CN110623924A

CN110623924A - Hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nano micelle and preparation and application thereof

Info

Publication number: CN110623924A
Application number: CN201910899943.5A
Authority: CN
Inventors: 杜子秀; 彭佳惠; 徐宇虹; 殷瑜
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-09-23
Filing date: 2019-09-23
Publication date: 2019-12-31
Anticipated expiration: 2039-09-23
Also published as: CN110623924B

Abstract

The invention discloses a polycaprolactone-polyethylene glycol nano micelle carrying hydrophobic antibiotics, and preparation and application thereof, wherein the micelle comprises the hydrophobic antibiotics and a micelle carrier, and the nano micelle has a core-shell structure; the hydrophobic block polycaprolactone and the hydrophobic antibiotic in the micelle carrier form an inner core part together, and the hydrophilic block polyethylene glycol forms an outer shell part. The invention utilizes the easy crystallization property of polycaprolactone to tightly wrap the hydrophobic antibiotic, thereby ensuring the stability of the drug before reaching target cells; the micelle formed by the hydrophilic PEG shell and the amphiphilic polymer block copolymer has the particle size of about 200nm, is beneficial to avoiding the micelle from being identified by a reticuloendothelial system, and has a long circulation function. The structure of the antibiotic-loaded nano micelle can obviously improve the in vivo stability of the antibiotic, has certain targeting property and avoids the generation of side reaction.

Description

Hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nano micelle and preparation and application thereof

Technical Field

The invention belongs to the technical field of drug delivery, relates to a hydrophobic antibiotic-loaded nano micelle, and particularly relates to a hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nano micelle and preparation and application thereof.

Background

Clinically, commonly used antibiotics such as beta-lactams, aminoglycosides, macrolides, lincomycins, polypeptides, quinolones, sulfonamides and the like have the effect of inhibiting or killing pathogenic microorganisms and are important medicaments for preventing and treating infectious diseases. At present, antibiotics used as antibacterial drugs account for a considerable proportion in clinical medication, but the problems of poor water solubility and stability, low concentration of focus parts, strong toxic and side effects and the like of the antibiotics for treating bacterial infection are found at the same time, the defects seriously restrict the effective utilization of the antibacterial drugs, damage the health of human bodies, and side effects of bacterial drug resistance, drug-resistant bacterial infection and the like are generated. In order to overcome the above disadvantages of antibiotics, improve the bioavailability of in vivo drugs, reduce the dosage of antibiotics, avoid bacterial drug resistance and drug-resistant bacterial infection, etc., people are trying to utilize nanoparticles to realize safe and efficient delivery in vivo while continuously developing new and more effective antibiotic drugs. There are various antibiotic-loaded delivery systems currently used as nanoformulations of antibiotics, such as polymeric nanoparticles, solid lipid nanoparticles, lipid/polymer hybrid nanoparticles, as well as nanofibers/scaffolds, nanosheets, nanotubes, nanoemulsions, and the like, assembled from different materials. The nano delivery system prepared by the high molecular biological material is still at the beginning stage in the delivery of antibiotics, but has already embodied unique superiority.

In the macromolecular biomaterial, the amphiphilic segmented copolymer is easy to form a micelle structure, and the antibiotic with low water solubility is wrapped by a hydrophobic core and long-acting circulation in vivo is realized by a hydrophilic chain shell. The delivery system is characterized by stably existing in a physiological environment with pH of 7.4, and after reaching an acidic inflammation part eroded by bacteria, the delivery carrier can release antibiotics through degradation to enhance the purposes of specific delivery and sustained and controlled release of the drugs. In addition, the surface area of the nano particles is large, so that the nano particles are easily endocytosed by bacteria, the drug concentration of the focus can be improved, and the local toxic and side effects of antibiotics can be reduced. Among a plurality of amphiphilic block copolymers, polycaprolactone-polyethylene glycol (PCL-PEG) is widely applied to the field of medical biology due to the advantages of safety, no toxicity, good biocompatibility, no immunogenicity and the like. Polycaprolactone is easy to crystallize at the melting temperature of below 60 ℃, the Critical Micelle Concentration (CMC) is very low, and the contained alkyl branched chain is few, so that the steric hindrance is small, and a stable micelle is easier to form [ Duzi Xiu et al, appearance and macromolecules of the micelle formed by PCL-PEG segmented copolymer with a crystalline core in aqueous solution, 2007,40, 7633 and 7637 ]; by utilizing the amphipathy and biocompatibility of PCL-PEG and the high crystallinity of PCL, the PCL-PEG is usually used for coating hydrophobic antitumor drugs to form nano micelles [ Guchou et al, uses PCL-PEG nano target modified by low molecular weight MMP-2/9 protein to treat glioma and biological materials, 2013, 34, 196 and 208 ]. The PEGylated micelle surface also reduces the drug uptake of liver and kidney, and has the sustained drug release capability [ Wen-jen Lin et al, characterization of PEGylated micelle copolymer, in vivo pharmacokinetics and biodistribution research, biomedical materials research journal b biological application research materials, 2006,4,77(1):188-94 ].

Wherein, the nanometer conveying system prepared by PCL-PEG material is still in the beginning stage in the conveying of antibiotics.

Disclosure of Invention

In order to overcome the problems existing in the use of the existing antibiotic preparation, such as the instability of the existing hydrophobic antibiotic in vivo delivery system and the inability to play a targeting role, the invention aims to provide the polycaprolactone-polyethylene glycol nano micelle carrying the hydrophobic antibiotic, and the preparation and the application thereof.

Namely, a stable and uniform drug delivery system which can tightly wrap the hydrophobic antibiotic is provided; more specifically, the invention relates to a hydrophobic antibiotic-loaded nanoparticle constructed by using an amphiphilic crystalline block copolymer and polycaprolactone-polyethylene glycol (PCL)₂₀₀₀-MPEG₂₀₀₀And PCL₅₀₀₀-PEG₂₀₀₀) High-efficiency and safe nano micelle prepared by carrying macrolide antibiotic erythromycinThe system is also suitable for other crystalline amphiphilic block copolymer hydrophobic antibiotic-loaded nanoparticles.

The purpose of the invention is realized by the following technical scheme:

the invention provides a polycaprolactone-polyethylene glycol nano micelle loaded with hydrophobic antibiotics, which comprises the hydrophobic antibiotics and a micelle carrier, wherein the nano micelle has a core-shell structure; the hydrophobic block polycaprolactone and the hydrophobic antibiotic in the micelle carrier form an inner core part together, and the hydrophilic block polyethylene glycol forms an outer shell part.

Preferably, the nano-micelle is a rod-shaped micelle, the length of the nano-micelle is 30-200 nm under a transmission electron microscope, and the diameter of the nano-micelle is 10-20 nm. The stable rod-shaped nano micelle formed by the invention can improve the bioavailability of the drug, realize the functions of long-acting circulation and slow controlled release in vivo, and release the drug by self degradation after reaching the pathological cells, thereby increasing the solubility of the drug in the pathological cells, prolonging the detention time of the drug at the inflammation part, better playing the drug effect and reducing side effects.

Preferably, the micelle carrier is prepared from the following components in a mass ratio of (0-0.8): 1 PCL₂₀₀₀-MPEG₂₀₀₀And PCL₅₀₀₀-PEG₂₀₀₀。

Preferably, the micelle carrier is prepared from the following components in a mass ratio of (0.3-0.6): 1 PCL₂₀₀₀-MPEG₂₀₀₀And PCL₅₀₀₀-PEG₂₀₀₀. More preferably, the mass ratio of the two is 0.5: 1.

Preferably, the hydrophobic antibiotic is erythromycin.

The invention also provides a preparation method of the polycaprolactone-polyethylene glycol nano micelle loaded with the hydrophobic antibiotic, which comprises the following steps:

A. adding hydrophobic antibiotic into the micelle carrier, and co-dissolving in an organic solvent;

B. adding water into the solution formed in the step A, and uniformly mixing to form a uniform emulsion;

C. and removing the organic solvent and the unencapsulated hydrophobic antibiotic in the emulsion to obtain the nano micelle.

Preferably, in the step A, the adding amount of the hydrophobic antibiotic is 1-20% of the total mass of the micelle carrier; more preferably 5-10%.

Preferably, in step a, the organic solvent is selected from organic solvents such as tetrahydrofuran or chloroform that can dissolve the drug together with the carrier material.

Preferably, the volume ratio of the tetrahydrofuran to the water is (3-9): 1; the volume ratio of chloroform to water is 1: (5-10), otherwise, the prepared micelle has poor particle size uniformity or the drug loading is influenced.

The invention also provides application of the polycaprolactone-polyethylene glycol nano micelle carrying the hydrophobic antibiotic in preparation of a drug delivery system for treating infectious diseases.

Compared with the prior art, the invention has the following beneficial effects:

on the basis of the prior art, the invention creatively utilizes biodegradable high molecular materials to tightly wrap the hydrophobic antibiotics to prepare the medicinal carrier which is in vivo long-circulating, namely slow-release.

In particular, the amount of the solvent to be used,

(1) the invention utilizes the property of easy crystallization of polycaprolactone to tightly wrap the hydrophobic antibiotic;

(2) the PEG surface of the micelle carrier can realize the long circulation function of the carrier in vivo;

(3) the PEG tail end on the surface of the micelle carrier can be further used for connecting a targeting group so as to realize the targeted delivery of the medicine and increase the intake of the medicine.

In the invention, the hydrophobic antibiotic is tightly wrapped by utilizing the easy crystallization property of the polycaprolactone, so that the stability of the drug before reaching a target cell is ensured; the micelle formed by the hydrophilic PEG shell and the amphiphilic polymer block copolymer has the particle size of about 200nm, is beneficial to avoiding the micelle from being identified by a reticuloendothelial system, and has a long circulation function. The structure of the antibiotic-loaded nano micelle can obviously improve the in vivo stability of the antibiotic, has certain targeting property and avoids the generation of side reaction.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic diagram showing the construction of a nanomicelle in example 1 of the present invention;

FIG. 2 is a graph showing the particle size of blank micelles in example 1 of the present invention;

FIG. 3 is a graph showing the particle size of erythromycin-loaded micelles in example 2 of the present invention;

FIG. 4 is a graph showing the results of the stability of erythromycin-loaded serum in the effect verification example 1 of the present invention;

FIG. 5 is a morphology of a blank micelle under a bio-type transmission electron microscope in the validation example 2 of the effect of the present invention;

FIG. 6 is a morphology of erythromycin-loaded micelles under a biological transmission electron microscope in Effect verification example 3 of the present invention;

FIG. 7 is an in vitro release profile of erythromycin-loaded micelles in Experimental example 4 of the present invention;

FIG. 8 is a graph showing the toxicity of erythromycin-loaded micelles, blank micelles and erythromycin naked drug on SKBR-3 cells in the verification example 5 of the effects of the present invention.

Detailed Description

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods and preparation methods disclosed in the present invention all employ conventional techniques in the art.

The test methods used in the following examples are all conventional methods unless otherwise specified.

The material reagents and the like used in the following examples are commercially available unless otherwise specified.

The following examples are all specifically described with erythromycin as a model drug.

In the following examples, it is preferred that,

1. sources of experimental reagents

PCL₅₀₀₀-PEG₂₀₀₀And PCL₂₀₀₀-MPEG₂₀₀₀Purchased from sienna millennium biotechnology limited; erythromycin was purchased from biotechnology limited of yinaoka, beijing; alizarin was purchased from Shanghai Mobei Biotech, Inc.; phosphotungstic acid was purchased from Shanghai Merlin Biotechnology, Inc.; acetone and tetrahydrofuran are analytically pure and purchased from Shanghai national medicine group reagent Co., Ltd; SKBR-3 cell lines were purchased from Shanghai Mediterranean institute of cell culture; fetal bovine serum and RPMI-1640 medium were purchased from Gbico, USA; penicillin-streptomycin mixed solution (100 × double antibody) and trypsin solution were purchased from saimmer feishel science china ltd; the WST toxicity detection kit is purchased from Shanghai Biyuntian biotechnology limited company; staphylococcus aureus: ATCC 25923; LB liquid Medium (g/L): 10g of tryptone, 5g of yeast extract powder, 10g of NaCl and pH 7.4.

2. Laboratory apparatus

An Infine M200PRO multifunctional microplate reader: switzerland Ken company; FZ-A molecular hybridization instrument: taicano Hualida Experimental facilities, Inc.; a magnetic stirrer: shanghai Spire Instrument Co., Ltd; SHB-IIIS circulating water type multipurpose vacuum pump: zhengzhou great wall science and trade company, Inc.; R201B rotary evaporator: shanghai Shen Sheng Biotechnology, Inc.; KQ-400KDB type high power numerical control ultrasonic cleaner: kunshan ultrasonic instruments, Inc.; UPH-I-5TN ultrapure water machine: sichuan Yopu super pure science and technology, Inc.; LGJ-10D freeze-dryer: beijing four ring scientific Instrument plant, Inc.; tecnai G2 spiritBiotwin biotype transmission electron microscopy: FEI corporation, USA; Nano-ZS90 laser particle size analyzer: british malvern; copper mesh carbon support film: beijing, Mitsuoku technologies, Inc.

EXAMPLE 1 preparation of erythromycin-loaded micelles

The drug loading rate is the mass percentage of the erythromycin in the polymer carrier material.

The polymer carrier material PCL used in this example₂₀₀₀-MPEG₂₀₀₀And PCL₅₀₀₀-PEG₂₀₀₀The molecular weights are: PEG and MPEG are 2000Da, PCL is 2000Da and 5000 Da.

The synthetic construction scheme is shown in detail in FIG. 1.

The preparation method of the blank micelle comprises the following steps:

adding the PCL₂₀₀₀-MPEG₂₀₀₀And PCL₅₀₀₀-PEG₂₀₀₀According to the mass ratio of 0: 1. 0.1: 1. 0.3: 1. 0.5: 1. 0.8: 1. 1: 1. 1:0, dissolving in 10mL tetrahydrofuran, shaking to dissolve completely, and mixing uniformly; and then, according to the volume ratio of tetrahydrofuran to deionized water (3-9): 1 deionized water was added dropwise. And (3) rotationally evaporating to remove tetrahydrofuran in a round-bottom flask at the temperature of 30-50 ℃ under a vacuum condition to obtain a opalescent micelle solution, and then carrying out ultrasonic treatment in a water bath at the temperature of 65 ℃ for 10min to further form a uniform micelle system. After removing the unencapsulated drug (erythromycin) by filtration through a 0.22 μm microporous membrane, it was stored at 4 ℃ for subsequent experiments.

And (3) measuring the particle size of the nano micelle:

the particle size and polydispersity index (PDI) of the blank micelles and erythromycin-loaded micelles, respectively, were determined at room temperature using a laser particle sizer.

Particle size, Polydispersity (PDI) of blank micelle samples 1-4 prepared in the examples are shown in FIG. 2. As can be seen from FIGS. 2 and 3, the preferred micelle size after loading with drug is increased from about 200nm to about 220 nm. The particle size and polydispersity of the erythromycin-loaded micelles thus prepared are shown in Table 1. As can be seen from the results in Table 1, the blank micelles prepared in samples 1 to 4 had better particle size and dispersion coefficient than the other samples.

TABLE 1

EXAMPLE 2 preparation of erythromycin-loaded micelles

The preparation method of the erythromycin-carrying micelle comprises the following steps: adding the PCL₂₀₀₀-MPEG₂₀₀₀And PCL₅₀₀₀-PEG₂₀₀₀According to the mass ratio of 0.5:1, adding erythromycin accounting for 1%, 5%, 10%, 15%, 20% and 25% of the total mass of the polymer carrier material, dissolving in 2mL of chloroform, shaking to dissolve completely, and mixing uniformly; then, according to the volume ratio of the deionized water to the chloroform of 1: (5-10) dropwise adding deionized water. And rotationally evaporating in a round-bottom flask at 30-50 ℃ under a vacuum condition to remove chloroform to obtain a opalescent micelle solution, and then carrying out ultrasonic treatment in a water bath at 65 ℃ for 10min to further form a uniform micelle system. After removing the unencapsulated drug (erythromycin) by filtration through a 0.22 μm microporous membrane, it was stored at 4 ℃ for subsequent experiments.

The particle size and polydispersity of the erythromycin-loaded micelles prepared are shown in table 2. As can be seen from the results in Table 2, the particle size and dispersion coefficient of the erythromycin micelles prepared in sample 2-2 were better than those of the other samples. The particle diameter and polydispersity index (PDI) of the prepared erythromycin-loaded micelle (sample 2-2) are shown in FIG. 3.

TABLE 2

	Quality of erythromycin addition	Particle size (nm)	Coefficient of dispersion
				Sample 2-1	1％	178.6	0.254
Sample 2-2	5％	171	0.237
				Samples 2 to 3	10％	182.8	0.211
Samples 2 to 4	15％	187.5	0.260
				Samples 2 to 5	20％	183.6	0.247
Samples 2 to 6	25％	190.4	0.254

Comparative example 1

Adding PCL₅₀₀₀-MPEG₂₀₀₀Mixing with erythromycin accounting for 5% of the total mass of the high-molecular carrier material, dissolving in 10mL tetrahydrofuran, shaking to dissolve completely, and mixing uniformly; the other steps are the same as in example 1. The prepared erythromycin-carrying micelle has a particle size of 468.2nm and a polydispersity of 0.303.

Effect verification example 1 serum stability of erythromycin-carrying micelles

The stability of the erythromycin-carrying micelle in serum within three days was examined by using the particle size and polydispersity index (PDI) as indices to mimic the in vivo environment. Specifically, the erythromycin-loaded micelle solutions prepared in examples and comparative examples were mixed with fetal bovine serum uniformly in a volume of 1:1, and incubated in a 37 ℃ molecular hybridization apparatus at a constant temperature. The results of the particle size and PDI of the mixed solution are respectively measured in 0, 1, 2, 3, 4, 12, 24, 48 and 72h, and the results of the optimized erythromycin-loaded micelle sample 2-2 are shown in FIG. 4, so that the particle size does not change obviously within three days, and the carrier can still maintain the uniformly distributed micelle form, which indicates that the carrier has good stability.

The results of samples 2-1, 2-3 to 2-5 prepared in the examples show no significant change in particle size over three days, and the carrier is still able to maintain a uniformly distributed micelle morphology, indicating that the carrier is stable.

The results for samples 2-6 prepared in the examples show that the particle size changes greatly and is unstable after three days.

The results for the sample prepared in comparative example 1 showed an increase in particle size and an increase in PDI after three days of incubation, indicating poor stability.

Effect verification example 2 measurement of morphology under Transmission Electron Microscope (TEM) (including blank micelle and erythromycin-carrying micelle)

And carrying out morphological observation on the blank micelle and the erythromycin-loaded micelle prepared in the earlier stage by adopting a biological transmission electron microscope. The specific method comprises the steps of diluting the micelle to 0.5-1 mg/mL (carrier material concentration) by using deionized water as a dispersion medium, dripping 10 mu L of the diluted micelle onto a copper net paved with a carbon film, sucking the redundant liposome solution to dryness by using filter paper after 1min, dripping 10 mu L of phosphotungstic acid onto the copper net, re-dyeing the micelle on the copper net, sucking the micelle by using the filter paper after 1min, standing overnight, and observing by using a biological transmission electron microscope, wherein the measurement result is shown in a figure 5 (blank micelle sample 1-4) and a figure 6 (erythromycin-loaded micelle sample 2-2 prepared in example 1).

The two preparations are uniformly distributed, the blank micelle exists in a sphere-like shape, most of the erythromycin-carrying micelle is in a rod-like structure, and the length of the erythromycin-carrying micelle is within the range of 30-200 nm and the diameter of the erythromycin-carrying micelle is 10-20 nm under a transmission electron microscope.

Effect verification example 3 determination of encapsulation efficiency/drug-loading amount by alizarin charge-transfer method-ultraviolet spectrophotometry

In order to examine the encapsulation efficiency and the actual drug-loading rate of the erythromycin-loaded micelle, a rubiginine loading method-ultraviolet spectrophotometry method is established for determination. Alizarin and erythromycin can form a 1:1 type charge-transfer complex, and the absorption maximum is at 530 nm. The method comprises the following specific processes of precisely weighing a proper amount of freeze-dried powder carrying the erythromycin micelle, fully dissolving the freeze-dried powder with 2mL of acetone, adding 2mL of rubigins (1mg/mL) aqueous solution, adding water to a constant volume of 5mL, standing the mixture at room temperature for 30min, measuring an absorption value at 530nm, substituting a standard curve to calculate the erythromycin concentration, and substituting the following formula to calculate the erythromycin concentration:

encapsulation ratio (%) — erythromycin mass in micelle/erythromycin administration mass × 100

Erythromycin mass/total mass of drug-loaded micelle × 100

The results of the average drug loading and the average encapsulation efficiency of the erythromycin-loaded micelles are shown in Table 3, wherein the drug loading and the encapsulation efficiency of samples 1-1 to 1-7 are detected by using a high performance liquid phase after 5% of erythromycin is loaded.

TABLE 3

Effect verification example 4 in vitro Release of erythromycin-carrying micelles

0.1M PBS (pH7.4) was used as a release medium, and an ultra high performance liquid chromatography-triple quadrupole mass spectrometer (provided by the analytical test center of Shanghai university of transportation) was used as a detection means. The method comprises the following specific steps: 500. mu.L of a micellar solution containing 200. mu.g of erythromycin was taken and mixed with 3.5mL of a release medium (sample 2-2 in example 2), and placed in a dialysis tube of 10000MWCO, the tube was immersed in another 4mL of the release medium, a release test was performed at 37 ℃ and 100rpm, and 200. mu.L of an external dialysate was aspirated at predetermined time points (0.5h, 1h, 2h, 4h, 8h, 24h, 48h, and 72h) for measuring the drug content therein, and the cumulative release rate of the drug was calculated while immediately supplementing 200. mu.L of a fresh, preheated release medium. The result of the cumulative release rate of the erythromycin-loaded micelles at different time points is shown in fig. 7, and the slow release of erythromycin can be seen, and the release rate reaches about 80% in three days.

Effect verification example 5 measurement of cytotoxicity of erythromycin-carrying micelle

In order to explore the toxic effect of micelle preparations on cells, SKBR-3 breast cancer cells are taken as a model to carry out cytotoxicity determination of medicines, and a WST kit method is adopted for detection. Specifically, conventionally cultured SKBR-3 cells were digested with trypsin and counted in a 1X 10 order³The individual cells/well were plated on a 96-well plate and divided into erythromycin naked drug groups, erythromycin loaded micelle groups (sample 2-2 from example 2) and blank micelle groups. And continuously performing conventional culture for 24h, sucking out the culture solution, adding 60 mu L of liquid medicine diluted by sterile 0.1M PBS with the pH value of 7.4, wherein the drug concentrations of the erythromycin naked drug group and the erythromycin-loaded drug group are 4 mu g/mL, 8 mu g/mL, 20 mu g/mL, 40 mu g/mL and 80 mu g/mL in sequence, the erythromycin-loaded micelle and the blank micelle group have the same concentration of PCL-PEG, and each concentration is provided with three multiple holes. After incubation for 4h, the serum-free medium containing the drug was aspirated, 200. mu.L of fresh medium containing 10% serum was replaced, the culture was continued for 20h, 20. mu.L of WST reagent was added to each well, and finally incubation was carried out at 37 ℃ for 30min, and the absorbance at 450nm was measured for each well, and the cell activity was calculated. The results are shown in FIG. 8, and it can be seen that at different drug concentrations, neither the materials used nor erythromycin were toxic to the cells.

Effect test example 6 measurement of anti-Staphylococcus aureus Activity

Preparation of test sample mother liquor and concentration gradient: the preferred erythromycin-loaded micelles (sample 2-2 prepared using example 2) were divided into two parts: one part was left at 37 ℃ in 0.1M PBS (pH7.4) for 72 hours and then MIC (minimum inhibitory concentration) was measured, and the other part was immediately subjected to MIC measurement, with blank micelles and erythromycin naked drug as controls. In order to keep consistent with the concentration of the erythromycin-loaded micelle sample and the blank micelle material in the control group, the erythromycin-loaded micelle sample is diluted by PBS until the concentration of the erythromycin is 0.32mg/mL and the concentration of PEG-PCL is 3 mg/mL. The two-fold dilution procedure for the test sample solution was as follows: a sterile 96-well plate was used and 300. mu.L was added to well 1. 150. mu.L of sterile PBS was added to each of the test sample solutions obtained above at wells 2 to 8. Add 150. mu.L from well 1 to well 2 and mix well. Add 150. mu.L from well 2 to well 3, repeat to well 6, discard 150. mu.L, and add only 150. mu.L sterile PBS to a final well as a growth control. Thus, the sample solution to be tested with a series of gradient concentrations is obtained.

Determination of MIC (minimum inhibitory concentration) of sample: measured using the standard liquid micro-gradient dilution method recommended by CLSI 2013 edition. Inoculating single staphylococcus aureus colony separated and purified on solid plate into 3mL LB liquid culture medium, culturing overnight at 37 deg.C, diluting with corresponding liquid culture medium to obtain the final product with a bacterial concentration of about 10⁷CFU/mL of bacterial suspension. A sterile 96-well culture plate was prepared, and 20. mu.L of test sample dilutions of different gradient concentrations prepared in example 2 were added to each well, followed by addition of 180. mu.L of each bacterial suspension. The erythromycin solution and the PCL-PEG material solution with the same concentration are used as controls. After mixing, placing the mixture in an incubator at 37 ℃, and standing and culturing the mixture for 20 hours to judge the result. The effective concentration of the erythromycin is 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0 mu g/mL respectively; the effective concentration of PCL-PEG is 300, 150, 75, 37.5, 18.75, 9.4 and 0 mu g/mL. The same test sample dilution was set as 3 sets of parallel replicates. The MIC was determined by reference to the recommended standard (7.6.3) in the 2013 version of CLSI, with the lowest drug concentration that completely inhibited bacterial growth in the wells being the MIC.

As shown in Table 4, the MIC of the newly prepared drug-loaded micelle was much higher than that of the erythromycin nude drug (i.e., erythromycin control), but when the preparation was stored at 37 ℃ for three days and then subjected to the anti-Staphylococcus aureus activity assay test, the MIC of the drug-loaded micelle was found to be 2 times that of the erythromycin nude drug at the same concentration, while the carrier material showed no bacteriostatic effect.

TABLE 4

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Claims

1. The polycaprolactone-polyethylene glycol nano micelle loaded with the hydrophobic antibiotic is characterized by comprising the hydrophobic antibiotic and a micelle carrier, wherein the nano micelle has a core-shell structure; the hydrophobic block polycaprolactone and the hydrophobic antibiotic in the micelle carrier form an inner core part together, and the hydrophilic block polyethylene glycol forms an outer shell part.

2. The hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nanomicelle according to claim 1, wherein the nanomicelle is a rod-like micelle, and has a length of 30 to 200nm and a diameter of 10 to 20nm as shown under a transmission electron microscope.

3. The hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nanomicelle according to claim 1, wherein the micelle carrier is prepared by mixing the following components in a mass ratio of (0-0.8): 1 PCL₂₀₀₀-MPEG₂₀₀₀And PCL₅₀₀₀-PEG₂₀₀₀。

4. The hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nanomicelle according to claim 3, wherein the micelle carrier is prepared by mixing the following components in a mass ratio of (0.3-0.6): 1 PCL₂₀₀₀-MPEG₂₀₀₀And PCL₅₀₀₀-PEG₂₀₀₀。

5. The hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nanomicelle according to claim 1, wherein the hydrophobic antibiotic is erythromycin.

6. A method for preparing the hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nanomicelle according to any one of claims 1 to 5, comprising the following steps:

7. The preparation method of the hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nano micelle as claimed in claim 6, wherein in the step A, the addition amount of the hydrophobic antibiotic is 1-20% of the total mass of the micelle carrier.

8. The method for preparing hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nanomicelle according to claim 6, wherein in step A, the organic solvent is selected from tetrahydrofuran or chloroform.

9. The preparation method of the hydrophobic antibiotic-loaded polycaprolactone-polyethylene glycol nanomicelle according to claim 8, wherein the volume ratio of tetrahydrofuran to water is (3-9): 1; the volume ratio of chloroform to water is 1: (5-10).

10. An application of polycaprolactone-polyethylene glycol nano micelle carrying hydrophobic antibiotics in preparing a drug delivery system for treating infectious diseases.