CN116350588A - Antibiotic-carrying microorganism vesicle and preparation method and application thereof - Google Patents
Antibiotic-carrying microorganism vesicle and preparation method and application thereof Download PDFInfo
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- CN116350588A CN116350588A CN202310057617.6A CN202310057617A CN116350588A CN 116350588 A CN116350588 A CN 116350588A CN 202310057617 A CN202310057617 A CN 202310057617A CN 116350588 A CN116350588 A CN 116350588A
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- antibiotic
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- staphylococcus epidermidis
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
The invention belongs to the technical field of biological medicine, and particularly relates to an antibiotic-carrying microbial vesicle, and a preparation method and application thereof. The antibiotic-carrying microorganism vesicles comprise staphylococcus epidermidis extracellular vesicles and antibiotics wrapped in inner cavities of the staphylococcus epidermidis extracellular vesicles. The antibiotic-carrying microorganism vesicle takes the extracellular vesicle of staphylococcus epidermidis as a carrier, has nano-attribute and can be used as a nutrient substance to promote the growth of bacteria so as to increase the intake of the bacteria, prevent the antibiotic-carrying microorganism vesicle from generating drug resistance, further kill the bacteria at a low dose, and achieve the technical effects of effectively sterilizing and promoting the healing of skin wounds after being combined with antibiotics.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to an antibiotic-carrying microbial vesicle, and a preparation method and application thereof.
Background
Bacteria release nanoscale extracellular vesicles with diameters of 20-400nm, which carry a variety of biological components including lipopolysaccharide, peptidoglycan, cell membrane, periplasm, cytoplasmic proteins, toxins, nucleic acids, and the like. Bacterial extracellular vesicles affect a variety of biological processes including virulence, horizontal gene transfer, phage infection, cellular metabolite transport, and bacterial-bacterial or bacterial-host interactions. In terms of drug delivery, bacterial extracellular vesicles are similar to synthetic nanocarrier liposomes, both of which comprise biocompatible lipid bilayers. The nanocarrier of lipids provides a multifunctional platform for drug encapsulation, which allows for clinical conversion of a variety of drug formulations. Similarly, bacterial extracellular vesicles can transport a variety of bioactive molecules to recipient cells, thereby altering the physiological state of these cells. Notably, bacterial-derived extracellular vesicles have great potential to be novel drug delivery platforms due to their drug carrying capacity, immunostimulatory capacity, ease of modification, and good intercellular interactions.
In recent decades, research in the art on extracellular vesicles of gram-negative bacteria has been a long-standing development; however, few studies have been conducted on gram-positive extracellular vesicles. The main reason for the lack of interest of researchers in gram-positive extracellular vesicles is that their thick cell walls block extracellular vesicle release. Until 2009 Lee et al provided for the first time, conclusive evidence that the gram-positive bacterium staphylococcus aureus could naturally produce extracellular vesicles. Notably, gram-positive bacterial extracellular vesicles do not contain endotoxins specific to gram-negative bacterial extracellular vesicles, which makes them more suitable as drug delivery vehicles for effective intercellular interactions.
Wound infection is an important factor in the difficulty of healing a patient's wound. Antibacterial drugs are the first choice for clinical treatment of infectious diseases and have a very positive effect on improving the condition of patients. In the prior researches, cases of combining antibiotics and microbial extracellular vesicles to carry medicines exist, yang Yi and the like indicate that 'the antibiotic treatment not only damages normal flora of a human body to cause dysbacteriosis, but also can cause generation of a large number of vesicles to cause chronic inflammatory reaction' (the mechanism of the antibiotics inducing generation of gram-negative bacteria outer membrane vesicles and playing physiological roles [ J ]. Microorganisms and infection 2022,17 (2): 94-101), but most of the antibiotics induce skin inflammation, so that microbial resistance is increased, the effective antibacterial effect cannot be achieved, and the difficulty of wound repair is increased.
Disclosure of Invention
The invention aims to provide an antibiotic-carrying microorganism vesicle which can effectively kill various pathogenic bacteria, prevent the increase of bacterial drug resistance and promote the healing of skin wound surfaces.
The invention provides an antibiotic-carrying microorganism vesicle, which comprises an extracellular vesicle of staphylococcus epidermidis and antibiotics wrapped in an inner cavity of the extracellular vesicle of staphylococcus epidermidis.
Preferably, the antibiotic comprises a quinolone antibiotic or a macrolide antibiotic.
The invention also provides a preparation method of the antibiotic-carrying microorganism vesicle, which comprises the following steps: combining the staphylococcus epidermidis extracellular vesicles with antibiotics by a freeze thawing method to obtain the antibiotic-carrying microorganism vesicles.
Preferably, the staphylococcus epidermidis extracellular vesicles are used in the form of a staphylococcus epidermidis extracellular vesicle suspensionThe number of particles of the staphylococcus epidermidis extracellular vesicles in the staphylococcus epidermidis extracellular vesicle suspension is 2.33X10 × 10 11 ～10.37×10 11 particles/mL.
Preferably, the antibiotic is used in the form of an aqueous antibiotic solution, and the concentration of the aqueous antibiotic solution is 1/8MIC to saturation concentration.
Preferably, the volume ratio of the staphylococcus epidermidis extracellular vesicle suspension and the antibiotic aqueous solution is 1:1.
preferably, the freeze-thawing method comprises: mixing an extracellular vesicle of staphylococcus epidermidis with an antibiotic for freeze thawing treatment, wherein the procedure of the freeze thawing treatment is as follows: freezing at-196 deg.c or-80 deg.c for 5-30 min, refrigerating at-8-0 deg.c for 20-40 min, and thawing at 37-75 deg.c for 5-20 min; and repeating the cycle for 2 to 5 times.
The invention also provides the antibiotic-carrying microorganism vesicle in the technical scheme or the application of the antibiotic-carrying microorganism vesicle in the preparation of the medicine for resisting bacteria and/or promoting wound healing.
The invention also provides an antibacterial and/or wound healing promoting drug, which comprises antibiotic-carrying microorganism vesicles and auxiliary materials;
the antibiotic-carrying microorganism vesicle is the antibiotic-carrying microorganism vesicle according to the technical scheme or the antibiotic-carrying microorganism vesicle prepared by the preparation method according to the technical scheme.
Preferably, the effective dose of the antibiotic-carrying microbial vesicles in the medicament is at least 9.92 x 10, based on the particle concentration of the extracellular vesicles of staphylococcus epidermidis 9 particles/mL.
The beneficial effects are that:
the invention provides an antibiotic-carrying microorganism vesicle, which comprises an extracellular vesicle of staphylococcus epidermidis and antibiotics wrapped in an inner cavity of the extracellular vesicle of staphylococcus epidermidis. The antibiotic-carrying microorganism vesicle takes the extracellular vesicle of staphylococcus epidermidis as a carrier, has nano-attribute and can be used as a nutrient substance to promote the growth of bacteria so as to increase the intake of the bacteria, prevent the antibiotic-carrying microorganism vesicle from generating drug resistance, further kill the bacteria at a low dose, and achieve the technical effects of effectively sterilizing and promoting the healing of skin wounds after being combined with antibiotics.
Meanwhile, the invention also provides a preparation method of the antibiotic-carrying microorganism vesicle, which combines the epidermic grape bacteria extracellular vesicle with the antibiotic to prepare the antibiotic-carrying microorganism vesicle by a freeze thawing method, has high drug loading capacity and simple operation, and is easy to apply and produce.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a graph of SE-EVs nanoparticle tracking analysis in example 1;
FIG. 2 shows the non-inflammatory effect of SE-EVs on HaCaT cells in example 2;
FIG. 3 is a transmission electron microscope image of Lev@SE-EVs in example 3;
FIG. 4 is a graph showing average particle diameters of Lev@SE-EVs and Cal@SE-EVs in examples 4 and 6;
FIG. 5 is a potential diagram of Lev@SE-EVs and Cal@SE-EVs in example 4 and example 6;
FIG. 6 is a SDS-PAGE electrophoretogram of Lev@SE-EVs and Cal@SE-EVs in example 4 and example 6;
FIG. 7 is a graph of Lev concentration versus absorbance standard for example 4;
FIG. 8 is a transmission electron microscope image of Cal@SE-EVs in example 5;
FIG. 9 is a chemiluminescent diagram of Vibrio cholerae in application example 1;
FIG. 10 is a result of the Lev@SE-EVs inhibiting the chemiluminescence of Vibrio cholerae in application example 1;
FIG. 11 is a graph showing the dose response of cocultures of Lev@SE-EVs with Staphylococcus aureus at different particle concentrations in application example 1;
FIG. 12 is a graph showing the healing effect after treatment of infected wounds in mice with sterile SE-EVs solution, lev solution and Lev@SE-EVs, respectively, in application example 2;
FIG. 13 is a graph showing the number of residual Staphylococcus aureus after infection of wounds in mice treated with sterile SE-EVs solution, lev solution and Lev@SE-EVs, respectively, in application example 2.
Detailed Description
The invention provides an antibiotic-carrying microorganism vesicle, which comprises an extracellular vesicle of staphylococcus epidermidis and antibiotics wrapped in an inner cavity of the extracellular vesicle of staphylococcus epidermidis.
In the present invention, the antibiotic preferably includes a quinolone antibiotic or a macrolide antibiotic; the quinolone antibiotics preferably include, but are not limited to, levofloxacin, cinnoxacin, norfloxacin, enoxacin, ciprofloxacin, lomefloxacin, pefloxacin, fleroxacin, tosufloxacin, sparfloxacin, moxifloxacin, clinafloxacin, gemifloxacin, or sitafloxacin, further preferably levofloxacin; the macrolide antibiotic preferably includes, but is not limited to, azithromycin, clarithromycin, erythromycin or roxithromycin, and more preferably clarithromycin or roxithromycin.
In the present invention, the staphylococcus epidermidis extracellular vesicles are preferably secreted by staphylococcus epidermidis, preferably staphylococcus epidermidis ATCC12228. In the examples of the present invention, the particle size of the extracellular vesicles of Staphylococcus epidermidis secreted from Staphylococcus epidermidis ATCC12228 was 109.3.+ -. 1.1nm, and the ratio of the extracellular vesicles of Staphylococcus epidermidis in the range of 30 to 200nm was 97.6%.
The invention also provides a preparation method of the staphylococcus epidermidis extracellular vesicle, which comprises the following steps: sequentially performing activation culture and expansion culture on staphylococcus epidermidis to obtain fermentation liquor;
performing first centrifugation and suction filtration on the fermentation broth to obtain sterile filtrate;
concentrating the sterile filtrate and performing secondary centrifugation to obtain bacterial precipitate, namely the staphylococcus epidermidis extracellular vesicles.
The invention sequentially carries out activation culture and expansion culture on staphylococcus epidermidis to obtain bacterial liquid. The invention preferably inoculates staphylococcus epidermidis on LB solid culture medium, picks single colony in LB liquid culture medium for activating culture, and obtains seed liquid. The temperature of the activation culture according to the present invention is preferably 25 to 37℃and more preferably 37 ℃. The activation culture according to the present invention is preferably overnight culture, that is, preferably 8 to 12 hours, more preferably 12 hours.
After the seed liquid is obtained, the seed liquid is preferably inoculated into an LB liquid culture medium for expansion culture to obtain fermentation liquor. The volume ratio of the seed liquid to the LB liquid culture medium is preferably 1:100. the temperature of the expansion culture is preferably 25-37 ℃, more preferably 37 ℃; the time is preferably 24 hours. The amplification culture is preferably shake culture, and the rotation speed of shake culture is preferably 200rpm.
After the seed liquid is obtained, the seed liquid is preferably subjected to first centrifugation; the temperature of the first centrifugation is preferably 2-8 ℃, more preferably 4 ℃; the rotation speed is preferably 10000 to 14000g, more preferably 14000g; the time is preferably 20 to 60 minutes, more preferably 30 minutes. The number of the first centrifugation is preferably 1 to 3, more preferably 3.
After the first centrifugation, the invention performs the first suction filtration on the obtained filtrate and then performs the second suction filtration to obtain the sterile filtrate. The pore diameter of the filter membrane used for the first suction filtration is preferably 0.45 mu m; the pore size of the filter membrane used for the second suction filtration is preferably 0.22 μm.
After the sterile filtrate is obtained, the sterile filtrate is preferably concentrated. The concentration is preferably carried out using a tangential flow ultrafiltration device having a molecular weight cut-off of preferably 3 to 100kDa, more preferably 100kDa.
After the concentration, the present invention preferably subjects the obtained concentrated solution to a second centrifugation, and the supernatant is discarded, whereby the obtained bacterial pellet is the extracellular vesicles of staphylococcus epidermidis. The temperature of the second centrifugation is preferably 2-8 ℃, more preferably 4 ℃; the rotation speed is preferably 10000 to 20000g, more preferably 15000g; the time is preferably 1.5 to 3 hours, more preferably 2 hours.
The invention preferably further comprises the steps of cleaning the bacterial precipitate, mixing the cleaned precipitate with PBS solution, and repeatedly blowing to obtain the staphylococcus epidermidis extracellular vesicles which are uniformly dispersed, namely the staphylococcus epidermidis extracellular vesicles. The washing is preferably performed with a PBS solution, and the number of washing is preferably 2. The method of the repeated blowing is not particularly limited, and the extracellular vesicles of staphylococcus epidermidis may be dispersed in a PBS solution by a conventional repeated blowing method in the art.
The invention also provides a preparation method of the antibiotic-carrying microorganism vesicle, which comprises the following steps: combining the staphylococcus epidermidis extracellular vesicles with antibiotics by a freeze thawing method to obtain the antibiotic-carrying microorganism vesicles.
In the present invention, the staphylococcus epidermidis extracellular vesicles are preferably used in the form of staphylococcus epidermidis extracellular vesicle suspensions, and the preparation method is the same as above, and no description is given. The particle number content of the staphylococcus epidermidis extracellular vesicles in the staphylococcus epidermidis extracellular vesicle suspension is preferably 2.33X10 11 ～10.37×10 11 particles/mL, more preferably 6.20X10 11 particles/mL. The dispersing agent in the extracellular vesicle suspension of staphylococcus epidermidis according to the invention preferably comprises a sterile PBS solution, the pH value of which is preferably 7.4.
In the present invention, the binding of the antibiotic is preferably performed in the form of an aqueous antibiotic solution, the concentration of which is preferably 1/8MIC to saturated concentration, more preferably MIC to saturated concentration; the MIC represents the minimum inhibitory concentration. In one embodiment of the invention, the concentration of the aqueous solution of levofloxacin is 256g/mL and in another embodiment, the concentration of the aqueous solution of clarithromycin is 50. Mu.g/mL.
After the staphylococcus epidermidis extracellular vesicle suspension and the antibiotic aqueous solution are obtained, the invention preferably takes the staphylococcus epidermidis extracellular vesicle suspension and the antibiotic aqueous solution as raw materials, and prepares the antibiotic-carrying microorganism vesicle mixture by a freeze thawing method, wherein the antibiotic-carrying microorganism vesicle mixture comprises the antibiotic-carrying microorganism vesicle.
The volume ratio of the staphylococcus epidermidis extracellular vesicle suspension and the antibiotic aqueous solution is preferably 1:1 to 10, more preferably 1:1.
the freeze thawing method preferably comprises the steps of mixing the extracellular vesicle suspension of staphylococcus epidermidis and the aqueous solution of antibiotics, freezing at-196 ℃ or-80 ℃ for 5-30 min, refrigerating at-8-0 ℃ for 20-40 min, and thawing at 37-65 ℃ for 5-15 min; and repeating the cycle for 2 to 5 times. The freezing time is preferably 5min when the freezing temperature is preferably-196 ℃; the freezing temperature is preferably-80 ℃, and the freezing time is preferably 20-30 min. The temperature of the refrigeration according to the invention is preferably-1.4℃and the time is preferably 30 minutes. The thawing temperature according to the invention is preferably 65℃and the time is preferably 10 minutes. The number of cycle iterations of the present invention is preferably 3.
After completion of the freeze-thawing process, the present invention preferably further comprises removing antibiotics from the antibiotic-carrying microbial vesicle mixture that are not loaded into the extracellular vesicles of staphylococcus epidermidis. The removal of the invention preferably comprises a third centrifugation of the antibiotic-carrying microorganism vesicle mixture in an ultrafiltration tube to obtain a concentrate; after adding the concentrated solution into the PBS solution, centrifuging again, and repeating the operation for 3 times, wherein the obtained concentrated solution contains the antibiotic-carrying microorganism vesicles. The rotation speed of the third centrifugation is preferably 4000-5000 g, more preferably 4500g; the time is preferably 10 to 20 minutes, more preferably 15 minutes. The molecular weight cut-off of the ultrafiltration tube according to the invention is preferably between 30 and 100kDa, more preferably 100kDa. The parameters of the secondary centrifugation are the same as those of the third centrifugation, and the repeated description is omitted.
In one embodiment of the invention, the particle size of the antibiotic-carrying microbial vesicles (the antibiotic is levofloxacin and is marked as Lev@SE-EVs) prepared by the preparation method is 119.3+/-1.538 nm; PDI is 0.258+/-0.007 nm; the Zeta potential is-41.6+/-1.27 mV; the mass ratio of levofloxacin to the number of extracellular vesicles of staphylococcus epidermidis in lev@se-EVs is preferably 6.76 μg:10 11 particles。
In another embodiment of the present invention, the antibiotic-carrying microbial vesicles (antibiotic is clarithromycin, noted as cal@se-EVs) prepared by the above preparation method have a particle size of 16.9± 1.711nm; PDI is 0.245+/-0.011 nm; the Zeta potential is preferably-39.5.+ -. 1.36mV;
the invention also provides the antibiotic-carrying microorganism vesicle in the technical scheme or the application of the antibiotic-carrying microorganism vesicle in the preparation of the medicine for resisting bacteria and/or promoting wound healing. The medicament of the invention is preferably an antibacterial and wound healing promoting medicament. The antibiotic-carrying microorganism vesicle takes the extracellular vesicle of staphylococcus epidermidis as a carrier, has nano attribute and can increase the intake of bacteria, and can be used as a nutrient substance to promote the growth of bacteria, so that the antibiotic-carrying microorganism vesicle can be prevented from generating drug resistance while killing the bacteria at low dosage, and the technical effects of effectively sterilizing and promoting the healing of skin wounds can be achieved after the antibiotic-carrying microorganism vesicle is combined with antibiotics.
The invention also provides an antibacterial and/or wound healing promoting drug, which comprises antibiotic-carrying microorganism vesicles and auxiliary materials; the invention has no special limitation on the types of auxiliary materials, and the auxiliary materials can be added conventionally according to pharmaceutical requirements. The effective dose of the antibiotic-carrying microbial vesicles in the medicament of the invention is preferably at least 9.92 x 10, based on the particle concentration of the extracellular vesicles of staphylococcus epidermidis 9 particles/mL.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were obtained from conventional biochemical reagent purchasing sources. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Example 1
The preparation method of the staphylococcus epidermidis extracellular vesicles (SE-EVs) comprises the following steps:
1) Culturing staphylococcus epidermidis fermentation broth:
after thawing the glycerol frozen skin symbiotic microorganism staphylococcus epidermidis (ATCC 12228), streaking on an LB plate, culturing at a constant temperature of 37 ℃ until single colonies grow, and culturing the single colonies in LB liquid culture overnight to obtain seed liquid. Inoculating the seed solution into a large culture bottle containing TSB culture solution according to the volume ratio of 1:100, performing expansion culture, and performing shake culture in a shaking table at 37 ℃ for overnight (200 rpm/min) to obtain fermentation liquor.
2) Preparation of staphylococcus epidermidis extracellular vesicles (SE-EVs):
centrifuging the fermentation broth obtained in the step 1) in a centrifuge tube (12,000Xg, 20 min), discarding the precipitate to collect supernatant, and repeating the operation for three times; the supernatant was then filtered through a 0.45 μm filter followed by a 0.22 μm filter to give a sterile filtrate. The sterile filtrate was concentrated using a tangential flow ultrafiltration device with a molecular weight cut-off of 100kDa to obtain a concentrate containing SE-EVs. The concentrate was centrifuged at ultra-high speed (150,000Xg, 2.5 h) at 4℃and the supernatant was discarded, the pellet was washed three times with sterile PBS (pH 7.4) to obtain a uniformly dispersed SE-EVs suspension, which was finally passed through a 0.22 μm sterile filter head to obtain a sterile SE-EVs solution.
3) Nanoparticle Tracking Analysis (NTA) characterization: and (3) diluting the SE-EVs prepared in the step (2) according to different proportions of the suspension (including 10 times, 100 times and 1000 times of concentration of the suspension), respectively taking 100 mu L of diluted suspension, placing the diluted suspension into a sample cell, and measuring and displaying the result based on the particle number concentration measured at 20-120 particles/frame in order to ensure the accuracy of the result, namely, using the diluted suspension (77.9+/-3.5 particles/frame) of 1000 times.
The results showed that the concentration of SE-EVs particles in the stock solution was 6.35X10 11 ±4.02×10 11 particles/mL; particle size of 109.3+ -1.1 nm (shown in FIG. 1); the particle concentration in the distribution range of 30-200nm is 6.20X1011 particles/mL, the ratio is 97.6%, and therefore, the SE-EVs prepared by the method are mostly concentrated in a narrower range, and the influence on the ingestion caused by uneven size can be avoided, and the sterilization effect is influenced.
Example 2
The SE-EVs were validated for their non-inflammatory effect on the skin as follows:
HaCaT cells were seeded in 6-well plates at 37℃with 5% CO 2 Culturing in an incubator until 70-80%, adding LB culture solution containing 25 μg/mL SE-EVs prepared in example 1, 2 μg/mL LLPS (lipopolysaccharide) and 25 μg/mL SA-EVs (Staphylococcus aureus extracellular vesicles, the preparation method is the same as SE-EVs) into a 6-well plate respectively, and incubating for 24h, and detecting the production of inflammatory factors TNF-alpha, IL-1 beta and IL-6 by using a Soxhlet ELISA kit, as shown in FIG. 2.
From fig. 2, it can be derived that: the SE-EVs obtained by the preparation method in the example 1 can not induce the HaCaT cells to generate inflammatory factors, and the SA-EVs and the pro-inflammatory substance LPS can obviously induce the HaCaT cells to generate inflammatory factors, so that the SE-EVs in the example 1 can not generate inflammatory effects on skin and have antibacterial advantages.
Example 3
A preparation method of levofloxacin-loaded microbial vesicles (Lev@SE-EVs) comprises the following steps:
mixing the sterile SE-EVs solution prepared in the step 2) with 256g/mL of Lev water solution with the same volume, then placing the mixed solution in liquid nitrogen at the temperature of minus 196 ℃ for 5min, taking out, placing in a refrigerator at the temperature of minus 1.4 ℃ for 30min, finally placing in a water bath at the temperature of 65 ℃ for 10min, and repeating the operation for three times to finish freeze-thawing medicine carrying.
Centrifuging (4500 Xg, 15 min) the frozen and thawed mixed solution in ultrafiltration centrifugation of 100kDa, adding PBS solution (pH 7.4) into the concentrated solution of the ultrafiltration tube, mixing uniformly, centrifuging again, repeating the operation for three times, removing antibiotics not loaded into extracellular vesicles to obtain extracellular vesicle solution of drug-loaded microorganism, and finally passing the extracellular vesicle solution through a 0.22 μm sterile filter head to obtain sterile Lev@SE-EVs solution.
Example 4
Characterization of Lev@SE-EVs obtained in example 3 was performed as follows:
1) TEM characterization: after the Lev@SE-EVs solution prepared in the example 3 is properly diluted, respectively dripping 10 mu L of diluted solution on a copper mesh, adsorbing for 30min, and then drying by using filter paper; after further staining with 1% phosphotungstic acid for 5min, the filter paper was blotted dry and air-dried overnight, and the size and morphology of Lev@SE-EVs were observed by TEM, and the results are shown in FIG. 3.
From fig. 3, it can be derived that: the Lev@SE-EVs prepared in example 3 all exhibited a completely regular spherical structure with a particle size of about 120nm.
2) Particle size and potential: 1mL of the SE-EVs solution prepared in example 1 and the Lev@SE-EVs solution prepared in example 2 were added to a cuvette and a potentiometric cell, respectively, and the particle size and Zeta potential were measured by DLS, and the results are shown in FIGS. 4 to 5.
From fig. 4, it can be derived that: the particle sizes of the SE-EVs prepared in example 1 and the Lev@SE-EVs prepared in example 2 were 109.2.+ -. 0.8393nm and 119.3.+ -. 1.538nm, respectively. The measured polymer dispersibility indices PDI were 0.223.+ -. 0.013 and 0.258.+ -. 0.007, respectively;
from fig. 5, it can be derived that: the Zeta potentials of the SE-EVs prepared in example 1 and the Lev@SE-EVs prepared in example 2 were-42.8.+ -. 1.45mV and-41.6.+ -. 1.27mV, respectively.
After drug loading, the SE-EVs particle size slightly increased, but the potential did not change significantly.
3) Protein analysis: the SE-EVs solution prepared in example 1 and the Lev@SE-EVs solution prepared in example 2 were concentrated by a 100kDa ultrafiltration tube, 50. Mu.L of the concentrate was mixed with 5 Xprotein loading buffer in equal volumes and boiled for 10min, 10. Mu.L of the boiled mixed sample was loaded onto 20% SDS-PAGE, and subjected to electrophoresis at 100V for 2h, followed by Coomassie brilliant blue staining and decolorization with a decolorizing solution. The results show that: after drug loading, there was no change in protein in SE-EVs, as shown in FIG. 6.
4) Lev content determination in Lev@SE-EVs: 100 mu L of 256 mu g/mL Lev solution is taken, diluted in equal proportion and added into a 96-well plate, the absorbance is measured at 287nm, and a concentration-absorbance standard curve (shown in figure 7) is established, R 2 ＝0.9971>0.99, showing good linearity.
Lev@SE-EVs were then incubated with 0.5MEDTA for 3h at 37℃followed by centrifugation to remove empty SE-EVs. 100. Mu.L of the supernatant was added to 3 wells and its absorbance at 287nm was measured to be 1.942, 1.939 and 1.937, respectively. The concentration was calculated to be 42.93.+ -. 0.16. Mu.g/mL based on the standard curve. SE thereforeEVs comprising Lev at a concentration of 6.76. Mu.g/10 11 particles。
Example 5
A preparation method of clarithromycin-loaded microbial vesicles (Cal@SE-EVs) comprises the following steps:
the sterile SE-EVs solution prepared in example 1 was mixed with an equal volume of 50. Mu.g/mL of Cal aqueous solution, then the mixture was placed in liquid nitrogen at-196℃for 5min, removed and placed in a refrigerator at-1.4℃for 30min, finally placed in a water bath at 65℃for 10min, and the procedure was repeated three times to complete the freeze-thawing. Centrifuging the frozen and thawed mixed solution in ultrafiltration centrifugation of 100kDa (4500 Xg, 15 min), adding PBS (pH 7.4) into the concentrated solution of the ultrafiltration tube, mixing, centrifuging again, repeating the operation for three times, and removing antibiotics not loaded into extracellular vesicles to obtain the extracellular vesicle solution of the drug-loaded microorganism. Finally, the mixture is filtered through a 0.22 mu m sterile filter head to obtain a sterile Cal@SE-EVs solution.
Example 6
Characterization of Cal@SE-EVs obtained in example 5 was performed as follows:
1) TEM characterization: after properly diluting the Cal@SE-EVs solution prepared in example 5, 5. Mu.L of diluted solution is respectively dripped on a copper mesh, adsorbed for 20min, and then sucked by filter paper; after staining with 1% phosphotungstic acid for 3min, the filter paper was blotted dry and air-dried overnight, and the size and morphology of cal@se-EVs were observed by TEM, and the results are shown in fig. 8.
The TEM results in fig. 8 show that: cal@SE-EVs all exhibit a completely regular spherical structure with a particle size of about 116nm.
2) Particle size and potential: 1mLCal@SE-EVs solution was added to a cuvette and a potentiometric cell, and the particle size and Zeta potential were measured by DLS. The results show that the particle size of Cal@SE-EVs is 116.9+ -1.711 nm (as shown in FIG. 4), and the PDI is 0.245+ -0.011; the Zeta potential was-39.5.+ -. 1.36mV (see FIG. 5). After drug loading, the SE-EVs particle size slightly increased, but the potential did not change significantly.
3) Protein analysis: concentrating Cal@SE-EVs solution by using a 100kDa ultrafiltration tube, respectively taking 50 mu L of concentrated solution, mixing with 5X protein loading buffer solution in equal volume, boiling for 10min, taking 10 mu L of boiled mixed sample, loading to 20% SDS-PAGE, electrophoresis for 2h under 100V voltage, then coomassie brilliant blue staining, and decolorizing by using decolorizing solution, wherein the result shows that: after drug loading, there was no change in protein in SE-EVs (FIG. 6).
Test example 1
The antibacterial conditions of Lev@SE-EVs prepared in example 2 were studied by using the gram-negative bacteria vibrio cholerae and the gram-positive bacteria staphylococcus aureus as indicator strains, and the steps are as follows:
1) Vibrio cholerae against streptomycin (v.cc 6706; smR) was prepared as electrotransformed vibrio cholerae competent cells, and 5 μl of anti-caliamycin and anti-tetracycline plasmids pPlac-lux (50 ng; kanR, tetR) was mixed with 50 μl of electrotransformed vibrio cholerae competent cells and ice-bathed; transferring the mixture into a precooled sterile 0.2mm electric rotating cup, ice-bathing for 2min, wiping, and shocking at the voltage of 1.8 kv; rapidly adding 1mL of precooled LB culture medium, and recovering culture at 100rpm at 37 ℃ for 1h; coating the obtained bacterial liquid on a resistance plate containing the calicheamicin and the streptomycin, and culturing at 37 ℃ overnight; single colonies on the resistance plates, positive colonies, were verified for lux chemiluminescence.
The results show that: the chemiluminescent Vibrio cholerae was prepared (as shown in FIG. 9; only live bacteria were able to chemiluminescent, but dead bacteria were unable).
2) Then, chemiluminescent positive colonies were picked up, inoculated into LB medium containing calicheamicin, and cultured overnight, and diluted to 1X 10 with LB medium containing calicheamicin 9 CFU/mL of Vibrio cholerae bacterial liquid was prepared by taking 25. Mu.L of 1X 10 bacteria liquid, respectively 9 Adding CFU/mL vibrio cholerae bacterial liquid into three centrifuge tubes, respectively adding 25 mu L of PBS solution, SE-EVs solution in example 1 and Lev@SE-EVs solution in example 2 into the centrifuge tubes, uniformly mixing, and detecting chemiluminescence; after incubation at 37℃for 4 hours at 100rpm, the chemiluminescent conditions were detected (as shown in FIG. 10).
The results show that: after 4 hours, the chemiluminescence of the centrifuge tube added with the PBS solution and the SE-EVs solution is unchanged, and the chemiluminescence of the centrifuge tube added with the Lev@SE-EVs solution is obviously darkened, which indicates that the Lev@SE-EVs can kill vibrio cholerae, but the SE-EVs cannot.
3) The sterile SE-EVs solution prepared in example 1 was added to the first column A1, B1 and C1 wells of a 96-well plate, and the Lev@SE-EVs prepared in example 2 was added to the first column D1, E1 and F1 wells of a 96-well plate; 100 μLPBS solution was added to the A-F rows of a 96-well plate, followed by an equal dilution from column 1 to column 11, column 12 serving as a control; a further 100. Mu.L of 1X 10 was added to each well of the A-F rows 5 CFU/mL staphylococcus aureus liquid, cover the plate and cover, place in enzyme labeling instrument with program, culture at 37deg.C, shake and measure OD every 20 minutes 600 Value, according to its OD after 15h 600 The values establish a dose response curve (as shown in figure 11).
The results show that: SE-EVs have no antibacterial activity, lev@SE-EVs can effectively kill skin pathogenic bacteria staphylococcus aureus, and the particle concentration of Lev@SE-EVs required by minimum bacteriostasis is 9.92 multiplied by 10 9 particles/mL.
Test example 2
The antibacterial condition and the wound healing promoting condition of Lev@SE-EVs are studied by taking gram positive bacteria staphylococcus aureus as an indicator strain, and the steps are as follows:
18-20g female mice were divided into A, B, C, D groups of 6 animals. Dehairing the backs of mice with depilatory cream, after 24h skin barrier recovery, skin wound molding was performed on the backs of each mouse with a 6mm skin biopsy punch, followed by 100 μl1×10 7 CFU/mL staphylococcus aureus suspension was dropped at the wound of the mice. After 1h, group A mice were used as a blank to drop 100. Mu.L of the PBS solution at the wound, group B was dropped 100. Mu.L of the sterile SE-EVs solution prepared in example 1, group C was dropped 100. Mu.L of the Lev aqueous solution having a concentration of 0.125. Mu.g/mL, and group D was dropped 100. Mu.L of the Lev@SE-EVs prepared in example 2. Mice were photographed every two days for wound healing (results are shown in fig. 12). On day 11, mice were anesthetized, sampled at the healing wound with a 6mm skin biopsy punch, and after skin trituration, plated for colony count (as shown in 13).
From fig. 12 to 13, it can be derived that: compared to the other three groups, after topical application of lev@se-EVs prepared in example 2, the infected wound healed rapidly and the bacteria remaining from the infected wound were significantly reduced.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. An antibiotic-carrying microbial vesicle, comprising an extracellular vesicle of staphylococcus epidermidis and an antibiotic encapsulated in an inner cavity of the extracellular vesicle of staphylococcus epidermidis.
2. The antibiotic-carrying microbial vesicle of claim 1, wherein the antibiotic comprises a quinolone antibiotic or a macrolide antibiotic.
3. A method for preparing the antibiotic-carrying microbial vesicle according to claim 1 or 2, comprising: combining the staphylococcus epidermidis extracellular vesicles with antibiotics by a freeze thawing method to obtain the antibiotic-carrying microorganism vesicles.
4. A method of preparation according to claim 3, wherein the staphylococcus epidermidis extracellular vesicles are used in the form of a staphylococcus epidermidis extracellular vesicle suspension, the number of particles of the staphylococcus epidermidis extracellular vesicles in the staphylococcus epidermidis extracellular vesicle suspension being 2.33 x 10 11 ～10.37×10 11 particles/mL.
5. The method according to claim 3 or 4, wherein the antibiotic is used in the form of an aqueous antibiotic solution having a concentration of 1/8MIC to a saturated concentration.
6. The method according to claim 5, wherein the volume ratio of the extracellular vesicle suspension of staphylococcus epidermidis and the aqueous antibiotic solution is 1:1.
7. a method of preparation according to claim 3, wherein the freeze-thawing method comprises: mixing an extracellular vesicle of staphylococcus epidermidis with an antibiotic for freeze thawing treatment, wherein the procedure of the freeze thawing treatment is as follows: freezing at-196 deg.c or-80 deg.c for 5-30 min, refrigerating at-8-0 deg.c for 20-40 min, and thawing at 37-75 deg.c for 5-20 min; and repeating the cycle for 2 to 5 times.
8. Use of an antibiotic-carrying microbial vesicle according to claim 1 or 2 or an antibiotic-carrying microbial vesicle according to any one of claims 3 to 7 for the preparation of an antibacterial and/or wound healing promoting medicament.
9. A medicament having antibacterial and/or wound healing promoting functions, characterized in that the medicament comprises antibiotic-carrying microbial vesicles and auxiliary materials;
the antibiotic-carrying microorganism vesicle is the antibiotic-carrying microorganism vesicle in claim 1 or 2 or the antibiotic-carrying microorganism vesicle prepared by the preparation method in any one of claims 3 to 7.
10. The medicament according to claim 9, wherein the effective dose of the antibiotic-carrying microbial vesicles in the medicament is at least 9.92 x 10, based on the particle concentration of the extracellular vesicles of staphylococcus epidermidis 9 particles/mL.
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