pH / neuraminidase dual-responsive drug-loaded nanoparticles and their preparation method and application

Abstract

The present invention discloses a pH / neuraminidase dual-responsive drug-loaded nanoparticle, a preparation method, and an application thereof, belonging to the technical field of pharmaceutical preparations. The drug-loaded nanoparticle comprises polysialic acid and an amino acid, wherein the carboxyl group on the polysialic acid side chain is linked to the amino group in the amino acid via an amide bond. The present invention connects the polysialic acid and the amino acid via an amide bond to obtain a drug-loaded nanoparticle with a stable structure and performance. The drug-loaded nanoparticle has the function of targeting neuraminidase, while having low cytotoxicity and high cell entry efficiency. After an antibiotic drug is linked to the drug-loaded nanoparticle via an ester bond, the drug-loaded nanoparticle can deliver the antibiotic drug to the swine Grasseria in the pig's body, release the antibiotic drug in the slightly acidic environment of the infected site in the pig, and thus effectively kill the swine Grasseria. Therefore, the drug-loaded nanoparticle has good application prospects in the preparation of drugs for preventing and / or treating swine Grasseria infections.

Description

Technical Field

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

[0002] Due to the increasing scale and density of modern pig farming, infectious diseases in pigs are becoming increasingly common. Bacterial infection is a major problem in large-scale pig farms. The challenges of bacterial disease prevention and control are the multiple serotypes, poor targeted prevention and control effectiveness, conditional or environmental pathogenicity, persistent disease, and widespread drug resistance. Furthermore, the multi-serotype nature of bacteria, coupled with the frequent emergence of drug resistance and the complex clinical manifestations and epidemiological patterns, present challenges for clinical immunotherapy and prevention.

[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] Numerous studies have shown that drug-loaded nanoparticles can improve the metabolic kinetics of poorly soluble drugs and mask their toxicity, offering promising insights into treating drug-resistant bacterial infections and drug delivery. However, existing drug delivery systems have issues with poor targeting, stability, and low cell entry efficiency when delivering antibiotics used to kill GPS. Summary of the Invention

[0005] The present invention aims to provide pH / neuraminidase dual-responsive drug-loaded nanoparticles, their preparation method, and applications, to address the problems of poor targeting, poor stability, and low cell entry efficiency in existing drug delivery systems for delivering antibiotics used to kill GPS.

[0006] In a first aspect, the present invention provides a pH / neuraminidase dual-responsive drug-loaded nanoparticle, which comprises polysialic acid and amino acids, wherein the carboxyl group on the side chain of polysialic acid is connected to the amino group in the amino acid through an amide bond; wherein the amino acid is selected from at least one of glycine, alanine, lysine, methionine, phenylalanine, leucine or isoleucine.

[0007] In the drug-loaded nanoparticles provided by the present invention, polysialic acid is linked to amino acids via amide bonds, thereby making the structure and performance of the drug-loaded nanoparticles stable. The drug-loaded nanoparticles have the function of targeting neuraminidase, while having low cytotoxicity and faster efficiency of entering cells.

[0008] In some embodiments, the drug-loaded nanoparticles further include antibiotics, which contain alcoholic hydroxyl groups. The alcoholic hydroxyl groups in the antibiotics are connected to the carboxyl groups in the amino acids via ester bonds.

[0009] In this invention, antibiotics are linked to drug-loaded nanoparticles via ester bonds, resulting in a relatively stable structure and preventing 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.

[0010] In some embodiments, the antibiotic drug includes at least one of tedilosin, tilmicosin, tylosin, and florfenicol.

[0011] In a second aspect, the present invention provides a pharmaceutical composition comprising any one of the above-mentioned pH / neuraminidase dual-responsive drug-loaded nanoparticles.

[0012] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0013] 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.

[0014] In a third aspect, the present invention provides a method for preparing pH / neuraminidase dual-responsive drug-loaded nanoparticles as described above, comprising the following steps: mixing an Fmoc amino acid derivative with oxalyl chloride, performing an acyl chloride reaction under the catalysis of N,N-dimethylformamide to obtain an acyl chloride product; mixing the acyl chloride product with an antibiotic drug, performing an esterification reaction under the catalysis of triethylamine, adding an Fmoc removing agent to the obtained product to obtain an amino acid-antibiotic drug intermediate; adding 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysuccinimide to polysialic acid in sequence and incubating the mixture, then adding the amino acid-antibiotic drug intermediate to carry out a condensation reaction, and purifying the obtained product to obtain drug-loaded nanoparticles.

[0015] In the preparation method provided by the present invention, the acyl chloride reaction, esterification reaction and condensation reaction are all carried out in an organic solvent. After the reaction is completed, the step of removing the organic solvent by rotary evaporation is also included; the organic solvent in the acyl chloride reaction can be, for example, dichloromethane (DCM), the organic solvent in the esterification reaction can be, for example, dichloromethane (DCM), and the organic solvent in the condensation reaction can be, for example, dimethyl sulfoxide (DMSO).

[0016] In some preferred embodiments, 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, and drying the obtained precipitate to obtain drug-loaded nanoparticles.

[0017] In some more preferred embodiments, the organic solvent includes dichloromethane and petroleum ether; the centrifugation includes: centrifugation at a temperature of 0-4°C and a rotation speed of 12,000×g for 10-20 minutes, and repeating the centrifugation twice; and the drying temperature is 30-40°C.

[0018] In the present invention, the amounts of dichloromethane and petroleum ether can be conventionally adjusted according to actual use needs. The amount of dichloromethane 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.

[0019] In some embodiments, during the preparation of the acyl chloride product, the molar ratio of the Fmoc amino acid derivative to oxalyl chloride is 1:(3-5); the amount of N,N-dimethylformamide added accounts for 0.1-1% (V / V) of the volume of the acyl chloride reaction system; and the acyl chloride reaction step specifically includes: first reacting at a temperature of -1-1°C for 10-20 minutes; then reacting at a temperature of 20-25°C for 1-2 hours.

[0020] In some preferred embodiments, the Fmoc amino acid derivative is selected from at least one of Fmoc glycine, Fmoc alanine, and Fmoc lysine.

[0021] In some embodiments, during the preparation of the amino acid-antibiotic intermediate, the molar ratio of the antibiotic to the Fmoc amino acid derivative is (0.8-1.2):1; the amount of triethylamine added accounts for 1-2% (V / V) of the volume of the esterification reaction system; the esterification reaction step specifically includes: reacting at a temperature of 20-25° C. for 6-10 hours; the volume percentage of the Fmoc remover is 15-25%, and the Fmoc remover is selected from at least one of pyridine, piperidine, and diethylamine.

[0022] In some embodiments, during the preparation of drug-loaded nanoparticles, the molar ratio of polysaliva, 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride, N-hydroxysuccinimide and Fmoc amino acid derivative is (0.8~1.2):(0.8~1.5):(0.8~1.5):1; the incubation step specifically includes: after adding 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride, incubating at a temperature of 20~25°C for 0.5~1.5 hours; after continuing to add N-hydroxysuccinimide, incubating at a temperature of 20~25°C for 0.5~1.5 hours; the condensation reaction step specifically includes: reacting at a temperature of 45~60°C for 48~72 hours.

[0023] In a fourth aspect, the present invention provides the use of any of the above-mentioned pH / neuraminidase dual-responsive 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.

[0024] The beneficial effects of the present invention are as follows: unlike the prior art, the present invention connects polysialic acid and amino acids through amide bonds to obtain drug-loaded nanoparticles with stable structure and performance, and the drug-loaded nanoparticles have the function of targeting neuraminidase, while having low cytotoxicity and faster cell entry efficiency; further, after antibiotics are connected to the drug-loaded nanoparticles through ester bonds, the drug-loaded nanoparticles can deliver antibiotics to suis (targeting neuraminidase) in pigs, release antibiotics in the acidic environment of the infected site in the pig, and thus efficiently kill suis. Therefore, the drug-loaded nanoparticles have good application prospects in the preparation of drugs for preventing and / or treating suis suis infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention are 1 HNMR spectrum;

[0026] Figure 2FT-IR spectrum of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention;

[0027] Figure 3 TEM electron microscope image of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention;

[0028] Figure 4 : is the particle size distribution spectrum of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention;

[0029] Figure 5 zeta potential distribution spectrum of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention;

[0030] Figure 6 This is the UV-visible spectrophotometric spectrum of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention;

[0031] Figure 7 This is a graph of the responsive release of drugs from drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention;

[0032] Figure 8 This is a graph showing the results of cell uptake of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention, n=3. ;

[0033] Figure 9 This is a graph showing the cytotoxicity results of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention, n=3. ;

[0034] Figure 10 This is a diagram showing the extracellular antibacterial effect of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention;

[0035] Figure 11 This is a diagram showing the intracellular antibacterial effect of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] The present invention provides a pH / neuraminidase dual-responsive drug-loaded nanoparticle, which comprises polysialic acid, amino acids and antibiotic drugs, wherein the carboxyl group on the side chain of the polysialic acid is connected to the amino group in the amino acid through an amide bond; and the alcoholic hydroxyl group in the antibiotic drug is connected to the carboxyl group in the amino acid through an ester bond.

[0039] The preparation method of the drug-loaded nanoparticles comprises the following steps: mixing an Fmoc amino acid derivative with oxalyl chloride, performing an acyl chloride reaction under the catalysis of N,N-dimethylformamide to obtain an acyl chloride product; mixing the acyl chloride product with an antibiotic drug, performing an esterification reaction under the catalysis of triethylamine, adding an Fmoc removing agent to the obtained product to obtain an amino acid-antibiotic drug intermediate; sequentially adding 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysuccinimide to polysialic acid, followed by incubation, and then adding the amino acid-antibiotic drug intermediate to carry out a condensation reaction. After purifying the obtained product, the drug-loaded nanoparticles are obtained.

[0040] For example, drug-loaded nanoparticles were synthesized using Fmoc-Gly and tediluosin (TD) as raw materials.

[0041] Example 1 Synthesis and structural characterization of drug-loaded nanoparticles (PSA-Gly-TD)

[0042] A method for preparing pH / neuraminidase dual-responsive drug-loaded nanoparticles (PSA-Gly-TD) comprises the following steps:

[0043] 1) Add 1 mmol of Fmoc-Gly and 4 mmol of oxalyl chloride to 5 ml of dichloromethane, mix well, and place in an ice bath (temperature 0°C);

[0044] 2) Add 30 μL of N,N-dimethylformamide to the solution in step 1);

[0045] 3) The solution in step 2) was reacted at 0°C for 15 minutes and then at 25°C for 2 hours;

[0046] 4) collecting the reaction solution from step 3) and rotary evaporating until no liquid component remains;

[0047] 5) Add 5 ml of dichloromethane to the product in step 4) to redissolve it;

[0048] 6) Dissolve 1 mmol of TD in dichloromethane, add 80 μL of triethylamine and mix, then slowly inject the solution from step 5) while stirring;

[0049] 7) The solution in step 6) was reacted at 25°C for 10 hours;

[0050] 8) collecting the reaction liquid obtained in step 7) and removing the liquid component by rotary evaporation;

[0051] 9) Add an appropriate amount of dichloromethane to redissolve the solid product obtained in step 8), add 20% (v / v) piperidine, and react at 25°C for 20 hours;

[0052] 10) collecting the reaction liquid obtained in step 9), and removing the liquid component by rotary evaporation to obtain the reaction intermediate product Gly-TD;

[0053] 11) Dissolve 1 mmol of PSA (calculated as sialic acid monomers) in 10 ml of dimethyl sulfoxide;

[0054] 12) Add 1 mmol of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride to the solution in step 11) and incubate at 25°C for 1 hour. Then, add 1 mmol of N-hydroxysuccinimide and continue incubating at 25°C for 1 hour.

[0055] 13) The product Gly-TD obtained in step 10) was dissolved in 5 ml of dimethyl sulfoxide, and then added to the reaction solution obtained in step 12);

[0056] 14) The solution in step 13) was reacted at 50°C for 60 hours;

[0057] 15) Add 2 times the volume of dichloromethane and 1 times the volume of petroleum ether to the reaction product obtained in step 14), mix well, and form a flocculent precipitate;

[0058] 16) Place the product from step 15) in a high-speed refrigerated centrifuge and centrifuge at 4°C and 12,000 × g for 20 minutes, then remove the supernatant.

[0059] 17) Repeat step 16) twice to remove impurities;

[0060] 18) The product from step 17) was placed in a vacuum drying oven and dried at 35°C overnight to obtain drug-loaded nanoparticles (PSA-Gly-TD).

[0061] The prepared drug-loaded nanoparticles (PSA-Gly-TD) were tested by H NMR, FTIR, TEM, hydrated particle size, ζ potential and UV-visible spectrophotometry. The results were as follows: Figure 1-6 shown.

[0062] from Figure 1 It can be seen that the characteristic active hydrogen in the drug tediluosin (TD) appears on the drug-loaded nanoparticles (PSA-Gly-TD). The results of nuclear magnetic resonance hydrogen spectrum show that the drug-loaded nanoparticles (PSA-Gly-TD, PGT) are successfully synthesized.

[0063] from Figure 2 It can be seen that Fmoc-Gly is connected to tediluosin (TD) by an ester bond; the Gly-TD intermediate is connected to the PSA main chain by an amide bond, and the glycosidic bond structure of the neuraminidase responsive site in the main chain is not destroyed. The FT-IR results show that the drug-loaded nanoparticles (PSA-Gly-TD) are successfully synthesized.

[0064] from Figure 3 It can be seen that the prepared drug-loaded nanoparticles (PSA-Gly-TD) are spherical particles with good dispersibility.

[0065] from Figure 4 It can be seen that the particle size distribution of the prepared drug-loaded nanoparticles (PSA-Gly-TD) is around 65 nm, while the drug tediluosin (TD) is easy to agglomerate and the particle size distribution is uneven.

[0066] from Figure 5 It can be seen that the surface ζ potential value of the prepared drug-loaded nanoparticles (PSA-Gly-TD) is -32.45mV, and the particle dispersion stability threshold in the aqueous phase is ±30mV. The results show that the drug-loaded nanoparticles (PSA-Gly-TD) are stably dispersed in water.

[0067] from Figure 6 It can be seen that the UV absorption peak shape of the prepared drug-loaded nanoparticles (PSA-Gly-TD) is similar to that of the drug tediluosin (TD). The results further show that the drug-loaded nanoparticles contain TD.

[0068] The above results show that the present invention successfully synthesized drug-loaded nanoparticles (PSA-Gly-TD) using Fmoc-Gly, TD and PSA as raw materials, and the structure and performance of the drug-loaded nanoparticles are stable.

[0069] Example 2 Responsive release of drugs from drug-loaded nanoparticles (PSA-Gly-TD)

[0070] In this example, the release behavior of the drug in drug-loaded nanoparticles (PSA-Gly-TD) in different pH and neuraminidase target systems was studied.

[0071] Specifically, appropriate amounts of TD and PSA-Gly-TD were weighed and dissolved in PBS buffer at pH 5 and 7, respectively, to a final concentration of 5 mg / ml (based on the final concentration of TD). The above solutions were treated as follows: Group 1: PSA-Gly-TD solution (pH 7); Group 2: PSA-Gly-TD solution (pH 5, containing Glaseria suis (Gps), with a bacterial concentration of 10 9 CFU / mL); Group 3: PSA-Gly-TD solution (pH 7, containing Grasseria suis (Gps), bacterial concentration 10 9 CFU / mL); Group 4: TD solution (pH 7); Group 5: TD solution (pH 5, containing Grasseria suis (Gps), bacterial concentration 10 9 CFU / mL); the sixth group of TD solution (pH value is 7, containing Grasseria suis (Gps), bacterial concentration is 10 9 CFU / mL); 1 mL of each of the above solutions was taken and added to a dialysis bag with a molecular weight cutoff of 1000. The dialysis bag was placed in a clean 50 mL centrifuge tube containing PBS solution, placed in a constant temperature water bath shaker, and shaken in a water bath at 37°C and 200 r / min. 1 mL of sample was taken between the dialysis bag and the centrifuge tube at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h, respectively. 1 mL of isothermal PBS buffer was immediately added 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:

[0072]

[0073] 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.

[0074] The results are as follows Figure 7 shown.

[0075] from Figure 7 It can be seen that under neutral conditions, the drug content released by the drug-loaded nanoparticles (PSA-Gly-TD) is extremely low, but after adding Glaseria suis (containing neuraminidase (NA) target), the drug is released to a certain extent, and under acidic conditions, the drug has a higher release rate; the results show that the drug-loaded nanoparticles in the present invention have pH / neuraminidase dual-responsive targeted release characteristics.

[0076] Example 3 Cellular Uptake of Drug-Loaded Nanoparticles (PSA-Gly-TD)

[0077] This example tests the ability of cells to take up drug-loaded nanoparticles (PSA-Gly-TD).

[0078] Specifically, each group of RAW264.7 cells was treated with 32 μg / mL (based on the final concentration of TD) of TD and PSA-Gly-TD, and three biological replicates were set up in each group. The culture medium of each group was discarded at 2, 4, and 6 hours, 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, and centrifuged at 12000 r / min, 4°C, for 10 minutes. The supernatant was taken for testing. The content detection method was based on the tediluosin detection method in the pharmacopoeia. The results are as follows Figure 8 shown.

[0079] from Figure 8 It can be seen that drug-loaded nanoparticles (PSA-Gly-TD) are easily taken up by cells, which is beneficial to increase the intracellular drug concentration level and improve the bioavailability of the drug.

[0080] Example 4 Cytotoxicity of drug-loaded nanoparticles (PSA-Gly-TD)

[0081] In this example, the cytotoxicity of drug-loaded nanoparticles (PSA-Gly-TD) was tested.

[0082] Specifically, RAW264.7 cells grown to the logarithmic phase were cultured at a density of 5×10 4 / mL plate. Transfer the culture plate to a CO2 incubator and culture for 12 hours to allow the cells to settle evenly. After the incubation is completed, discard the culture medium in turn, set up the drug concentration gradient of the experimental group and the negative control. Inoculate into a 96-well plate at 100μL / well. Place the well plate in the incubator and continue incubating for 8 hours. After the incubation is completed, discard the surface drug-containing culture medium and wash with PBS. Add pre-prepared DMEM complete culture medium containing 10% (V / V) CCK-8 to each well, and set up three groups of wells without cells as blank controls. Incubate at 37°C for 1 hour, then use an enzyme reader to detect the absorbance at 450nm, and calculate the cell viability. The calculation formula is as follows:

[0083]

[0084] Where, A blank is the absorbance of CCK-8 solution without cells; A sample is the absorbance of CCK-8 incubation solution in each experimental group; A control is the absorbance of the CCK-8 incubation solution of the blank cell group without adding drugs.

[0085] The results are as follows Figure 9 shown.

[0086] from Figure 9 It can be seen that drug-loaded nanoparticles (PSA-Gly-TD) have low toxicity to cells.

[0087] Example 5 Extracellular Antibacterial Effect of Drug-Loaded Nanoparticles (PSA-Gly-TD)

[0088] In this example, the extracellular antibacterial ability of drug-loaded nanoparticles (PSA-Gly-TD) was tested.

[0089] Specifically, the pre-prepared 1280μg / mL tediluoxin standard stock solution and PSA-Gly-TD solution (calculated as an equivalent amount of TD original drug) were gradiently diluted with TSB broth medium to concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25μg / mL. Take a sterile 96-well plate and add 100μL of diluted drug to wells 1-10 respectively. Then add 100μL of spare bacterial suspension (wild-type swine Grasseria and resistant swine Grasseria) to each well. Only diluted drug was added to well 11 as a negative control, and only bacterial suspension was added to well 12 as a positive control. After marking, transfer to a CO2 incubator and culture for 24 hours before observing the results. The lowest concentration corresponding to the inhibition of bacterial reproduction in the well plate is the MIC value. The results are as follows: Figure 10 shown.

[0090] from Figure 10It can be seen that there is no significant difference in the extracellular inhibitory ability of drug-loaded nanoparticles (PSA-Gly-TD) and TD original drug against wild-type swine Grasseria and drug-resistant swine Grasseria.

[0091] Example 6 Intracellular antibacterial effect of drug-loaded nanoparticles (PSA-Gly-TD)

[0092] In this example, the intracellular antibacterial ability of drug-loaded nanoparticles (PSA-Gly-TD) was tested.

[0093] Specifically, RAW264.7 cells in the logarithmic growth phase were taken and diluted to 5×10 4 1 mL / well was inoculated into a 24-well plate and incubated in an incubator for 12 h to starve the cells and make them more susceptible to bacterial invasion. After the incubation, the culture medium was discarded and wild-type and drug-resistant Grassera suis were diluted to a concentration of 10% with DMEM containing 10% FBS (V / V), 0.2% NAD (V / V) and no double antibody. 6 CFU / mL, inoculate 1mL / well into a 24-well plate, so that the strain concentration: cell concentration = 100:1, incubate in the incubator for 2h, add TD and PSA-Gly-TD to a concentration of 32μg / mL, and continue incubation for 6h. After incubation, discard the culture medium containing bacteria and wash 3 times with PBS. Collect the cells, add 1mL of pure water, let it stand for 30min, wait for the cells to swell, dilute 100 times and spread on the plate, incubate at 37℃ for 24h, observe the number and distribution of colonies, and the results are as follows Figure 11 shown.

[0094] from Figure 11 It can be seen that compared with the original drug TD, at the same concentration, the drug-loaded nanoparticles have a better clearance rate for intracellular pathogens, and the drug-loaded nanoparticles have a better clearance effect on intracellular drug-resistant strains.

[0095] 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.

[0096] 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 pH / neuraminidase dual-responsive drug-loaded nanoparticle, characterized in that: The drug-loaded nanoparticles include polysialic acid and amino acids, and the carboxyl group on the side chain of the polysialic acid is connected to the amino group in the amino acid through an amide bond; wherein the amino acid is selected from at least one of glycine, alanine, leucine or isoleucine; The drug-loaded nanoparticles further include an antibiotic drug, wherein the antibiotic drug contains an alcoholic hydroxyl group, and the alcoholic hydroxyl group in the antibiotic drug is connected to the carboxyl group in the amino acid via an ester bond; The antibiotic drug is at least one of tedilosin, tilmicosin and tylosin.

2. A pharmaceutical composition, characterized in that It includes the pH / neuraminidase dual-responsive drug-loaded nanoparticles 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 pH / neuraminidase dual-responsive drug-loaded nanoparticles according to claim 1, characterized in that: The steps include: The Fmoc amino acid derivative is mixed with oxalyl chloride, and an acyl chloride reaction is carried out under the catalysis of N,N-dimethylformamide to obtain an acyl chloride product; The acyl chloride product is mixed with an antibiotic drug, and an esterification reaction is carried out under the catalysis of triethylamine. An Fmoc removal agent is added to the obtained product to obtain an amino acid-antibiotic drug intermediate; 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysuccinimide are sequentially added to the polysialic acid and incubated, and then the amino acid-antibiotic drug intermediate is added to carry out a condensation reaction. The obtained product is purified to obtain drug-loaded nanoparticles.

5. The preparation method according to claim 4, characterized in that In the preparation process of the acyl chloride product, the molar ratio of the Fmoc amino acid derivative to the oxalyl chloride is 1:(3-5); the amount of the N,N-dimethylformamide added accounts for 0.1-1% of the volume of the acyl chloride reaction system; The acyl chloride reaction step specifically includes: first reacting at a temperature of -1 to 1°C for 10 to 20 minutes; then reacting at a temperature of 20 to 25°C for 1 to 2 hours.

6. The preparation method according to claim 4, characterized in that In the preparation process of the amino acid-antibiotic drug intermediate, the molar ratio of the antibiotic drug to the Fmoc amino acid derivative is (0.8-1.2):1; the amount of triethylamine added accounts for 1-2% of the volume of the esterification reaction system; The esterification reaction step specifically comprises: reacting at a temperature of 20-25° C. for 6-10 hours; The volume percentage of the Fmoc removing agent is 15-25%, and the Fmoc removing agent is selected from at least one of pyridine, piperidine and diethylamine.

7. The preparation method according to claim 4, characterized in that In the preparation process of the drug-loaded nanoparticles, the molar ratio of the polysialic acid, the 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride, the N-hydroxysuccinimide and the Fmoc amino acid derivative is (0.8-1.2): (0.8-1.5): (0.8-1.5): 1; The incubation step specifically includes: after adding the 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride, incubating at a temperature of 20-25° C. for 0.5-1.5 hours; after continuing to add the N-hydroxysuccinimide, incubating at a temperature of 20-25° C. for 0.5-1.5 hours; The condensation reaction step specifically includes: reacting at a temperature of 45-60° C. for 48-72 hours.

8. Use of the pH / neuraminidase dual-responsive drug-loaded nanoparticles according to claim 1, the pharmaceutical composition according to any one of claims 2 to 3, or the pH / neuraminidase dual-responsive 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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