CN107308118B - Lung-targeting cefquinome sulfate PLGA microspheres and preparation method thereof - Google Patents
Lung-targeting cefquinome sulfate PLGA microspheres and preparation method thereof Download PDFInfo
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- CN107308118B CN107308118B CN201710551086.0A CN201710551086A CN107308118B CN 107308118 B CN107308118 B CN 107308118B CN 201710551086 A CN201710551086 A CN 201710551086A CN 107308118 B CN107308118 B CN 107308118B
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
- A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
- A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
- A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
Abstract
The invention provides a lung-targeted cefquinome sulfate PLGA microsphere and a preparation method thereof, belonging to the technical field of antibiotic targeted preparations for animals, wherein the preparation method comprises the following steps: mixing the raw material medicine and the dispersing agent uniformly, carrying out ball milling, and volatilizing the liquid to obtain the processed raw material medicine cefquinome; weighing PLGA and PLA as carriers, dissolving the carriers in a mixed solvent, adding the raw material medicines after the carriers are completely dissolved, adding the glidants, uniformly mixing by ultrasonic waves, spray drying, simultaneously carrying out magnetic stirring, and then obtaining the microspheres through a vortex separator. The encapsulation rate of the microsphere is more than 95%, the drug loading is more than 20%, and more than 85% of the particle size of the microsphere is distributed in the range of 10-25 um; the operation method is simple and convenient, the microsphere preparation efficiency is high, and the obtained microsphere has good dispersibility, excellent slow release effect and lung targeting property. The established method for detecting the medicine in the tissue has the advantages of good specificity, high sensitivity and recovery rate and excellent precision.
Description
Technical Field
The invention relates to the technical field of antibiotic targeting preparations, in particular to lung targeting cefquinome sulfate PLGA microspheres and a preparation method thereof.
Background
Cefquinome (Cefquinome), also known as Cefquinome, is a special 4 th generation cephalosporin antibiotic for 1 st animal developed by Hoechst Roussel Vet company in germany, and is widely used at home and abroad for treating respiratory diseases of livestock due to its strong antibacterial activity, broad antibacterial spectrum and low drug resistance.
The research and development of cefquinome and its preparation in China are relatively late, only cefquinome sulfate raw material medicines and injection (suspension injection and injection powder) thereof are approved by Ministry of agriculture for production and sale permission at present, and the types of dosage forms matched with the cefquinome sulfate raw material medicines are less. Cefquinome is not well absorbed by oral administration, is relatively quickly absorbed by injection, and has a short half-life period in vivo, namely 1-3 hours. In order to achieve effective treatment concentration, multiple injections are needed, which brings great inconvenience to clinical application. Therefore, the development of formulations with targeted sustained release properties is imminent.
The lung targeting microspheres can be trapped in the lung, so that the loaded medicine is enriched in the lung, the treatment level of the medicine is effectively improved, and the toxic and side effects of the medicine on non-target organs are greatly reduced; meanwhile, the slow release performance of the microspheres can enable the medicament to achieve the purpose of long acting, thereby avoiding the inconvenience of frequent administration.
In order to solve the limitation of the common preparation of cefquinome sulfate for livestock and improve the application value of the cefquinome sulfate, a new preparation taking cefquinome as a raw material is deeply developed and applied to the actual production. The preparation of the lung-targeting cefquinome sulfate PLGA microspheres has some defects, and the application of the microspheres in effect needs to be further improved.
Disclosure of Invention
The invention aims to provide lung-targeted cefquinome sulfate PLGA microspheres and a preparation method thereof.
In order to achieve the above object, the present invention is realized by the following measures: a preparation method of lung-targeted cefquinome sulfate PLGA microspheres comprises the following steps:
the raw material medicine and the dispersing agent are mixed according to the proportion of 1 g: 2-5ml of the mixture is uniformly mixed, and the mixture is put into a variable frequency planetary ball mill for ball milling for 1-3h, the batch time is 0.5-1.5h, the mixture is ball milled for 1-3h again, and the liquid is volatilized to obtain the treated raw material drug cefquinome; weighing PLGA and PLA as a carrier according to a proportion, dissolving the carrier in 200ml of mixed solvent of dichloromethane and methanol, after complete dissolution, adding a cefquinome raw material drug according to a proportion, adding a flow aid, uniformly mixing by ultrasonic waves, carrying out low-power 300-plus 400-W ultrasonic waves for 1-1.5min, and high-power 800-plus 1000-W ultrasonic waves for 0.5-1min, then carrying out spray drying by using a two-fluid spray dryer, simultaneously carrying out magnetic stirring, wherein the spray drying sample injection speed is 8ml/min, and then obtaining the lung-targeted cefquinome sulfate PLGA microspheres by using a vortex separator.
Wherein, in the dispersant, ethanol: isopropyl alcohol: the volume ratio of the dichloromethane is 5-10:1-3: 0.5-2.
Wherein, the grinding machine is a variable frequency planetary ball mill.
Wherein the mass ratio of PLGA to PLA in the microsphere preparation carrier is 1:4-1: 10.
Wherein the condition parameters of the spray drying are as follows: the inlet temperature is 40-200 ℃, the ventilation rate is 400-1000 percent, and the outlet temperature is 20-50 ℃.
Wherein the volume fraction of dichloromethane and methanol in the mixed solvent is 1:3-1: 10.
Wherein the mass ratio of the cefquinome bulk drug to the carrier is 1:2-1: 10.
Wherein the glidant is magnesium stearate or talcum powder, and the mass ratio of the carrier to the glidant is 1-5: 0.01-0.1.
In addition, the invention also provides a preparation method of the lung-targeted cefquinome sulfate PLGA microspheres, wherein the preparation method comprises the following steps:
the raw material medicine and the dispersing agent are mixed according to the proportion of 1 g: 2-5ml of the mixture is uniformly mixed, and the mixture is put into a variable frequency planetary ball mill for ball milling for 1-3h, the batch time is 0.5-1.5h, the mixture is ball milled for 1-3h again, and the liquid is volatilized to obtain the treated raw material drug cefquinome; weighing PLGA and PLA as a carrier according to a proportion, dissolving the carrier in 200ml of mixed solvent of dichloromethane and methanol, after complete dissolution, adding a cefquinome raw material drug according to a proportion, adding a flow aid, uniformly mixing by ultrasonic waves, carrying out low-power 300-plus 400-W ultrasonic waves for 1-1.5min, and high-power 800-plus 1000-W ultrasonic waves for 0.5-1min, then carrying out spray drying by using a two-fluid spray dryer, simultaneously carrying out magnetic stirring, wherein the spray drying sample injection speed is 8ml/min, and then obtaining the lung-targeted cefquinome sulfate PLGA microspheres by using a vortex separator.
The invention has the beneficial effects that: the prepared microsphere preparation has the encapsulation rate of more than 95 percent, the drug-loading rate of more than 20 percent and the particle size distribution of more than 85 percent of microspheres within the range of 10-25 um, has the advantages of simple and convenient operation method, high microsphere preparation efficiency, mild preparation conditions and the like, has high reproducibility and stability, and the prepared cefquinome microspheres have good dispersibility and uniform appearance, and the in vitro release research and the rat in vivo tissue distribution research show that the prepared microsphere preparation has excellent slow release effect and lung targeting property.
Drawings
FIG. 1 is a distribution diagram of the particle size of microspheres;
FIG. 2 shows the drug dissolution rate of drug loaded PLGA microspheres;
FIG. 3 is a standard curve of tissue addition of cefquinome sulfate;
FIG. 4 shows the drug distribution in rat tissues after a single dose (6mg CEQ/kg B.W.) of intravenous injection of cefquinome sulfate injection;
FIG. 5 shows the drug distribution in rat tissues after a single dose (6mg CEQ/kg B.W.) of intravenous injection of cefquinome sulfate PLGA microspheres;
FIG. 6 is a pathological section of rat lung tissue and lung blank tissue after a single dose (6mg CEQ/kg B.W.) of intravenous injection of cefquinome sulfate PLGA microspheres.
Detailed Description
The invention is realized by the following measures: a preparation method of lung-targeted cefquinome sulfate PLGA microspheres comprises the following steps:
the raw material medicine and the dispersing agent are mixed according to the proportion of 1 g: 2-5ml of the mixture is uniformly mixed, and the mixture is put into a variable frequency planetary ball mill for ball milling for 1-3h, the batch time is 0.5-1.5h, the mixture is ball milled for 1-3h again, and the liquid is volatilized to obtain the treated raw material drug cefquinome; weighing PLGA and PLA as a carrier according to a proportion, dissolving the carrier in 200ml of mixed solvent of dichloromethane and methanol, after complete dissolution, adding a cefquinome raw material drug according to a proportion, adding a flow aid, uniformly mixing by ultrasonic waves, carrying out low-power 300-plus 400-W ultrasonic waves for 1-1.5min, and high-power 800-plus 1000-W ultrasonic waves for 0.5-1min, then carrying out spray drying by using a two-fluid spray dryer, simultaneously carrying out magnetic stirring, wherein the sample injection speed of the spray drying is 8ml/min, and then obtaining the lung-targeted cefquinome sulfate PLGA microspheres by using a vortex separator.
Wherein, in the dispersant, ethanol: isopropyl alcohol: the volume ratio of the dichloromethane is 5-10:1-3: 0.5-2.
Wherein, the grinding machine is a variable frequency planetary ball mill.
Wherein the mass ratio of PLGA to PLA in the microsphere preparation carrier is 1:4-1: 10.
Wherein the condition parameters of the spray drying are as follows: the inlet temperature is 40-200 ℃, the ventilation rate is 400-1000 percent, and the outlet temperature is 20-50 ℃.
Wherein the volume fraction of dichloromethane and methanol in the mixed solvent is 1:3-1: 10.
Wherein the mass ratio of the cefquinome bulk drug to the carrier is 1:2-1: 10.
Wherein the glidant is magnesium stearate or talcum powder, and the mass ratio of the carrier to the glidant is 1-5: 0.01-0.1.
In addition, the invention also provides a preparation method of the lung-targeted cefquinome sulfate PLGA microspheres, wherein the preparation method comprises the following steps:
the raw material medicine and the dispersing agent are mixed according to the proportion of 1 g: 2-5ml of the mixture is uniformly mixed, and the mixture is put into a variable frequency planetary ball mill for ball milling for 1-3h, the batch time is 0.5-1.5h, the mixture is ball milled for 1-3h again, and the liquid is volatilized to obtain the treated raw material drug cefquinome; weighing PLGA and PLA as a carrier according to a proportion, dissolving the carrier in 200ml of mixed solvent of dichloromethane and methanol, after complete dissolution, adding a cefquinome raw material drug according to a proportion, adding a flow aid, uniformly mixing by ultrasonic waves, carrying out low-power 300-plus 400-W ultrasonic waves for 1-1.5min, and high-power 800-plus 1000-W ultrasonic waves for 0.5-1min, then carrying out spray drying by using a two-fluid spray dryer, simultaneously carrying out magnetic stirring, wherein the spray drying sample injection speed is 8ml/min, and then obtaining the lung-targeted cefquinome sulfate PLGA microspheres by using a vortex separator.
And carrying out performance detection on the microspheres obtained by the invention.
Analysis of microsphere granularity, drug loading rate and encapsulation rate of obtained cefquinome sulfate microspheres
1.1 materials and instruments
1.1.1 materials and reagents
Materials and reagents required for the experiment are shown in Table 1-1.
TABLE 1-1 Experimental materials and reagents
| Name (R) | Manufacturer of the product | Specification of |
| Methylene dichloride | Chemical reagents of national drug group Co Ltd | Analytical purity |
| Glacial acetic acid | Chemical reagents of national drug group Co Ltd | Analytical purity |
| Anhydrous ethanol | Chemical reagents of national drug group Co Ltd | Analytical purity |
| Cefquinome standard | China institute for veterinary medicine | 82.6％ |
1.1.2 instruments and devices
The instruments and equipment required for the experiments are shown in tables 1-2.
TABLE 1-2 Experimental instruments and apparatus
1.2 Experimental methods
1.2.1 measurement of microsphere particle size
Weighing a proper amount of microsphere powder, dispersing the microsphere powder with physiological saline, dipping the microsphere powder with a glass rod, coating the glass rod on a glass slide, covering the glass slide with a cover glass, placing the glass slide under an optical microscope, moving the glass slide in a visual field according to a bow shape, measuring the particle sizes of 500 microspheres in the visual field, calculating the average particle size, and drawing a particle size distribution diagram.
1.2.2 determination of drug-loading rate and encapsulation efficiency of microspheres
The method for measuring the drug content in the drug-loaded PLGA microspheres adopts a method of ultrasonically crushing the microspheres by a probe, and comprises the following specific steps: accurately weighing 25mg of drug-loaded microsphere powder, placing the drug-loaded microsphere powder in a 50mL centrifuge tube, adding 1mL of dichloromethane, oscillating at 1000rpm for 3min, adding 30mL of mobile phase, ultrasonically crushing the microspheres for 45 cycles by using a probe, carrying out ultrasonic power of 800W, carrying out ultrasonic for 4s, stopping the operation for 5s, cooling by using a proper ice bath, placing the mixture in a shaking table, oscillating at room temperature for 1h to fully release the drug, transferring the drug to a 50mL volumetric flask to fix the volume of the mobile phase, taking supernatant, passing the supernatant through a 0.22 mu m filter membrane, carrying out liquid phase detection, combining a standard curve, and calculating the drug loading LE% and the encapsulation efficiency EE% according to the formula (1).
The drug loading rate is the mass of the drug in the microsphere/the mass of the drug-loaded microsphere is 100 percent;
the encapsulation rate is the mass of the drug in the microsphere/the mass of the added drug is 100 percent;
1.2.3 determination of dissolution Rate of microsphere drug
The in vitro drug dissolution rate of the microspheres is determined by dialysis, specifically, 50mg of each of three continuous batches of microspheres are accurately weighed and respectively placed into three dialysis bags (molecular weight cut-off of 8000-. The study of the drug dissolution rate was carried out using 250ml of PBS (pH7.4) as a buffer medium and a 37 ℃ constant temperature shaker at a rotation speed of 150 rpm. Sampling 1ml at 5min, 15min, 30min, 45min, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h and 72h respectively, immediately adding equal volume of blank buffer solution, and replacing dialysate outside the dialysis bag every 12 h. And (4) carrying out HPLC analysis on the sample, obtaining the drug dissolution amount according to a standard curve, and drawing a release curve graph of accumulated drug dissolution amount-time to obtain a dissolution curve.
1.3 Experimental results and analysis
1.3.1 microsphere size analysis
The results of particle size measurement of PLGA microspheres are shown in FIG. 1.
As shown in FIG. 1, the particle size distribution of the prepared PLGA microspheres is narrow, and the microspheres with the particle size of 7-35 μm account for more than 80.0%.
1.3.2 analysis of drug-loading and encapsulation efficiency of microspheres
TABLE 1-3 drug loading of PLGA microspheres
| Name (R) | Theoretical drug loading (%) | Actual drug loading (%) | Encapsulation efficiency (%) |
| PLGA microspheres | 20.0％ | 18.2％ | 91.0％ |
As can be seen from tables 1-3, the encapsulation efficiency of the PLGA microsphere exceeds 85%, and the prepared microsphere has better drug-loading performance.
1.3.3 analysis of drug dissolution Rate
The drug dissolution rate analysis of the PLGA microsphere is shown in fig. 2.
As can be seen from fig. 2, the dissolution rate of the PLGA microsphere drug is significantly slowed, the drug release is 16.4% in 0.5h and 97.8% in 48h, while the drug release is 98.9% in 4h, as a result, the PLGA microsphere has a better sustained-release effect than the cefquinome drug.
1.4 summary
The following conclusions were obtained from the analysis in this section: (1) the microsphere granularity result proves that the prepared PLGA microsphere has uniform size, more concentrated distribution between 7 and 35 mu m (2) drug loading rate, and the encapsulation rate result proves that the prepared PLGA microsphere has higher drug loading rate and encapsulation rate, the drug loading rate is between 10 and 20 percent, and the encapsulation rate is between 80 and 95 percent. (3) The microsphere drug dissolution rate test result proves that the prepared PLGA microsphere has a slow release effect, has an obvious slow release effect compared with the cefquinome raw material drug, and the PLGA microsphere is completely released in 36 hours.
Secondly, research on in vivo lung tissue selective distribution of prepared cefquinome sulfate microspheres
In the experiment, Wistar rats are taken as objects to perform in vivo lung tissue selective distribution test of cefquinome sulfate microspheres.
2.1 materials of the experiment
2.1.1 materials and reagents
Materials and reagents required for the experiment are shown in Table 2-1.
TABLE 2-1 Experimental materials and reagents
2.1.2 instruments and apparatus
The instruments and equipment required for the experiment are shown in Table 2-2.
TABLE 2-2 Experimental instruments and apparatus
| Name (R) | Manufacturer of the product | Model number |
| Liquid chromatography mass spectrometer | Limited agilent technologyCompany(s) | BC-J80S |
| High-speed refiner | Wuxi Voxin instruments Co Ltd | FSH-2A |
| Analytical balance | Mettler-Tollido Switzerland | ME203E |
| Centrifugal machine | Shanghai' an pavilion | TDL-40C |
| Micro-pipette | Eppendorf | 5415R |
2.2 Experimental methods
2.2.1 establishment of UPLC/MS/MS detection methodology
2.2.1.1 Standard solution preparation
Weighing a proper amount of cefquinome sulfate reference substance into a 25ml brown volumetric flask by adopting a decrement method, fixing the volume to a scale by using redistilled water, uniformly mixing, preparing a 1000ppm standard stock solution, and storing the stock solution in a refrigerator at the temperature of-20 ℃ for later use. The standard working solution diluted by the mobile phase into a certain concentration is taken out before use. The stock solution is available within 1 month.
2.2.1.2 chromatographic conditions
Chromatographic conditions are as follows:
ACQUITY UPLC BEH C18 chromatographic column (50 mm. times.2.1 mm,1.7um)
2.2.1.3 tissue sample pretreatment
Taking 1.0g of rat tissue, adding a proper amount of 0.1% formic acid solution, uniformly mixing by using an adjustable high-speed homogenizer, taking 0.3g of homogenate liquid, placing in a 1.5ml centrifuge tube, shaking uniformly, adding 0.8ml of extracting solution (acetonitrile: water 95:5), vortexing for 30s, ultrasonically extracting for 15min, centrifuging at 4 ℃ and 12000rpm for 10min at a high speed, extracting residues for 2 times again, combining the extracting solutions, drying by nitrogen, adding 1ml of redistilled water and 0.5ml of n-hexane, pouring off an n-hexane layer, and freeze-drying. Adding 1ml (acetonitrile: 0.1% formic acid 15:85) for re-dissolving, filtering with 0.22 μm filter, and detecting by HPLC/MS/MS injection.
2.2.1.4 preparation of Standard Curve
Taking 5 parts of blank lung tissue, sequentially adding a certain volume of cefquinome sulfate standard working solution to ensure that the drug concentration in the lung tissue is respectively 3, 10, 50, 100 and 500ug/kg, carrying out HPLC detection according to the processing method under the item 2.2.1.3, and recording a chromatogram. And taking the measured peak area as a vertical coordinate (Y) and the concentration of cefquinome sulfate as a horizontal coordinate (X) to obtain a linear regression equation and calculate a correlation coefficient.
2.2.1.5 detection and quantitation limits
1, 3ng/g standard addition samples were prepared from the blank tissue, and 3 replicates of each sample were tested according to the procedure under "2.2.1.3". The S/N ratio is 3 as the limit of detection (LOD), and S/N ratio is 10 as the limit of quantification (LOD).
2.2.1.6 accuracy test
Adding high, medium and low 3 concentration standard solutions into the blank tissue sample to make the concentrations of the standard solutions in the blank tissue respectively 10, 20 and 100ug/kg, and dividing the peak area of cefquinome sulfate in the sample by the response value of the standard sample according to the processing method under the item of '2.2.1.3', thereby obtaining the extraction recovery rate.
2.2.1.7 precision test
Adding high, medium and low 3 concentration standard solutions into the blank lung tissue sample to make the concentrations of the standard solutions in the blank tissue respectively 10, 20 and 100 mug/kg, and repeating each concentration for 5 times to obtain the precision in the day; samples from 5 analytical batches were prepared and assayed consecutively on different days to obtain batch-to-batch precision.
2.2.2 rat tissue distribution Experimental design.
288 rats with the body weight of 180-. After fasting but free drinking of water for 12h, animals were sacrificed by caudal vein injection of 12.0mg/kg cefquinome sulfate, respectively, with 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 36, 48h post-administration, by bleeding from femoral artery. Immediately taking the heart, liver, spleen, lung and kidney, and detecting the drug content in each tissue.
2.2.3 histopathological analysis of rats.
The left lung was collected by dissecting the rat at 48H and fixed in 4% paraformaldehyde, embedded in paraffin, cut into 3 μm sections, and the sections stained with hematoxylin and eosin (H and E). Identification of pathological changes by means of a Vectra3.0 automated quantitative pathology imaging System
2.3 Experimental results and analysis
2.3.1 preparation of cefquinome sulfate Standard Curve
Through measurement, the drug addition concentration in the lung tissue is between 3 ppb and 500ppb, and the linear relation between the concentration and the peak area is good. Wherein, the correlation coefficient R2 of the drug in the lung tissue is 0.9999, and the regression equation is y 6.1112x + 4.9902. The standard curve is shown in fig. 3.
2.3.2 measurement of detection Limit and quantitation Limit of Cefquinome sulfate
The detection limit of cefquinome sulfate in tissues is 1 mu g/kg (S/N >3), the quantification limit is 3 mu g/kg (S/N >10)
2.3.3 accuracy test
Taking a blank tissue sample, adding a certain volume of cefquinome sulfate standard working solution to ensure that the drug concentration in the sample is respectively 10 ng/g, 20 ng/g and 100ng/g, each concentration is 5 parts in parallel, processing according to the item of '2.2.1.3', carrying out UPLC/MS/MS detection, and calculating according to peak area ratio to obtain the recovery rate shown in tables 2-3 to 2-7.
TABLE 2-3 recovery of cefquinome sulfate in Heart tissue
TABLE 2-4 recovery of cefquinome sulfate in liver tissue
TABLE 2-5 recovery of cefquinome sulfate in spleen tissue
TABLE 2-6 recovery of cefquinome sulfate in lung tissue
TABLE 2-7 recovery of cefquinome sulfate in renal tissue
2.3.4 precision test
A blank tissue sample is taken, a certain volume of cefquinome sulfate standard working solution is added, so that the drug concentration in the sample is respectively 10 ng/g, 20 ng/g and 100ng/g, each concentration is 5 parts in parallel in the same working day (in days), 5 batches (in days) are processed in different working days, UPLC/MS/MS detection is carried out after the treatment according to the method under the item 2.2.1.3, and the relative standard deviation in days and days is calculated (see tables 2-8 to 2-12).
TABLE 2-8 precision of cefquinome sulfate in cardiac tissue
TABLE 2-9 precision of cefquinome sulfate in liver tissue
TABLE 2-10 precision of cefquinome sulfate in spleen tissue
TABLE 2-11 precision of cefquinome sulfate in lung tissue
TABLE 2-12 precision of cefquinome sulfate in renal tissue
2.3.4 concentration of cefquinome sulfate in tissue
The concentrations of the drug in the different tissues after single dose administration in the experimental rats are shown in tables 2-13 to 2-15.
Tables 2-13 drug concentration in rat tissues after single dose (6mg CEQ/kg B.W.) of intravenous cefquinome sulfate injection
Tables 2-14 Single dose (6mg CEQ/kg B.W.) drug concentration in rat tissues after intravenous injection of cefquinome sulfate PLGA microspheres
2.3.5 tissue distribution map
The profiles of the different drug formulations in each tissue after single dose administration in the experimental rats are shown in figures 4 to 5.
As can be seen from fig. 4 and 5, after injecting the cefquinome sulfate PLGA microsphere preparation, compared with the cefquinome sulfate injection of the positive control group (fig. 4), the drug concentration in the lung tissue is high relative to the drug concentration in other organs, and the targeting and slow-release effects are obvious.
2.3.6 histopathological section analysis
No pathological changes were observed in the experimental group compared with the control group 48h after the rats were administered via the tail vein.
2.4 summary
The experiment shows that after rats are injected with the cefquinome sulfate PLGA microspheres, the targeting property and the slow release effect of cefquinome sulfate medicaments are effectively enhanced.
In conclusion, the lung-targeting cefquinome sulfate microsphere preparation prepared by the invention greatly improves the targeting property and the slow-release effect of cefquinome sulfate, and compared with a positive control medicament in an animal body, the obtained cefquinome sulfate microsphere preparation has obvious targeting property and slow-release effect and has consistency in vitro and in vivo. The preparation method is efficient and stable, and successfully obtains the lung-targeting cefquinome sulfate microsphere preparation.
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1
The preparation method comprises the following steps:
uniformly mixing 3.7g of the raw material medicine with 15ml of a dispersing agent, putting the mixture into a variable frequency planetary ball mill, carrying out ball milling for 2 hours, carrying out intermittent 1 hour, carrying out ball milling for 2 hours again, and volatilizing the liquid to obtain the processed raw material medicine cefquinome; weighing PLGA and PLA as carriers, dissolving the carriers in 200ml of mixed solvent of dichloromethane and methanol, adding cefquinome raw material medicine after complete dissolution, adding 0.15g of glidant, uniformly mixing by ultrasonic waves, carrying out low-power 300w ultrasonic wave for 1min, carrying out high-power 800w ultrasonic wave for 0.5min, carrying out spray drying by using a two-fluid spray dryer, carrying out magnetic stirring simultaneously, carrying out spray drying at a sample injection speed of 8ml/min, and obtaining the lung-targeted cefquinome sulfate PLGA microspheres by using a vortex separator.
Wherein, in the dispersant, ethanol: isopropyl alcohol: the volume ratio of the dichloromethane is 5:2: 1.
Wherein, the grinding machine is a variable frequency planetary ball mill.
Wherein PLGA12g and PLA3g are contained in the microsphere preparation carrier.
Wherein the condition parameters of the spray drying are as follows: the inlet temperature is 50 ℃, the ventilation rate is 700% and the air outlet temperature is 30 ℃.
Wherein the volume fraction of dichloromethane and methanol in the mixed solvent is 1: 5.
Wherein the glidant is magnesium stearate.
The average particle size of the prepared microspheres is 13.45 micrometers, and 88.43 percent of microspheres are distributed in the range of 10-25 micrometers; the scanning electron microscope shows that the appearance is round and smooth, and the surface is compact and smooth; the drug loading rate is 16.86 percent, the encapsulation rate is 84.3 percent, and the in vitro release of the drug can reach more than 24 hours.
Example 2
The preparation method comprises the following steps:
uniformly mixing 4g of the raw material medicine with 20ml of a dispersing agent, putting the mixture into a variable frequency planetary ball mill, carrying out ball milling for 2 hours, carrying out intermittent 1 hour, carrying out ball milling for 2 hours again, and volatilizing liquid to obtain the processed raw material medicine cefquinome; weighing PLGA10g and PLA2g according to a proportion as carriers, dissolving the carriers in 200ml of mixed solvent of dichloromethane and methanol, after completely dissolving, adding cefquinome raw material medicines according to a proportion, then adding 0.3g of flow aid, uniformly mixing by ultrasonic waves, carrying out low-power 350w ultrasonic wave for 1min and high-power 900w ultrasonic wave for 1min, then carrying out spray drying by adopting a double-fluid spray dryer, carrying out magnetic stirring simultaneously, carrying out spray drying at a sample injection speed of 8ml/min, and then obtaining the lung-targeted cefquinome sulfate PLGA microspheres by a vortex separator.
Wherein, in the dispersant, ethanol: isopropyl alcohol: the volume ratio of the dichloromethane is 5:2: 1.
Wherein, the grinding machine is a variable frequency planetary ball mill.
Wherein the condition parameters of the spray drying are as follows: the inlet temperature is 60 ℃, the ventilation rate is 600%, the sample introduction rate is 8ml/min, and the air outlet temperature is 30 ℃.
Wherein the volume fraction of dichloromethane and methanol in the mixed solvent is 1: 5.
Wherein the glidant is talcum powder.
The average particle size of the prepared microspheres is 14.56 micrometers, and 79.73% of the microspheres are distributed in the range of 10-25 micrometers; the scanning electron microscope shows that the appearance is round and smooth, and the surface is compact and smooth; the drug loading rate is 19.84%, the encapsulation rate is 79.36%, and the in vitro release of the drug can reach more than 36 h.
Example 3
The preparation method comprises the following steps:
uniformly mixing 3g of the raw material medicine and 15ml of dispersing agent, putting the mixture into a variable frequency planetary ball mill, carrying out ball milling for 2 hours, carrying out intermittent 1 hour, carrying out ball milling for 2 hours again, and volatilizing the liquid to obtain the processed raw material medicine cefquinome; weighing PLGA14g and PLA1.4g as carriers, dissolving the carriers in 200ml of mixed solvent of dichloromethane and methanol, after complete dissolution, adding cefquinome raw material medicine in proportion, then adding 0.205g of flow aid, uniformly mixing by ultrasonic waves, carrying out low-power 300w ultrasonic for 1min and high-power 800w ultrasonic for 1min, then carrying out spray drying by adopting a double-fluid spray dryer, carrying out magnetic stirring simultaneously, carrying out spray drying at the sample injection speed of 8ml/min, and then obtaining the lung-targeted cefquinome sulfate PLGA microspheres by a vortex separator.
Wherein, in the dispersant, ethanol: isopropyl alcohol: the volume ratio of the dichloromethane is 5:3: 1.
Wherein, the grinding machine is a variable frequency planetary ball mill.
Wherein the condition parameters of the spray drying are as follows: the inlet temperature is 100 ℃, the ventilation rate is 800%, the sample introduction rate is 8ml/min, and the air outlet temperature is 30 ℃.
Wherein the volume fraction of dichloromethane and methanol in the mixed solvent is 1: 7.
Wherein the glidant is magnesium stearate.
The average particle size of the prepared microspheres is 16.03 microns, and 88.24% of the microspheres are distributed in the range of 10-25 microns; the scanning electron microscope shows that the appearance is round and smooth, and the surface is compact and smooth; the drug loading rate is 11.65%, the encapsulation rate is 69.88%, and the in vitro release of the drug can reach more than 24 h.
Example 4
The preparation method comprises the following steps:
uniformly mixing 6g of the raw material medicine with 24ml of a dispersing agent, putting the mixture into a variable frequency planetary ball mill, carrying out ball milling for 3 hours, carrying out intermittent 1 hour, carrying out ball milling for 2 hours, and volatilizing the liquid to obtain the processed raw material medicine cefquinome; weighing PLGA16g and PLA2g as carriers, dissolving the carriers in 200ml of mixed solvent of dichloromethane and methanol, adding cefquinome raw material medicine after complete dissolution, then adding 0.324g of glidant, uniformly mixing by ultrasonic waves, carrying out low-power 350w ultrasonic wave for 1min and high-power 900w ultrasonic wave for 1min, then carrying out spray drying by adopting a double-fluid spray dryer, carrying out magnetic stirring simultaneously, carrying out spray drying at the sample injection speed of 8ml/min, and then obtaining the lung-targeted cefquinome sulfate PLGA microspheres by a vortex separator.
Wherein, in the dispersant, ethanol: isopropyl alcohol: the volume ratio of the dichloromethane is 5:2: 2.
Wherein, the grinding machine is a variable frequency planetary ball mill.
Wherein the condition parameters of the spray drying are as follows: the inlet temperature is 60 ℃, the ventilation rate is 750 percent, the sample introduction rate is 8ml/min, and the air outlet temperature is 30 ℃.
Wherein the volume fraction of dichloromethane and methanol in the mixed solvent is 1: 6.
Wherein the glidant is magnesium stearate.
The average particle size of the prepared microspheres is 16.03 microns, and 88.24% of the microspheres are distributed in the range of 10-25 microns; the scanning electron microscope shows that the appearance is round and smooth, and the surface is compact and smooth; the drug loading rate is 24.22%, the encapsulation rate is 96.88%, and the in vitro release of the drug can reach more than 48 h.
Example 5
The preparation method comprises the following steps:
uniformly mixing 2.8g of the raw material medicine and a dispersing agent according to 24ml, putting the mixture into a variable frequency planetary ball mill, carrying out ball milling for 2 hours, carrying out intermittent 1 hour, carrying out ball milling for 2 hours again, and volatilizing the liquid to obtain the processed raw material medicine cefquinome; weighing PLGA20g and PLA2.5g as carriers, dissolving the carriers in 200ml of mixed solvent of dichloromethane and methanol, adding cefquinome raw material drug after complete dissolution, then adding 0.02g of glidant, uniformly mixing by ultrasonic waves, carrying out low-power 400w ultrasonic for 1min and high-power 900w ultrasonic for 1min, then carrying out spray drying by adopting a two-fluid spray dryer, carrying out magnetic stirring simultaneously, wherein the spray drying sample injection speed is 8ml/min, and then obtaining the lung-targeted cefquinome sulfate PLGA microspheres by a vortex separator.
Wherein, in the dispersant, ethanol: isopropyl alcohol: the volume ratio of the dichloromethane is 6:2: 1.
Wherein, the grinding machine is a variable frequency planetary ball mill.
Wherein the condition parameters of the spray drying are as follows: the inlet temperature is 100 ℃, the ventilation rate is 600%, the sample introduction rate is 8ml/min, and the air outlet temperature is 30 ℃.
Wherein the volume fraction of dichloromethane and methanol in the mixed solvent is 1: 5.
Wherein the glidant is magnesium stearate.
The average particle size of the prepared microspheres is 16.03 microns, and 78.32% of the microspheres are distributed in the range of 10-25 microns; the scanning electron microscope shows that the appearance is round and smooth, and the surface is compact and smooth; the drug loading rate is 7.72 percent, the encapsulation rate is 69.53 percent, and the in vitro release of the drug can reach more than 36 hours.
The technical features of the present invention which are not described in the above embodiments may be implemented by or using the prior art, and are not described herein again, of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples, and variations, modifications, additions or substitutions which may be made by those skilled in the art within the spirit and scope of the present invention should also fall within the protection scope of the present invention.
Claims (1)
1. A lung-targeted cefquinome sulfate PLGA microsphere is characterized in that the preparation method comprises the following steps:
uniformly mixing 6g of the raw material medicine with 24ml of a dispersing agent, putting the mixture into a variable frequency planetary ball mill, carrying out ball milling for 3 hours, carrying out intermittent 1 hour, carrying out ball milling for 2 hours, and volatilizing the liquid to obtain the processed raw material medicine cefquinome;
weighing PLGA16g and PLA2g as carriers, dissolving the carriers in 200ml of mixed solvent of dichloromethane and methanol, adding the processed raw material cefquinome after complete dissolution, adding 0.324g of glidant, uniformly mixing by ultrasonic waves, performing low-power 350w ultrasonic for 1min and high-power 900w ultrasonic for 1min, performing spray drying by using a two-fluid spray dryer, performing magnetic stirring at the same time, wherein the spray drying sample injection speed is 8ml/min, and obtaining lung-targeted cefquinome sulfate PLGA microspheres by using a vortex separator;
wherein, in the dispersant, ethanol: isopropyl alcohol: the volume ratio of the dichloromethane is 5:2: 2;
wherein, the grinding machine is a variable frequency planetary ball mill;
wherein the condition parameters of the spray drying are as follows: the inlet temperature is 60 ℃, the ventilation rate is 750 percent, the sample introduction rate is 8ml/min, and the air outlet temperature is 30 ℃;
wherein the volume fraction of dichloromethane and methanol in the mixed solvent is 1: 6;
wherein the glidant is magnesium stearate.
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Effective date of registration: 20240116 Address after: Building 6, Building 304A, Blue Biomedical Industry Park, No. 368 Hedong Road, High tech Zone, Qingdao, Shandong Province, 266109 Patentee after: Qingdao Deji Agricultural Biotechnology Co.,Ltd. Address before: 266109 No. 700, the Great Wall Road, Chengyang District, Shandong, Qingdao Patentee before: Qingdao Agricultural University |