Preparation method and application of baicalein and cefixime combined nano drug delivery system

Abstract

The invention discloses a preparation method and application of a baicalein and cefixime combined nano drug delivery system, and relates to the technical field of pharmaceutical preparations, the key point of the technical scheme is that the preparation method comprises the following steps: S1, weighing a carrier material, putting the carrier material into a container, adding a 1% glacial acetic acid solution, and stirring with a magnetic stirrer until the carrier material is completely dissolved; s2, dropwise adding a baicalein solution and a cefixime solution, simultaneously continuing magnetic stirring, and then performing ultrasonic treatment by using a probe to ensure that the solutions are fully mixed; and S3, centrifuging the mixed solution to remove impurities, and filtering the mixed solution through a 0.45 mu m microporous filter membrane to obtain the baicalein and cefixime combined nano drug delivery system. The water solubility of baicalein is remarkably improved through a drug carrier material, the in-vivo absorption and delivery efficiency of the drug is improved through a nano drug delivery system, baicalein and cefixime synergistically enhance the activity of resisting methicillin-resistant staphylococcus aureus, and a good biological effect and clinical application potential are shown.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and more specifically, to a preparation method and application of a nano drug delivery system of baicalein combined with cefixime. Background Art

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that is resistant to multiple antibiotics and plays an important role in hospital infections and implantable medical device infections.

[0003] Currently, antibiotics are usually the first choice for treating MRSA infections. However, due to the widespread use and abuse of antibiotics, bacteria have developed resistance to a variety of antibiotics. In particular, when bacteria form biofilms, the effectiveness of traditional antibiotics is significantly reduced, resulting in weakened efficacy, prolonged treatment cycles, and increased patient suffering. In recent years, the toxic side effects and drug resistance of antibiotic treatment have attracted widespread attention, so the development of new and more efficient treatment options is urgent.

[0004] Traditional Chinese medicine has shown good application prospects in antibacterial aspects. Scutellaria baicalensis is considered to be a potential Chinese medicine resource for the treatment of bacterial infections, especially MRSA infections, due to its significant antibacterial and antiviral properties. Baicalein, the main active ingredient of Scutellaria baicalensis, shows strong antibacterial activity against a variety of bacteria, including MRSA. However, due to its poor water solubility, the clinical application of baicalein is limited. Nanotechnology provides an innovative method for drug delivery. By loading drugs into nanoscale carriers to prepare nano drug delivery systems, the solubility, bioavailability and targeting of drugs can be effectively improved. Nano drug delivery systems utilize their high surface area to significantly improve the solubility of hydrophobic drugs such as baicalein and increase their absorption rate in the body.

[0005] The baicalin-cefixime nano drug delivery system successfully prepared by the present invention can be used clinically for the treatment of anti-MRSA biofilm-related infections. This not only solves the problem of low water solubility of baicalin, but also enhances the antibacterial activity and clinical efficacy of the drug through nanotechnology, providing a new treatment plan for MRSA infections, especially stubborn infections caused by biofilms. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation method and application of a nano drug delivery system of baicalin combined with cefixime, so as to solve the problems of low solubility of baicalin and severe drug resistance of MRSA to antibiotics in the prior art.

[0007] The above technical objectives of the present invention are achieved through the following technical solutions: A method for preparing a nano drug delivery system of baicalin combined with cefixime, comprising the following steps:

[0008] S1: weigh the carrier material and put it into a container, add 1% glacial acetic acid solution, and stir with a magnetic stirrer until it is completely dissolved;

[0009] S2: Add baicalein solution and cefixime solution dropwise while continuing magnetic stirring, and then use probe ultrasound to ensure that the solutions are fully mixed;

[0010] S3: The mixed solution is centrifuged to remove impurities, and filtered through a 0.45 μm microporous filter membrane to obtain a nano drug delivery system of baicalin combined with cefixime.

[0011] The present invention is further configured as follows: the carrier material in step S1 is chitosan chitosan cyclodextrin polymer CS-CD, gelatin cyclodextrin polymer Gel-CD and D-α-tocopherol polyethylene glycol 1000 succinate TPGS; the weighed weights of the CS-CD, Gel-CD and TPGS carrier materials are 15 mg, 15 mg and 5 mg respectively.

[0012] The present invention is further configured as follows: in the nano drug delivery system, the ratio of the carrier material to baicalein and cefixime is 15:1.

[0013] The present invention is further configured as follows: the magnetic stirring time in step S2 is 5 hours, and the probe ultrasonic treatment time is 15 minutes.

[0014] The present invention is further configured as follows: the centrifugal speed in step S3 is 4000 rpm.

[0015] Another object of the present invention is to provide a nano drug delivery system of baicalin combined with cefixime prepared according to the above preparation method.

[0016] The present invention is further configured as follows: the average particle size of the nano drug delivery system of baicalin combined with cefixime is 260±5 nm, the PDI value is 0.236±0.012, and the Zeta potential is 29.5±0.4 mV.

[0017] Another object of the present invention is to provide a nano drug delivery system of baicalin combined with cefixime for use in inhibiting MRSA infection in mice.

[0018] By adopting the above technical scheme, the present invention significantly improves the water solubility of baicalin by using chitosan cyclodextrin polymer (CS-CD), gelatin cyclodextrin polymer (Gel-CD) and D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) as carrier materials; CS-CD and Gel-CD form inclusion complexes with baicalin through the hydrophobic inner cavity of the grafted cyclodextrin, increasing the solubility of the drug, while the hydrophilic outer edge makes the entire inclusion system more stable in water. In particular, the presence of Gel-CD not only improves the solubility, but also has good biocompatibility and biodegradability; TPGS further improves the solubility of baicalin by reducing the surface tension of water, so that baicalin can be more effectively dissolved in water in the nano drug delivery system, thereby improving its solubility and absorption efficiency in the body.

[0019] The nano drug delivery system prepared by the present invention has a nanometer-scale particle size (260±5nm), and this size characteristic enables the drug to effectively penetrate biological barriers, such as the blood-brain barrier and the mucosal barrier, thereby increasing the delivery efficiency in the body. Thanks to the small size, these nanoparticles can prolong the blood circulation time of the drug in the body, and help the drug passively target target tissues such as infection sites or tumors, and use the EPR effect of tumors and infected tissues to enhance retention. This delivery ability ensures that the drug can accumulate to a sufficient concentration in the target area, while reducing unnecessary exposure to healthy tissues, thereby improving the therapeutic effect and reducing side effects.

[0020] The nano drug delivery system prepared by the present invention demonstrated significant antibacterial effects in experiments, and its ability to combat methicillin-resistant Staphylococcus aureus was particularly prominent. Baicalein and cefixime acted together to enhance the antibacterial activity through a synergistic mechanism. Baicalein affected the expression of autolytic enzymes in bacteria, and cefixime was able to more effectively attack the formation of bacterial cell walls, thereby enhancing the antibacterial efficacy of the drug. In a mouse infection model, the use of the nanosystem significantly reduced the infection level and reduced the inflammatory response, showing good biological effects in treatment and potential clinical application value.

[0021] In summary, the present invention has the following beneficial effects:

[0022] 1. The present invention combines chitosan cyclodextrin polymer CS-CD, gelatin cyclodextrin polymer Gel-CD and D-α-tocopherol polyethylene glycol 1000 succinate TPGS as carrier materials, which significantly improves the water solubility of baicalin. These carrier materials enhance the solubility and biocompatibility of baicalin through inclusion and formation of nanoparticles, thereby improving its absorption efficiency in the body;

[0023] 2. The particle size of the nano drug delivery system prepared by the present invention is about 260±5nm, which enables the drug to effectively penetrate biological barriers, prolong its circulation time in the blood, and achieve passive targeting to specific tissues such as infection sites or tumors. This efficient drug delivery capability ensures that the drug concentration in the target area is sufficient while reducing damage to healthy tissues;

[0024] 3. The nano drug delivery system prepared by the present invention shows significant effects in fighting methicillin-resistant Staphylococcus aureus. The system greatly enhances the antibacterial activity through the synergistic effect of baicalin and cefixime, by affecting the expression of drug-resistant proteins, the expression of autolytic enzymes and the ability of bacteria to take up drugs, and effectively reduces the infection level in the mouse model, reflecting good biological effects and potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the MIC colony count of bacteria 43300 and the nano drug delivery system in Example 1 of the present invention;

[0026] in, Figure 1 A: Bacterial control group, diluted 10 5 times the number of colonies; Figure 1 B: MIC concentration of raw material group, diluted 10 3 times the number of colonies; Figure 1 C: MIC concentration nano drug delivery system group, the number of colonies diluted 50 times;

[0027] Figure 2 is a bar graph of the colony counts of bacteria 43300 and the MIC concentration of the nano drug delivery system in Example 1 of the present invention;

[0028] Figure 3 is a morphological diagram of MRSA observed under a scanning electron microscope in Example 2 of the present invention;

[0029] in, Figure 3 A: MRSA bacteria control group; Figure 3 B: API control group; Figure 3 C: Nano drug delivery system group;

[0030] Figure 4 This is a statistical bar graph of the colony count in Example 3 of the present invention to investigate the inhibitory effect of the drug on MRSA in mice;

[0031] Figure 5 This is a schematic diagram of HE staining of lung pathological histology in Example 3 of the present invention;

[0032] Figure 6 is the fluorescence intensity of methicillin-resistant Staphylococcus aureus observed by LSCM at different concentrations in Example 4 of the present invention;

[0033] in, Figure 6 A: Coumarin API group; Figure 6 B: Coumarin CS-CD nanoformulation; Figure 6 C: coumarin CS-CD / Gel-CD / TPGS;

[0034] Figure 7 is the fluorescence intensity of drug uptake by methicillin-resistant Staphylococcus aureus observed by LSCM at different times in Example 4 of the present invention;

[0035] in, Figure 7 A: Coumarin API group; Figure 7 B: Coumarin CS-CD nanoformulation group; Figure 7 C: coumarin CS-CD / Gel-CD / TPGS group;

[0036] Figure 8 This is a schematic diagram of the effect of the nano drug delivery system in Example 5 of the present invention on the expression of the drug resistance gene mecA in MRSA bacteria;

[0037] Fig. 9 Schematic diagram of the effect of the nano drug delivery system in Example 6 of the present invention on the expression of drug resistance protein PBP2a in MRSA bacteria;

[0038] Fig.10 This is a schematic diagram of the effect of the nano drug delivery system in Example 7 of the present invention on the expression of autolytic enzymes in MRSA bacteria. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0040] Example 1: A method for preparing a nano drug delivery system of baicalin combined with cefixime, comprising the following steps:

[0041] S1: Prepare and weigh the following carrier materials: chitosan cyclodextrin polymer (CS-CD) 15 mg; gelatin cyclodextrin polymer (Gel-CD) 15 mg; D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) 5 mg;

[0042] Place the weighed carrier material into a 25 ml beaker, add 5 ml of 1% glacial acetic acid solution into the beaker, and stir with a magnetic stirrer until all the materials are completely dissolved. The solution needs to be kept under uniform conditions for a period of time to ensure that each component is completely dissolved.

[0043] S2: Under continuous stirring, add baicalein reference solution and cefixime reference solution dropwise to the solution, keep the solution constant under stirring to ensure that the drug is gradually dispersed in the carrier solution, and continue to stir with a magnetic stirrer for a total of 5 hours. Subsequently, the solution is treated with a probe ultrasonic device for 15 minutes. Ultrasound can help further refine the drug particles and promote uniform mixing;

[0044] S3: The mixed solution is transferred to a centrifuge tube and centrifuged at 4000 rpm to remove undissolved impurities and other possible particles. The supernatant after centrifugation is used for subsequent treatment; the supernatant after centrifugation is filtered through a 0.45 μm microporous filter membrane. The purpose of the filtration is to ensure that there are no large particles or impurities in the resulting solution, so as to obtain a uniform nanoparticle dispersion.

[0045] The final nano drug delivery system is light yellow with opalescence, indicating that baicalin and cefixime can be made into a nano drug delivery system. The nano drug delivery system includes the following basic characteristics: average particle size of 260±5nm; polydispersity index (PDI) value of 0.236±0.012; Zeta potential of 29.5±0.4mV.

[0046] Example 2: In vitro antibacterial effect of nano drug delivery system

[0047] Determination of MIC of nano drug delivery system: The experimental groups were set up as follows:

[0048] Culture medium control group: does not contain any drugs and is used to determine whether the culture medium is sterile;

[0049] Bacterial control group: contains bacteria but no drugs, used to confirm the growth status of bacteria;

[0050] API group: contains bacteria and APIs, used to evaluate the inhibitory effect of APIs on bacteria;

[0051] Nano drug delivery system group: includes bacteria and nano drug delivery system, used to evaluate the inhibitory effect of nano drug delivery system on bacteria;

[0052] Dilute the bacterial culture solution in the logarithmic growth phase to a concentration of 1×10 6 CFU / ml, add 100μl of diluted bacterial solution to each well of a 96-well plate, dilute the drug using the doubling dilution method, and add it to the corresponding bacterial solution wells in the 96-well plate, and place the 96-well plate in a 37°C constant temperature incubator for 18 hours; after the incubation, add 10μl of glacial acetic acid to each well, and determine the drug concentration at which bacterial growth is completely inhibited (i.e., the clearest in the well) by visual observation. This concentration is the MIC value.

[0053] The observed MIC values ​​are recorded in a table to facilitate comparison of MIC differences between different groups. Through the above experimental steps, the MIC values ​​of different drug groups can be obtained, thereby evaluating the advantages of the nano drug delivery system over the raw material in antibacterial effect. The lower the MIC value, the smaller the required drug concentration and the better the antibacterial effect. The experimental results can provide important reference data for subsequent drug development and application. The experimental results are shown in Table 1 below.

[0054] Colony count: After 18 hours of culture, the culture medium was aspirated from the MIC of the nano drug delivery system in the 96-well plate, and the average number of bacteria in each group was determined and calculated using the plate colony count method. Figure 1-2 shown.

[0055] Table 1 Results of determination of MIC of bacteria 43300-nano drug delivery system

[0056]

[0057] Observe bacterial morphology using a scanning electron microscope: Take 5 ml of bacteria in the logarithmic growth phase, centrifuge at 5000 r / min for 5 min, discard the supernatant, resuspend the bacteria with a nano-drug delivery system solution at 1 / 2 times the MIC concentration, and design a bacterial control group and a raw material drug control group, culture in a 37°C shaking incubator for 1.5 h, fix with 3% glutaraldehyde, rinse with PBS, dehydrate with 30%, 50%, 70%, 80%, 90%, 95%, and 100% series of gradient alcohol, dehydrate for 10 min each gradient, dry, and ion sputter plate, and observe the morphology of Staphylococcus xylosus using a scanning electron microscope. The experimental results are as follows: Figure 3 shown.

[0058] The morphology of MRSA cell membrane was observed by scanning electron microscopy. Figure 3 As shown, Figure 3 A is untreated MRSA, Figure 3 B is MRSA treated with raw materials. The bacteria are spherical with complete and smooth structure. Figure 3 C is MRSA treated with the nano drug delivery system. The MRSA morphology has changed, the surface is rough, and some bacteria are even completely broken. There are also fine particles of about 300 nm adhering to the surface, which may be due to the adhesion of the nano drug delivery system to the bacterial surface, which is related to the positive charge of the nano drug delivery system. It can be seen that the in vitro antibacterial effect of the baicalin-cefixime nano drug delivery system is stronger than that of the raw material, which is consistent with the MIC test results and colony count results.

[0059] Example 3: Anti-MRSA effect of nano drug delivery system in vivo

[0060] Lung colony count determination: The plate colony count method was used to examine the inhibitory effect of the preparation on MRSA infection in mice. The lungs of MRSA-infected mice after administration were sterilely taken, weighed, homogenized, and cultured on agar for 24 hours, and the number of colonies was counted. Figure 4 The results showed that the preparation group could significantly enhance the inhibitory effect of the drug on MRSA in mice.

[0061] Lung pathological histological observation: The lungs of MRSA-infected mice were removed after administration, fixed with formalin, embedded in paraffin, and sectioned. After HE staining, pathological changes of the tissues were observed under a microscope. Figure 5 As shown, the lung tissue structure of mice in the normal saline group was intact, and there was no abnormality in the alveolar cavity, no edema, no inflammatory cell infiltration and congestion. The lung tissue structure of mice in the pneumonia model group was fuzzy, the alveoli could not be identified, the bronchiolar epithelial cells were partially necrotic, there were a large number of inflammatory cells in the cavity, and there was edema around the blood vessels; the alveolar morphology of most of the lung tissues of mice in the baicalin-cefixime API solution group could not be identified, the inflammatory cells ruptured, the tissue fluid exuded, and there was edema around the blood vessels. The alveolar cavity of some of the lung tissues of mice in the nanoformulation group was fused, and the alveolar wall capillaries showed slight expansion and infiltration of inflammatory cells. It had a better therapeutic effect than the model group and the API group.

[0062] Through the above experimental steps, the therapeutic effects of different preparations on MRSA-infected mouse lungs can be evaluated. Lung colony count determination can quantitatively analyze the number of MRSA, while pathological histological observation can qualitatively evaluate the degree of lung inflammation and damage. The experimental results show that the nanoformulation has a significant inhibitory effect on MRSA infection in vivo and is better than the raw drug solution in reducing lung pathological damage.

[0063] Example 4: Study on the mechanism of enhancing drug efficacy of baicalin-mediated β-lactam anti-MRSA nano-drug delivery system

[0064] Laser confocal microscopy (LSCM) to investigate bacterial uptake:

[0065] 43300 bacterial colonies were selected and inoculated into TSB liquid medium, incubated at 37°C, and the bacterial solution in the logarithmic growth phase was diluted to 1x10 6 CFU / ml, add 500μl diluted bacterial solution to a sterile glass-bottomed culture dish and incubate at a constant temperature for 24h. Discard the bacterial solution, add 500μl of coumarin nano-drug delivery system with a concentration of 60μg / ml, 30μg / ml and 15μg / ml, respectively, incubate at 37℃ for 1h, and set up a coumarin bulk drug group and a coumarin CS-CD nano-preparation group. Wash with sterile PBS for 3 times, and use LSCM to examine the bacterial uptake of coumarin. The experimental results are as follows Figure 6 shown.

[0066] Using LSCM, we observed the effect of nano-drug delivery system on bacterial drug uptake. Figure 6 It can be seen from the fluorescence intensity that the fluorescence intensity of the nano drug delivery system groups with different coumarin concentrations was higher than that of the CS-CD nano preparation group and the raw material drug group after incubation for 1 hour at 37°C.

[0067] 43300 bacterial colonies were selected and inoculated into TSB liquid medium, incubated at 37°C, and the bacterial solution in the logarithmic growth phase was diluted to 1x10 6 CFU / mL, add 500μL diluted bacterial solution to a sterile glass-bottomed culture dish and incubate at a constant temperature for 24h; aspirate the bacterial solution in the culture dish, add 500μL of nanoformulation (30μg / mL), incubate at 37℃ for 0.5h, 1h, 3h, and set up a coumarin bulk drug group and a coumarin CS-CD nanoformulation group, wash with sterile PBS three times, and use LSCM to examine the bacterial uptake of coumarin at different times. The experimental results are as follows Figure 7 shown.

[0068] from Figure 6-7 It can be seen from the fluorescence intensity that the fluorescence intensity of the nanodrug delivery system group increased with the increase of incubation time, and its fluorescence intensity was higher than that of the CS-CD nanoformulation group and the raw material drug group.

[0069] Example 5: Effect of Nano-drug Delivery System on Reverse Transcription of MRSA Bacterial MecA Gene

[0070] Preparation of bacterial samples: Take 1 ml of bacteria in the logarithmic growth phase, add 50 ml of TSB culture medium, add the nano drug delivery system to make the final concentration of 1 / 2MIC, and set up a drug control group. Culture at 37°C until the OD value is about 1, collect the bacteria by centrifugation at 4500 rpm for 15 min, wash three times with sterile PBS, and store at -80°C for later use.

[0071] Extraction of total bacterial RNA: Referring to the kit, add freshly prepared TE buffer containing lysozyme (0.4 mg / ml) to the centrifuged bacteria, 200 μl per group, repeatedly invert the centrifuge tube to mix, incubate at room temperature for 5 min, add 600 μl lysis buffer containing β-mercaptoethanol, mix well, then add 360 μl anhydrous ethanol, mix well, add to GeneJETTM RNA purification column with collection tube, centrifuge at 12000 rpm for 1 min, discard the penetration solution, replace with a new collection tube, add 700 μl of rinse solution 1, centrifuge at 12000 rpm for 1 min, discard the penetration solution, add 600 μl of wash solution 2, centrifuge at 12000 rpm for 1 min, discard the penetration solution, add 250 μl of wash solution 2, centrifuge at 12000 rpm for 2 min, and discard the penetration solution. Place the purification column into a sterile nuclease-free collection tube, add 50 μl of nuclease-free water to the center of the purification column membrane, centrifuge at 12,000 rpm for 1 min, repeat once, discard the purification column, and the liquid in the collection tube is the total bacterial RNA. Use an RNA concentration instrument to measure RNA concentration and purity.

[0072] Removal of genomic DNA: Add the relevant components according to Table 3 below to an RNase-free reaction tube and incubate at 37°C for 30 min. Add 1 μl of 50 mM EDTA and incubate at 65°C for 10 min to remove genomic DNA from the RNA sample.

[0073] Table 3 Solvent ratios required for removing genomic DNA from total RNA

[0074]

[0075] First-strand cDNA synthesis: Add the reagents in Table 4 below to a sterile RNase-free reaction tube in order, seal the tube with a sealing cap, centrifuge briefly, incubate at 42°C for 30 min, heat at 95°C for 5 min, and store at -20°C for later use.

[0076] Table 4 RNA reverse transcription cDNA system

[0077]

[0078]

[0079] qPCR amplification: Add relevant components to eight-tube concatenation for RT-PCR according to Table 5 below, cover the tube, centrifuge briefly, place the sample in a PCR amplification instrument, and amplify according to the set program (reaction conditions: 95°C, 30s, 1 cycle; 95°C, 5s, 60°C, 30s, 40 cycles). Set 6 replicate wells for each group of target genes and internal reference genes. The primers for the target genes and internal reference genes are designed as shown in Table 6 below.

[0080] Table 5 cDNA amplification system

[0081]

[0082] Table 6 Primer design

[0083]

[0084] RT-PCR was used to investigate the effect of the preparation on mecA gene transcription. The absorbance values ​​of the total RNA extracted in this experiment were A260 / A280>2 and A260 / A230>2, indicating that the RNA purity was high and could be used for the experiment. Figure 8 It showed that under the conditions of the nanodrug delivery system, the level of mecA gene RNA was significantly lower than that in the raw material drug group.

[0085] Example 6: Determination of PBP2a protein by Western blot

[0086] Extraction of total bacterial protein: Take 1 ml of bacteria in logarithmic growth phase, add 100 ml of TSB medium, add nanoparticles to a final concentration of 1 / 2 MIC, culture at 37°C until the OD value is about 1, collect bacteria by centrifugation at 4500 rpm for 15 min, wash 3 times with sterile PBS, add 2 ml of pre-cooled RIPA lysis buffer, ultrasonically break in an ice bath, centrifuge at 12000 rpm for 15 min at 4°C, the supernatant is the total bacterial protein extract, and set up a bulk drug control group. The protein concentration of each group was determined using the BCA protein concentration determination kit method.

[0087] Protein denaturation: Add 5× loading buffer to the extracted protein at a ratio of 4:1, mix well, heat in a 100°C metal bath for 10 min to denature the protein, and store at -20°C for later use.

[0088] SDS-PAGE gel electrophoresis: refer to the instructions of the gel kit, prepare 10% separation gel, pour it into the glass template prepared in advance, add pure water to flatten the gel, wait for the lower separation gel to solidify, add 5% upper concentrated gel prepared according to the kit, and insert the comb. After the upper gel solidifies, unplug the comb, install the prepared gel into the vertical electrophoresis tank, add electrophoresis buffer, add 2μl rainbow marker to the first well, add 10μg denatured protein to the remaining wells, and perform electrophoresis at 60V for 30min, and at 90V until the bromophenol blue reaches the bottom, then stop the electrophoresis.

[0089] Activate the 0.45μm PVDF membrane in methanol for 20 minutes, and use a current of 300mA to transfer the proteins on the gel to the PVDF membrane. After the transfer, block the PVDF membrane in 5% skimmed milk powder for 3 hours, and wash it three times with TBST buffer, each time for 5 minutes. Add the primary antibody to the washed PVDF membrane, incubate it on a shaker at 4°C overnight, wash it three times with TBST buffer, each time for 5 minutes, add HRP-labeled goat anti-rabbit secondary antibody, incubate it on a shaker at room temperature for 3 hours, and wash it three times with TBST buffer, each time for 10 minutes. After washing, add ultra-sensitive ECL chemiluminescent liquid for development under light-proof conditions, and collect WB bands with an all-purpose imaging analyzer. The experimental results are shown in Fig. 9 shown.

[0090] Western blot was used to determine the effect of the nano-drug delivery system on PBP2a protein. Fig. 9 As shown, when the amount of protein loaded is the same, the grayscale of the protein band of the nano-drug delivery system is lighter than that of the raw material drug, which is consistent with the RT-PCR results.

[0091] Example 7: Effect of Nano-drug Delivery System on MRSA Autolytic Enzyme

[0092] Crude cell wall extract: Take 1 ml of bacteria in the logarithmic growth phase and add it to 100 ml of TSB medium. Cultivate the bacteria until OD600 is about 1, collect the bacteria, add 50 ml of 8% SDS solution, heat in a 100°C water bath for 30 min, immediately cool in an ice bath, centrifuge at 5000 r / min for 15 min, remove the supernatant, wash with sterile distilled water 3 times, centrifuge at 12000 r / min for 10 min, collect the precipitate, and freeze-dry to obtain a crude cell wall extract.

[0093] Autolytic enzyme extraction: set up blank control, raw material drug, and drug delivery system groups, inoculate 1 ml of logarithmically growing bacteria into 50 ml TBS culture medium with 1 / 2 MIC preparation concentration, culture overnight, collect the bacteria, wash once with sterile PBS buffer, add 4% SDS, let stand at room temperature for 30 min, centrifuge at 12000r / min for 10 min, collect the supernatant, and determine the protein concentration by BCA method.

[0094] SDS-PAGE gel electrophoresis: Prepare 10% polyacrylamide gel solution according to the kit, add 1 mg of crude bacterial cell wall for every 1 ml of polyacrylamide gel solution, and after the gel solidifies, load 20 μg of autolytic enzyme extract and perform electrophoresis at 20 mA. After the electrophoresis, soak the gel in sterile distilled water for 30 minutes, and change the sterile distilled water every 10 minutes. Gently shake the gel at 37°C in an autolytic enzyme refolding buffer containing 1% Triton X-100, 10 mM CaCl2, 10 mM MgCl2, and 50 mM Tris-Cl (PH=7.0) for 36 hours to allow the autolytic enzyme to fully dissolve the cell wall components in the gel. See the experimental results. Fig.10 shown.

[0095] The experimental results show that the effect of the nano-drug delivery system on autolytic enzymes was determined by autolytic enzyme spectrum method. Compared with the blank group, both the nano-drug delivery system and the raw material drug can increase the amount and type of autolytic enzymes.

[0096] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to this embodiment as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A method for preparing a nano drug delivery system of baicalin combined with cefixime, characterized in that: The following steps are involved: S1: weigh the carrier material and put it into a container, add 1% glacial acetic acid solution, and stir with a magnetic stirrer until it is completely dissolved; S2: Add baicalein solution and cefixime solution dropwise while continuing magnetic stirring, and then use probe ultrasound to ensure that the solutions are fully mixed; S3: The mixed solution is centrifuged to remove impurities, and filtered through a 0.45 μm microporous filter membrane to obtain a nano drug delivery system of baicalin combined with cefixime.

2. The method for preparing the nano drug delivery system of baicalin combined with cefixime according to claim 1, characterized in that: The carrier materials in step S1 are chitosan-cyclodextrin polymer (CS-CD), gelatin cyclodextrin polymer (Gel-CD) and D-α-tocopherol polyethylene glycol succinate (TPGS); the weights of the CS-CD, Gel-CD and TPGS carrier materials are 15 mg, 15 mg and 5 mg respectively.

3. The method for preparing the nano drug delivery system of baicalein combined with cefixime according to claim 1, characterized in that: In the nano drug delivery system, the ratio of the carrier material to baicalein and cefixime is 15:

1.

4. The method for preparing the nano drug delivery system of baicalein combined with cefixime according to claim 1, characterized in that: The magnetic stirring time in step S2 is 5 hours, and the probe ultrasonic treatment time is 15 minutes.

5. The method for preparing the nano drug delivery system of baicalin combined with cefixime according to claim 1, characterized in that: The centrifugal speed in step S3 is 4000 rpm.

6. A nano drug delivery system of baicalin combined with cefixime prepared by the method of any one of claims 1 to 5.

7. The nano drug delivery system of baicalin combined with cefixime according to claim 6, characterized in that: The average particle size of the nano drug delivery system of baicalin combined with cefixime is 260±5nm, the PDI value is 0.236±0.012, and the Zeta potential is 29.5±0.4mV.

8. Use of the nano drug delivery system of baicalin combined with cefixime according to any one of claim 6 in inhibiting methicillin resistant Staphylococcus aureus (MRSA) infection in mice.

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