an easily detectable analog substitute (C6) for intracellular localization detection. C6 is a fluorescent dye that is commonly used for in vivo tracking of active molecule drug delivery systems, and the study of cellular uptake and transport mechanisms.

[0054] 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 sealed with anti-quenching mounting media, and examined under a fluorescence microscope. The results are shown in Fig. 9 shown.

[0055] from Fig. 9 As can be seen, fluorescence appears in the cells, and the results further show that cells can take up drug-loaded nanoparticles (PSA@PLGA-TD / SMT) more efficiently.

[0056] Example 6 Extracellular antibacterial activity of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) In this example, the extracellular antibacterial ability of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) was studied.

[0057] Specifically, Glaseria porcine (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 shown in Fig.10 and 11 shown.

[0058] from Fig.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.

[0059] Example 7 Regulation of Cellular Inflammation by Drug-Loaded Nanoparticles (PSA@PLGA-TD / SMT) In this example, the cell inflammation regulation of drug-loaded nanoparticles (PSA@PLGA-TD / SMT) was studied.

[0060] Specifically, a clean cell slide treated with sterile polylysine was placed at the bottom of the well plate in advance. The cell slides were divided into six groups. The first group was treated with PBS. In the second to sixth groups, each group of cells was first infected with a bacterial solution with an MOI of 100, and then the third to sixth groups of cells were treated with TD, PSA@PLGA (the concentration was consistent with the shell concentration of PSA@PLGA-TD), PSA@PLGA-TD, and PSA@PLGA-TD / SMT at a concentration of 32 μg / mL (based on the final concentration of TD or SMT). After the treatment, the culture medium containing drugs and strains was discarded, and each well of cells was washed with PBS buffer until clean. Switch to 4% PFA to fix the cells at room temperature for 20 minutes. After the end, wash with PBS until clean, and then add a cell permeabilization solution containing 0.1% Triton X-100 to permeabilize at room temperature for 10 minutes. Continue to wash with PBS and block in 5% BSA TBST solution for two hours. After washing with PBS, add ARG1, NOS2, COX2 primary antibodies and incubate at 4°C overnight. After washing with PBS, add fluorescent secondary antibodies and incubate at room temperature in the dark for 2 hours. After washing with PBS, add anti-fluorescence quenching sealing agent to seal the slices, and use fluorescence microscope for fluorescence microscopy. The results are as follows Fig.12 shown.

[0061] from Fig.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 subside inflammation and repair cell tissues and organs.

[0062] 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 matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0063] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A drug-loaded nanoparticle targeting neuraminidase, characterized in that: The drug-loaded nanoparticles include polysialic acid and polylactic-co-glycolic acid; 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.

2. The drug-loaded nanoparticles targeting neuraminidase according to claim 1, characterized in that: The drug-loaded nanoparticles also include antibiotic drugs, which are dispersed in the drug-loaded nanoparticles; the mass ratio of the antibiotic drugs to the drug-loaded nanoparticles is (0.3-0.6):1, and the antibiotic drugs include at least one of tedilosin, tilmicosin, clarithromycin, roxithromycin and tylosin.

3. The drug-loaded nanoparticles targeting neuraminidase according to claim 2, characterized in that: The drug-loaded nanoparticles also include an inflammatory regulator, which is adsorbed on the surface of the drug-loaded nanoparticles through 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 isothiocarboxylate.

4. A pharmaceutical composition, characterized in that The drug-loaded nanoparticles targeting neuraminidase according to any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, characterized in that Also included are pharmaceutically acceptable carriers.

6. A method for preparing drug-loaded nanoparticles targeting neuraminidase according to any one of claims 1 to 3, characterized in that: The steps include: Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine to the polysialic acid aqueous solution to fully dissolve the polysialic acid, then adding the polylactic acid hydroxy organic solution to carry out a condensation reaction, and purifying the obtained product to obtain a polysialic acid-polylactic acid glycolic acid intermediate compound; dissolving the polysialic acid-polylactic-co-glycolic acid intermediate compound and the inflammation regulator in water to obtain an aqueous phase solution; Dissolving antibiotic drugs 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 subjected to ultrasonic oscillation, and then stirred. The obtained product is centrifuged, washed and freeze-dried to obtain drug-loaded nanoparticles.

7. The preparation method according to claim 6, 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 is 8~12mM; and the condensation reaction step specifically includes: reacting for 15~20h at a temperature of 35~45°C.

8. The preparation method according to claim 6, 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 cationic surfactant includes hexadecyltrimethylammonium bromide, and the organic solvent includes chloroform.

9. The preparation method according to claim 6, 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.

10. Use of the drug-loaded nanoparticles targeting neuraminidase according to any one of claims 1 to 3, the pharmaceutical composition according to any one of claims 4 to 5, or the drug-loaded nanoparticles targeting neuraminidase prepared by the preparation method according to any one of claims 6 to 9 in the preparation of drugs for preventing and / or treating infection with Glaseria suis.

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