PH / neuraminidase dual-response drug-loaded nanoparticles as well as preparation method and application of pH / neuraminidase dual-response drug-loaded nanoparticles

Abstract

The invention discloses pH / neuraminidase dual-response drug-loaded nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The drug-loaded nanoparticle comprises polysialic acid and amino acid, wherein carboxyl on a side chain of the polysialic acid is connected with amino in the amino acid through an amido bond; polysialic acid and amino acid are connected through an amido bond 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 low cytotoxicity and high efficiency of entering cells; according to the present invention, after the antibiotic drug is connected to the drug-loaded nanoparticle through the ester bond, the drug-loaded nanoparticle can deliver the antibiotic drug to the pig Gleisseria in the pig body, and the antibiotic drug is released in the acidic environment at the infection site in the pig body so as to efficiently kill the pig Gleisseria, such that the drug-loaded nanoparticle can be used for preparing the drug-loaded nanoparticle, the compound has a good application prospect in preparation of drugs for preventing and / or treating swine Gleisseria infection.

Description

Technical Field

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

[0002] Due to the increase in the scale of production and stocking density of modern pig farming, pig infectious diseases are becoming more and more frequent. Bacterial infection is a major chronic disease in large-scale pig farms. The difficulty in preventing and controlling bacterial diseases is that there are many serotypes, the targeted prevention and control effect is poor, conditional or environmental pathogens, persistent pathogens, and strong general drug resistance. In addition, because bacteria have multiple serotypes, drug resistance is frequent, and clinical manifestations and epidemic situations are complicated, which brings difficulties to clinical immune prevention and control.

[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] At present, many studies have shown that nanoparticles can improve the metabolic kinetics of poorly soluble drugs and mask drug toxicity, which has positive significance for the treatment of drug-resistant bacterial infections and drug delivery. However, the existing drug delivery systems have problems such as poor targeting, poor stability and low efficiency in entering cells when delivering antibiotics used to kill GPS. Summary of the invention

[0005] The purpose of the present invention is to provide a pH / neuraminidase dual-responsive drug-loaded nanoparticle and a preparation method and application thereof, which are used to solve the problems of poor targeting, poor stability and low efficiency of entering cells when delivering antibiotic drugs used to kill GPS in existing drug delivery systems.

[0006] In the first aspect, the present invention provides a pH / neuraminidase dual-responsive drug-loaded nanoparticle, the drug-loaded nanoparticle comprising polysialic acid and amino acids, wherein the carboxyl group on the side chain of the polysialic acid is connected to the amino group in the amino acid via 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 connected to amino acids through amide bonds, 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 lower cytotoxicity and faster efficiency of entering cells.

[0008] In some embodiments, the drug-loaded nanoparticles further include antibiotic drugs, the antibiotic drugs contain alcoholic hydroxyl groups, and the alcoholic hydroxyl groups in the antibiotic drugs are connected to the carboxyl groups in the amino acids through ester bonds.

[0009] In the present invention, antibiotic drugs are connected to drug-loaded nanoparticles through ester bonds, and the structure is relatively stable, which prevents 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), and release antibiotic drugs in the acidic environment of the infected site in the pig body, thereby efficiently 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 includes a pharmaceutically acceptable carrier.

[0013] 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.

[0014] In a third aspect, the present invention provides a method for preparing any of the pH / neuraminidase dual-responsive drug-loaded nanoparticles as described above, comprising the following steps: mixing an Fmoc amino acid derivative with oxalyl chloride, and 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, and performing an esterification reaction under the catalysis of triethylamine, and 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 for 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, the esterification reaction and the 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: centrifuging for 10-20 min at a temperature of 0-4°C and a rotation speed of 12000×g, repeating the centrifugation twice; and the drying temperature is 30-40°C.

[0018] In the present invention, the amount of dichloromethane and petroleum ether can be conventionally adjusted according to actual use needs. The amount of dichloromethane can be, for example, 2 times the volume of the product, and the amount of petroleum ether 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; 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, 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% (V / V) of the volume of the esterification reaction system; the esterification reaction step specifically includes: reacting for 6~10 hours at a temperature of 20~25°C; 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 for 0.5~1.5h at a temperature of 20~25°C; after continuing to add N-hydroxysuccinimide, incubating for 0.5~1.5h at a temperature of 20~25°C; the condensation reaction step specifically includes: reacting at a temperature of 45~60°C for 48~72h.

[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: different from 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, and at the same time have lower cytotoxicity and faster efficiency of entering cells; further, after antibiotics are connected to the drug-loaded nanoparticles through ester bonds, the drug-loaded nanoparticles can deliver antibiotics to Glaseria suis (targeting neuraminidase) in pigs, and release antibiotics in the acidic environment of the infected site in the pig, thereby efficiently killing Glaseria suis. Therefore, the drug-loaded nanoparticles have good application prospects in the preparation of drugs for preventing and / or treating Glaseria 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; Figure 2FT-IR spectrum of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention; Figure 3 This is a TEM electron microscope image of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention; Figure 4 This is the particle size distribution spectrum of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention; Figure 5 is the zeta potential distribution spectrum of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention; Figure 6 is the UV-visible spectrophotometric spectrum of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention; Figure 7 This is a responsive release diagram of the drug in the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention; Figure 8 This is a result diagram of the cell uptake of the drug-loaded nanoparticles (PSA-Gly-TD) prepared in Example 1 of the present invention, n=3, ; Fig. 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, ; Fig.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; Fig.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

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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, incubating, then adding the amino acid-antibiotic drug intermediate to carry out a condensation reaction, and purifying the obtained product to obtain the drug-loaded nanoparticles.

[0030] Exemplarily, drug-loaded nanoparticles are synthesized using Fmoc-Gly and tediluosin (TD) drugs as raw materials.

[0031] Example 1 Synthesis and structural characterization of drug-loaded nanoparticles (PSA-Gly-TD) A method for preparing pH / neuraminidase dual-responsive drug-loaded nanoparticles (PSA-Gly-TD) comprises the following steps: 1) Take 5 ml of dichloromethane, add 1 mmol of Fmoc-Gly and 4 mmol of oxalyl chloride, mix well, and place in an ice bath (temperature is 0°C); 2) Add 30 μL of N,N-dimethylformamide to the solution in step 1); 3) The solution in step 2) was reacted at 0°C for 15 minutes and then at 25°C for 2 hours; 4) collecting the reaction solution in step 3) and rotary evaporating until there is no liquid component; 5) Add 5 ml of dichloromethane to the product in step 4) to redissolve it; 6) Dissolve 1 mmol TD in dichloromethane, add 80 μL triethylamine and mix, then slowly inject the solution in step 5) while maintaining stirring; 7) reacting the solution in step 6) at 25°C for 10 hours; 8) collecting the reaction liquid obtained in step 7), and removing the liquid component by rotary evaporation; 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; 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; 11) Dissolve 1 mmol PSA (based on sialic acid monomer) in 10 ml dimethyl sulfoxide; 12) Add 1 mmol of 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride to the solution in step 11), incubate at 25°C for 1 hour, then add 1 mmol of N-hydroxysuccinimide, and continue incubating at 25°C for 1 hour; 13) Dissolve the product Gly-TD obtained in step 10) in 5 ml of dimethyl sulfoxide, and then add it to the reaction solution obtained in step 12); 14) reacting the solution in step 13) at 50°C for 60 hours; 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 make the product produce flocculent precipitation; 16) placing the product in step 15) in a high-speed refrigerated centrifuge, centrifuging at 4°C and 12,000×g for 20 min, and removing the supernatant; 17) Repeat step 16) twice to remove impurities; 18) The product in step 17) was placed in a vacuum drying oven and dried at 35° C. overnight to obtain drug-loaded nanoparticles (PSA-Gly-TD).

[0032] The prepared drug-loaded nanoparticles (PSA-Gly-TD) were tested by H NMR, FT-IR, TEM, hydrated particle size, ζ potential and UV-visible spectrophotometry. The results were as follows: Figure 1-6 shown.

[0033] 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) were successfully synthesized.

[0034] 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.

[0035] from Figure 3It can be seen that the prepared drug-loaded nanoparticles (PSA-Gly-TD) are spherical particles and have good dispersibility.

[0036] 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.

[0037] from Figure 5 It can be seen that the surface ζ potential value of the prepared drug-loaded nanoparticles (PSA-Gly-TD) is -32.45 mV, and the particle dispersion stability threshold in the aqueous phase is ±30 mV. The results show that the drug-loaded nanoparticles (PSA-Gly-TD) are stably dispersed in water.

[0038] from Figure 6 It can be seen that the prepared drug-loaded nanoparticles (PSA-Gly-TD) have a similar UV absorption peak shape to that of the drug tediluosin (TD). The results further show that the drug-loaded nanoparticles contain TD.

[0039] 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.

[0040] Example 2 Responsive release of drugs from drug-loaded nanoparticles (PSA-Gly-TD) 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.

[0041] Specifically, appropriate amounts of TD and PSA-Gly-TD were weighed and dissolved in PBS buffers with pH values ​​of 5 and 7, respectively, to make the 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 value of 7); Group 2: PSA-Gly-TD solution (pH value of 5, containing Glasella suis (Gps), bacterial concentration of 10 9 CFU / mL); Group 3: PSA-Gly-TD solution (pH 7, containing Glasella suis (Gps), bacterial concentration 10 9 CFU / mL); Group 4: TD solution (pH 7); Group 5: TD solution (pH 5, containing Glasella suis (Gps), bacterial concentration 10 9 CFU / mL); the sixth group of TD solution (pH 7, containing Grassella suis (Gps), bacterial concentration 10 9CFU / mL); Take 1mL of each of the above solutions and add it 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. And take 1mL of samples between the dialysis bag and the centrifuge tube 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:

[0042] In the formula, V 0 is the total volume of the drug release medium, and its value 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 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.

[0043] The results are as follows Figure 7 shown.

[0044] 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, and 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.

[0045] Example 3 Cellular Uptake of Drug-Loaded Nanoparticles (PSA-Gly-TD) In this example, the ability of cells to take up drug-loaded nanoparticles (PSA-Gly-TD) was tested.

[0046] 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 was discarded at 2, 4, and 6 hours in each group, and PBS was washed three times. Pure water was added and placed at -20°C, and repeated freezing and thawing three times. The supernatant was centrifuged at 12000r / min and 4°C for 10 minutes, and the content detection method was referred to the detection method of tediluosin in the pharmacopoeia. The results are shown in Figure 8 shown.

[0047] 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.

[0048] Example 4 Cytotoxicity of drug-loaded nanoparticles (PSA-Gly-TD) In this example, the cytotoxicity of drug-loaded nanoparticles (PSA-Gly-TD) was tested.

[0049] 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 to incubate for 8 hours. After the incubation is completed, discard the surface drug-containing culture medium, wash with PBS, add the 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 its absorbance at 450nm, and calculate the cell survival rate. The calculation formula is as follows:

[0050] In the formula, 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 It is the absorbance of CCK-8 incubation solution of blank cell group without adding drugs.

[0051] The results are as follows Fig. 9 shown.

[0052] from Fig. 9 It can be seen that the drug-loaded nanoparticles (PSA-Gly-TD) have low toxicity to cells.

[0053] Example 5 Extracellular antibacterial activity of drug-loaded nanoparticles (PSA-Gly-TD) In this example, the extracellular antibacterial ability of drug-loaded nanoparticles (PSA-Gly-TD) was tested.

[0054] Specifically, the pre-prepared 1280μg / mL tediloxane stock solution and PSA-Gly-TD solution (calculated as an equivalent amount of TD original drug equivalent) 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, and then add 100μL of spare bacterial suspension (wild-type swine Glaserella and resistant swine Glaserella) 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 shown in the following table. Fig.10 shown.

[0055] from Fig.10 It can be seen that there is no significant difference in the inhibitory ability of drug-loaded nanoparticles (PSA-Gly-TD) and TD original drug against wild-type swine Glaseriae and resistant swine Glaseriae in vitro.

[0056] Example 6 Intracellular antibacterial effect of drug-loaded nanoparticles (PSA-Gly-TD) In this example, the intracellular antibacterial ability of drug-loaded nanoparticles (PSA-Gly-TD) was tested.

[0057] Specifically, RAW264.7 cells in the logarithmic growth phase were taken and diluted to 5×10 4 1 mL / mL was inoculated into a 24-well plate and incubated in an incubator for 12 h to starve the plate and make it more susceptible to bacterial invasion. After the incubation, the culture medium was discarded and the wild-type and drug-resistant Glaseria suis were diluted to a concentration of 10 with DMEM medium containing 10% FBS (V / V), 0.2% NAD (V / V) and no double antibody. 6 CFU / mL, inoculate into 24-well plates at 1mL / well, so that the strain concentration: cell concentration = 100:1, incubate in the incubator for infection 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 with PBS 3 times. Collect the cells, add 1mL of pure water, let stand for 30min, wait for the cells to swell, dilute 100 times and apply to the plate, incubate at 37℃ for 24h, observe the number and distribution of colonies, and the results are as follows Fig.11 shown.

[0058] from Fig.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.

[0059] 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.

[0060] 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 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, lysine, methionine, phenylalanine, leucine or isoleucine.

2. The pH / neuraminidase dual-responsive drug-loaded nanoparticles according to claim 1, characterized in that: The drug-loaded nanoparticles also include antibiotic drugs, which contain alcoholic hydroxyl groups. The alcoholic hydroxyl groups in the antibiotic drugs are connected to the carboxyl groups in the amino acids through ester bonds.

3. The pH / neuraminidase dual-responsive drug-loaded nanoparticles according to claim 2, characterized in that: The antibiotic drugs include at least one of tedilosin, tilmicosin, tylosin and florfenicol.

4. A pharmaceutical composition, characterized in that The invention comprises the pH / neuraminidase dual-responsive drug-loaded nanoparticles 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 pH / neuraminidase dual-responsive drug-loaded nanoparticles according to any one of claims 1 to 3, 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, and an Fmoc removing 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 for incubation, and then the amino acid-antibiotic drug intermediate is added for condensation reaction. The obtained product is purified to obtain drug-loaded nanoparticles.

7. The preparation method according to claim 6, 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 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.

8. The preparation method according to claim 6, 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 for 6 to 10 hours at a temperature of 20 to 25°C; 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.

9. The preparation method according to claim 6, 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 h; after continuing to add the N-hydroxysuccinimide, incubating at a temperature of 20-25° C. for 0.5-1.5 h; The condensation reaction step specifically includes: reacting for 48 to 72 hours at a temperature of 45 to 60°C.

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

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