CN113350294A

CN113350294A - Preparation method of coated quorum sensing inhibitor composite particles and product thereof

Info

Publication number: CN113350294A
Application number: CN202110627216.0A
Authority: CN
Inventors: 孙亚娟; 邓涵越; 杨成; 吕妍
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2021-09-07
Anticipated expiration: 2041-06-04
Also published as: CN113350294B

Abstract

The invention discloses a preparation method of a composite particle for wrapping a quorum sensing inhibitor and a product thereof, wherein the preparation method comprises the steps of preparing a mixed solution of montmorillonite and sodium alginate as a water phase; preparing an oil phase containing a quorum sensing inhibitor; adding the oil phase into the water phase, and shearing and mixing to obtain a microemulsion; and cooling and diluting the obtained microemulsion to obtain an aqueous dispersion of the emulsion, dropwise adding an ionic cross-linking agent solution, stirring and crosslinking after dropwise adding, standing, washing the particles with deionized water, and freeze-drying to obtain the coated quorum sensing inhibitor composite particles. The invention takes the montmorillonite-based Pickering emulsion as a template to prepare particles, has lower biological toxicity, obviously improves the encapsulation rate and stability of the medicament, and greatly reduces the leakage of active ingredients in the storage process.

Description

Preparation method of coated quorum sensing inhibitor composite particles and product thereof

Technical Field

The invention belongs to the technical field of antibacterial agent preparation, and particularly relates to a preparation method of coated quorum sensing inhibitor composite particles and a product thereof.

Background

Carvacrol is a monoterpene compound and is found in several aromatic plants such as oregano (Origanum vulgare), piper nigrum (Lepidium flavum), thyme (Thymus vulgaris) and the like. According to research reports, the sub-inhibitory concentration of carvacrol can reduce the motility and the invasion of bacteria and can also reduce the biofilm formation of staphylococcus and salmonella. The results indicate that carvacrol interferes with quorum sensing signaling mechanisms between bacterial cells, thereby reducing the ability to form biofilms.

Carvacrol is a highly lipophilic phenolic substance, has low solubility in water, has high volatility, and has a strong odor of essential oils, so that it is difficult to use it in a pure form in practical applications.

Currently, pharmaceutical carriers have limitations in protecting active substances from adverse environmental conditions, increasing the solubility, bioavailability, and improving controlled release of lipid soluble substances. Such as: when the liposome is used as an entrapment system, because the outer membrane body of the liposome is thin, the content is easy to leak, the application range of the polymer nanoparticles is limited because the organic solvent used in the preparation process can not be completely removed, the solid lipid nanoparticles are easy to have polycrystalline transformation, the drug loading rate is low, and unpredictable gelation tendency can occur.

Therefore, there is a need in the art for a method of preparing composite microparticles with high encapsulation efficiency and stability of the drug, and reduced leakage of the active ingredient during storage, and products thereof.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.

Accordingly, it is an object of the present invention to overcome the deficiencies of the prior art and to provide a method for preparing coated quorum sensing inhibitor composite particles.

In order to solve the technical problems, the invention provides the following technical scheme: a method for preparing composite particles for wrapping quorum sensing inhibitor comprises,

preparing a montmorillonite and sodium alginate mixed solution as a water phase;

preparing an oil phase containing a quorum sensing inhibitor;

adding the oil phase into the water phase, and shearing and mixing to obtain a microemulsion;

and cooling and diluting the obtained microemulsion to obtain an aqueous dispersion of the emulsion, dropwise adding an ionic cross-linking agent solution, stirring and crosslinking after dropwise adding, standing, washing the particles with deionized water, and freeze-drying to obtain the coated quorum sensing inhibitor composite particles.

As a preferable embodiment of the method for preparing the coated quorum sensing inhibitor composite particles, the method comprises the following steps: the montmorillonite and sodium alginate mixed solution is prepared by the following steps,

adding sodium alginate into deionized water at 60 ℃, and stirring until the sodium alginate is completely dissolved to prepare a sodium alginate aqueous solution;

adding montmorillonite powder into sodium alginate aqueous solution, mixing, cooling to 25 deg.C, stirring for 1h, standing overnight to discharge bubbles, and making into montmorillonite and sodium alginate mixed solution.

As a preferable embodiment of the method for preparing the coated quorum sensing inhibitor composite particles, the method comprises the following steps: the montmorillonite and sodium alginate mixed solution comprises 1-2.5 wt% of montmorillonite and 0.25-2.5 wt% of sodium alginate.

As a preferable embodiment of the method for preparing the coated quorum sensing inhibitor composite particles, the method comprises the following steps: the mass fraction of the montmorillonite is 2 wt%, and the mass fraction of the sodium alginate is 2 wt%.

As a preferable embodiment of the method for preparing the coated quorum sensing inhibitor composite particles, the method comprises the following steps: the formulation comprises an oil phase containing a quorum sensing inhibitor, including,

heating and dissolving solid lipid at 65 deg.C, adding quorum sensing inhibitor after completely dissolving, and mixing to obtain oil phase containing quorum sensing inhibitor.

As a preferable embodiment of the method for preparing the coated quorum sensing inhibitor composite particles, the method comprises the following steps: the solid lipid comprises beeswax and monoglyceryl stearate, and the quorum sensing inhibitor comprises carvacrol; the mass ratio of the quorum sensing inhibitor to the oil phase is 1: 1-2.

As a preferable embodiment of the method for preparing the coated quorum sensing inhibitor composite particles, the method comprises the following steps: adding the oil phase into the water phase, and shearing and mixing to obtain the microemulsion, wherein the oil-water ratio is 1: 1-5, the shearing and mixing speed is 8000-25000 rpm, and the shearing and mixing time is 2-4 min.

As a preferable embodiment of the method for preparing the coated quorum sensing inhibitor composite particles, the method comprises the following steps: cooling and diluting the microemulsion, wherein the cooling temperature of the microemulsion is 1-10 ℃, and the dilution multiple is 5-100 times; the ionic crosslinking agent is any one or a mixture of two of zinc chloride, barium chloride, calcium dihydrogen phosphate, calcium sulfate, calcium hydrogen hydrochloride and calcium lactate aqueous solution, the concentration of the ionic crosslinking agent is 0.05-5 mol/L, and the volume ratio of the ionic crosslinking agent to the emulsion aqueous dispersion is 1: 3.

As a preferable embodiment of the method for preparing the coated quorum sensing inhibitor composite particles, the method comprises the following steps: and stirring and crosslinking, wherein the stirring and crosslinking time is 30-60 min, and the stirring rotating speed is 100 rpm.

It is a further object of the present invention to overcome the deficiencies of the prior art and to provide coated quorum sensing inhibitor composite particles prepared by the method.

The invention has the beneficial effects that:

(1) the preparation method takes the montmorillonite-based Pickering emulsion as a template to prepare particles, has low biological toxicity, obviously improves the encapsulation rate and stability of the medicament, and greatly reduces the leakage of active ingredients in the storage process; meanwhile, the study on the anti-pseudomonas aeruginosa quorum sensing activity of the carvacrol particles, the study on the contents of pyocin, the bacterial motility, the influence on bacterial cell biomembranes and the like is carried out, and the thought is widened for the practical application of the carvacrol particles.

(2) The carvacrol particles prepared by taking the montmorillonite-based Pickering emulsion as the template have better anti-quorum sensing activity, and the action mechanism is as follows: firstly, after the solid oil is compounded with the carvacrol essential oil, the carvacrol is not easy to leak, and the available concentration is guaranteed; after the Alg of the shell layer is gelled, carvacrol can be better stored in the shell layer; secondly, because the particle size of the particles is smaller, the transportation resistance of the active substances can be efficiently reduced, and the passive absorption of bacteria is enhanced. In addition, the smaller size can also increase the adhesion of particles on the surface of bacteria, and better block signal communication between bacteria, thereby achieving the anti-quorum sensing property.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a microscopic image of Pickering emulsion prepared from different types of oil phases in an example of the present invention, wherein (a) liquid paraffin; (c) beeswax; (e) GMS; and a micrographic image of the fine particles, wherein (b) liquid paraffin; (d) beeswax; (f) GMS.

FIG. 2 is a microscope photograph of emulsion of microparticles prepared with different concentrations of Alg according to the present invention, wherein (a) is 1.0%, (b) is 1.5%, (c) is 2.0%, (d) is 2.5%.

FIG. 3 is a microscopic image of microparticles prepared at different oil-water ratios in examples of the present invention, wherein (a) is 1:1, (b) is 1:3, and (c) is 1: 5.

FIG. 4 is a graph comparing the effect of GMS to carvacrol ratio on microparticle preparation in examples of the invention, wherein (a) is 2:1 and (b) is 1: 1.

FIG. 5 is a scanning microscope image of encapsulated quorum sensing inhibitor composite particles made in accordance with an embodiment of the present invention.

FIG. 6 is a comparative test chart of MIC determination of carvacrol particles in the embodiment of the invention, which is 850, 700, 550, 400, 250, 125 and 62.5 μ g/mL from left to right in sequence from 1 to 6; and (5) controlling.

FIG. 7 is a graph comparing the results of the montmorillonite-based Pickering emulsion particles inhibiting the pyocin production of Pseudomonas aeruginosa in the examples of the present invention.

FIG. 8 is a graph comparing the inhibition of Pseudomonas aeruginosa biofilms by sub-inhibitory concentrations of samples according to the examples of the present invention.

FIG. 9 is a graph comparing the inhibition of Pseudomonas aeruginosa (a) swimming and (b) colonization by samples of different concentrations in the examples of the present invention.

FIG. 10 is a comparison of the observation of bacterial adhesion in examples of the present invention in which (a-c) bacteria treated with carvacrol particles; (d-f) blank particle treated bacteria; (g) untreated bacteria; (h) adsorbing the bacteria by the particles; (i) a damaged bacterium.

FIG. 11 is a comparison of bacterial biofilms in accordance with an embodiment of the present invention, wherein (a-c) bacterial biofilms (1/2MIC, 1/4MIC, 1/8MIC) were treated with carvacrol microparticles; (d) untreated biofilms.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

And (3) observing the appearance of the particles: scanning electron microscopy was used to observe the surface topography of the microparticles. The method comprises the following specific steps: firstly, a layer of conductive adhesive is pasted on a sample table, then, a toothpick is used for picking up trace particle powder, the trace particle powder is uniformly scattered on the surface of the conductive adhesive, redundant particle powder is blown off by an ear washing ball, and the surface appearance of the particles is observed after gold spraying.

Particle size analysis of the microparticles: photographs were taken under a scanning microscope and the average particle size of the prepared microparticles was counted using Nano Measure 1.2 software.

Preparing a culture medium and culturing bacteria:

LB (Luria-Bertani) Medium configuration: weighing 1% tryptone, 0.5% yeast extract and 1% NaCl respectively, placing in a beaker, adding distilled water into the beaker, stirring with a magnetic stirrer until the drug is completely dissolved, pouring the solution into a conical flask, adding water to the required volume, and adjusting the pH to 7.2 with 1.0mol/L NaOH solution. If preparing solid culture medium, adding 2% agar, heating to melt, packaging, wrapping, and sterilizing with high pressure steam at 121 deg.C for 20 min. Preparation of PDP medium: weighing 2% tryptone, 1.4% MgCl2 and 1% K2SO4, dissolving in warm water, adjusting pH to 7.3 +/-0.1 with 1mol/L NaOH solution, adding 10mL glycerol, adding pure water to a constant volume of 1000mL, and sterilizing at 121 ℃ under high pressure for 20 min.

The swimming culture medium is prepared by the following method: 0.3% of agar, 1.0% of tryptone and 0.5% of NaCl; the formula of the cluster culture medium is as follows: 0.5% of agar, 1.0% of tryptone, 0.5% of NaCl and 0.5% of glucose.

The microorganism is Pseudomonas aeruginosa (ATCC 9027), commonly commercially available. The strains were stored at-80 ℃ in LB medium containing 25% glycerol. Inoculating the strain suspension on LB slant culture medium at 4 deg.C for storage, inoculating the strain on LB solid culture medium, aerobic culturing at 37 deg.C, inoculating the single strain on LB liquid culture medium after vigorous bacterial flora grows out, culturing at 37 deg.C for 24 hr to obtain strain suspension, and repeating the process each time.

Determination of Minimum Inhibitory Concentration (MIC): the Minimum Inhibitory Concentration (MIC) of carvacrol was determined with minor modification according to the double dilution method of the clinical and laboratory standards association. Specifically, the bacteria were activated overnight, cultured at 8000rpm for 5min to remove biofilm, diluted with LB medium to a turbidity of 0.1 for measurement, and MIC test was performed using 96-well microplate to determine absorbance. First 100 μ L of medium was added to each well, a stock solution of carvacrol was diluted and transferred to the first well, after which the carvacrol concentration in each well was 1/2 from the previous well, and all wells were flushed 20 times with a pipette to ensure that the solutions were well mixed. The 96-well plate was incubated in an incubator at 37 ℃ for 24 h. The MIC was determined using a microplate reader to detect absorbance at OD 600. The lowest concentration that showed complete inhibition of visible growth represents the minimum inhibitory concentration. Three replicates of each concentration were run, and a negative control (medium only without added broth) and a positive control (medium only without added active drug) were run on the same plate.

The minimum inhibitory concentration of the drug-loaded emulsion or drug-loaded microparticles was measured using a tetramethylazozolium (MTT) colorimetric method. The slightly modified fold-dilution method was used to prepare 6 gradient drug concentrations of 1000. mu.g/mL, 850. mu.g/mL, 700. mu.g/mL, 550. mu.g/mL, 400. mu.g/mL, 250. mu.g/mL and blank control, respectively. Each 100. mu.L of the suspension was put into a 96-well plate, and 100. mu.L of the bacterial suspension and 100. mu.L of 0.5% MTT were added thereto, followed by incubation at 37 ℃ in an incubator for 24 hours. MIC was determined according to colorimetric method.

Determination of pyocyanin content:

1/2MIC, 1/4MIC and 1/8MIC were selected for the analysis of pyocin release, with three sets in parallel for each concentration and a blank set. The pseudomonas aeruginosa is cultured overnight by using a PDP culture medium until the OD600 is 0.1, and then the culture medium with or without carvacrol and emulsion or drug-loaded particles are uniformly mixed with bacteria and cultured for 24 hours at 37 ℃. Subsequently, the suspension was centrifuged at 10000rpm for 5min to remove bacteria. Thereafter, the pyocin in the supernatant was extracted with 1mL of dichloromethane, then extracted again with 400. mu.L of 0.2mol/L HCI, and the liquid obtained after the extraction was placed in a 96-well plate and the absorbance was measured at OD 520. The inhibition rate was calculated from the following formula:

inhibition rate (%) (control OD)₅₂₀Test OD₅₂₀) /(control OD)₅₂₀)]×100。

And (3) determining the content of the biological membrane by a crystal violet staining method: the effect of carvacrol and the drug-loaded emulsion on the formation of pseudomonas aeruginosa biofilms was reflected by quantitative measurements by crystal violet assay. Briefly, the OD value of pseudomonas aeruginosa was diluted to 0.1 with LB medium, inoculated into a well plate, then carvacrol at 1/2, 1/4, 1/8MIC concentrations and a drug-loaded emulsion or drug-loaded microparticles were added to a 96 well plate, flushed with a pipette, and incubated in an incubator at 37 ℃ for 24 h. After 24h incubation, the upper medium was aspirated and carefully washed three times with sterile Phosphate Buffered Saline (PBS, pH 7.0) to remove free bacteria. Drying in an oven at 45 ℃, adding 0.1% by mass of crystal violet staining solution, standing and dyeing for 30 min. After the staining solution is removed by suction, the biological membrane is washed for 3 times by using sterile PBS solution again to remove excessive staining agent, and then the treated biological membrane is decolorized for 30min by using ethanol solution with the volume fraction of 95%. The absorbance was measured at 570nm using a microplate reader. Three replicates of each group were run and a blank control was set. The inhibition rate was calculated from the following formula:

inhibition rate (%) (control OD)₅₇₀OD of sample₅₇₀₎/(control OD)₅₇₀)]×100

Detection of swimming and cluster movements:

after mixing the bacterial suspension and carvacrol or drug-loaded emulsion or drug-loaded microparticles well, 5 μ L of the liquid was transferred to the center of a prepared electrophoresis agar plate (0.3% agar) to assess the motility of pseudomonas aeruginosa in semi-solid agar. For cluster motility (0.5% agar), 5 μ L of inoculum was pipetted into the center of the agar surface to visualize motility on the agar surface. The diameters of bacterial migration and colonization were measured after incubation at 37 ℃. Each group was measured three times and a blank control group was set.

Observing the adhesion of bacteria by a microscope: pseudomonas aeruginosa with OD600 of 0.1 and active substances or emulsion or drug-loaded particles with different concentrations are mixed uniformly and inoculated into a 6-well plate containing a sterile glass sheet, and the mixture is cultured for 24 hours at 37 ℃. After 24h, the coverslips were carefully rinsed with sterile PBS buffer to remove loosely adhering bacteria. The coverslips were then fixed with 2.5% glutaraldehyde for 60min and washed again 3 times with sterile PBS buffer. Thereafter, ethanol (10%, 30%, 50%, 70%, 90%, 100%) was dehydrated for 15min using different concentration gradients, respectively. The treated cover glass is dried at room temperature, sprayed with gold by an ion sputtering device, and observed under a scanning electron microscope after completion.

And (3) observing a bacterial biofilm: pseudomonas aeruginosa with OD600 ═ 0.1 and different concentrations of active or microparticles were mixed well and inoculated into 6-well plates containing sterile glass slides and incubated at 37 ℃ for 24 h. After 24h, the coverslips were carefully rinsed with sterile phosphate buffer to remove loosely adhering bacteria. And taking out the glass sheet, dyeing the glass sheet with crystal violet for 30min, and observing the biofilm under a microscope with ultra-depth of field.

Example 1

The embodiment provides a preparation method of coated quorum sensing inhibitor composite particles, which comprises the following steps:

(1) adding sodium alginate into deionized water at 60 ℃, and stirring until the sodium alginate is completely dissolved to prepare a sodium alginate aqueous solution; adding montmorillonite powder into sodium alginate aqueous solution, mixing, cooling to 25 deg.C, stirring for 1h, standing overnight to discharge bubbles, and making into montmorillonite and sodium alginate mixed solution as water phase for use, wherein the montmorillonite accounts for 2 wt%, and the sodium alginate accounts for 2 wt%;

(2) heating and dissolving monoglyceride stearate at 65 ℃, adding carvacrol after the monoglyceride stearate and the carvacrol are completely dissolved, and uniformly mixing the monoglyceride stearate and the carvacrol to obtain an oil phase, wherein the mass ratio of the monoglyceride stearate to the carvacrol is 2: 1;

(3) heating the water phase prepared in the step (1) to 70 ℃, pouring the water phase into a beaker filled with the oil phase in the step (2), and continuously emulsifying for 2min at 12000rpm under a high-speed shearing machine to form uniform Pickering emulsion, wherein the volume ratio of the oil phase to the water phase is 1: 3;

(4) cooling the emulsion obtained in the step (3) in ice water at 4 ℃, taking out 1g of the emulsion after the emulsion is cooled, adding the emulsion into 199g of deionized water, and stirring at 100rpm until the emulsion is completely dispersed to obtain an aqueous dispersion of the emulsion;

adding CaCl at 4 deg.C₂Dripping the solution into the aqueous dispersion of the emulsion, continuously stirring at 100rpm for 1h after dripping, stopping stirring after solidification, and standing at room temperature for 1h, wherein the CaCl is₂The concentration of the solution is 2.5mol/L, CaCl₂The volume ratio of the solution to the aqueous emulsion dispersion was 1: 3.

(5) And washing the obtained particles with deionized water, centrifuging, and freeze-drying to obtain the coated quorum sensing inhibitor composite particles.

Example 2

Under the conditions of example 1, the control conditions were: the effect of the oil phase of liquid paraffin, beeswax and GMS (mono glyceryl stearate) on the preparation of microparticles was investigated in the same manner as in example 1 except that the MMT (montmorillonite) content was 2% (w/w), the Alg (sodium alginate) content was 2% (w/w), the oil: carvacrol (mass ratio) was 2:1, and the oil-water ratio was 1:5 (volume ratio).

The results are shown in FIG. 1.

Fig. 1(a) and (b) are a Pickering emulsion microscopic image and a microparticle SEM image prepared using liquid paraffin as an oil phase. It can be seen that the particle size of the emulsion is about 20 μm, the particle size difference after curing is large, and the shape is not fixed. The reason is that the preparation process of the particles needs to undergo a large amount of steps of dilution, stirring, centrifugation and the like, the Pickering emulsion prepared by the liquid paraffin can be demulsified and coalesced, more oil phase cannot be wrapped due to the loss of the template, and MMT particles and Alg form aggregates and sink. Therefore, fine particles having a good quality cannot be obtained after curing.

Fig. 1(c) and (d) are microscope images and particle SEM images of Pickering emulsion prepared by using beeswax as an oil phase, and since beeswax is easily crystallized and a higher temperature is required in an emulsification process, the particle size distribution of the prepared Pickering emulsion is wider, the emulsion stability is weakened, and a demulsification phenomenon is increased, so that ineffective crosslinking of Alg is increased, and particles with better sphericity cannot be obtained.

FIGS. 1(e) and (f) are microscope images and microparticle SEM images of Pickering emulsions prepared using GMS as the oil phase. As can be seen from the figure, the particle size of the emulsion is about 1-3 μm, and the Pickering emulsion prepared by using GMS as an oil phase has better dispersibility under the same dilution degree.

In addition, GMS has better emulsification performance and the prepared emulsion is more stable. When the prepared Pickering emulsion is placed in cold water for cooling, GMS serving as an oil core is rapidly solidified when meeting cold, active substances are effectively wrapped inside, and MMT and Alg on the outer layer are fixed on the outer layer of the oil core. During curing, the outer layer of Alg and MMT can better form a shell layer on the outside, so that the particles with better sphericity and higher entrapment performance can be prepared.

GMS is therefore preferred as the oil phase in the present invention.

Example 3

Influence of Alg mass fraction: under the conditions of example 1, the control conditions were: the effect of Alg mass fractions of 1%, 1.5%, 2.0%, and 2.5% on the preparation of fine particles was examined for an MMT (montmorillonite) content of 2% (w/w), GMS: carvacrol of 2:1, and an oil-water ratio of 1:5, and the other conditions were the same as in example 2.

The results are shown in FIG. 2, in which (a) is 1.0%, (b) is 1.5%, (c) is 2.0%, and (d) is 2.5%.

It can be seen that at lower Alg content, there is not enough Alg and Ca²⁺Crosslinking may result in partial shell layers of the cured particles not being cured into a network or shell layers being thin, leaving the outer portion of the particle insufficiently hard and rigid to withstand external forces. And the fragile shell layer can be broken during curing and dispersion, the oil phase cannot be well wrapped, and the particles are greatly agglomerated. On the other hand, when the Alg concentration is high, Ca is contained during solidification because the continuous phase contains a large amount of free Alg²⁺The Alg on the particle shell and the Alg of the continuous phase are connected together and cannot be separated, so that the formed particles are distributed in a colony pattern, and the figure also shows that more free Alg solidified bodies exist. In addition, too much wall material cannot be adhered to the fine particlesThe surface, also causes the waste of wall material, so the invention selects 2% (w/w) of Alg as the optimum concentration.

Example 4

Influence of oil-water ratio: under the conditions of example 1, the control conditions were: when the content of MMT (montmorillonite) was 2% (w/w), the content of Alg was 2% (w/w), and GMS: carvacrol was 2:1, the effect on the microparticle preparation was investigated when the oil-water ratio was 1:5, 1:3, and 1:1, respectively.

The results are shown in FIG. 3, in which: (a)1:1, (b)1:3 and (c)1:5, and it can be seen that when the oil phase content is higher, the viscosity of the whole system is rapidly increased, a larger rotating speed is required to form an emulsion during homogeneous emulsification, and an excessively high rotating speed generates larger heat, so that the quality and the activity of carvacrol are reduced.

Further, the morphology of the fine particles is more affected by external force due to the increase of lipid, and the regularity is deteriorated by random deposition of the fine particles, thereby generating fine particles having different shapes. In addition, when the oil phase ratio is increased, the droplets formed by the GMS emulsification are smaller in size, and part of the droplets are adhered to the surface of the particles and washed away during washing, thereby causing loss. At lower oil phase levels, the encapsulation efficiency of the microparticles may be reduced.

Comprehensively, when the oil-water ratio is 1:3, the oil-water ratio is the optimal oil-water ratio for preparing the particles.

Example 5

Effect of GMS to carvacrol ratio:

the effect of GMS to carvacrol ratios of 2:1 and 1:1 on microparticle preparation was investigated at an MMT content of 2% (w/w), an Alg content of 2% (w/w), and an oil-to-water ratio of 1:3 under the conditions of example 1. The results are shown in FIG. 4, (a)2:1, (b)1: 1.

It can be seen that when the proportion of carvacrol is large, the mixed solid lipid cannot form harder particles, and is easier to peel off lipid fragments, even to cause particle dent, and the content flows out (as shown in figure 4b), which has a great influence on the solidification of subsequent particles.

When too much carvacrol is contained, GMS may not be capable of completely wrapping carvacrol in the inner core of the lipid, and a part of carvacrol is distributed in the outer layer of the lipid, so that the carvacrol is easier to leak. And because the mixed lipid is too soft, the particle particles are more easily adhered together to form larger aggregates which are not easy to separate during centrifugal washing.

Example 6

Scanning microscope analysis of the encapsulated quorum sensing inhibitor composite particles prepared in example 1 was performed, and the results are shown in fig. 5, wherein (a) and (b) are scanning microscope images under the same conditions.

FIG. 5 shows a scanning electron micrograph of montmorillonite-based Pickering emulsion particles, which are not very regular spheres and have obvious surface wrinkles. When not dried, the sphericity of the microparticles observed with an optical microscope is better. This is probably due to the fact that the microparticles produced are more hydrated and shrink more on drying. At the same time, Ca²⁺The cross-linking with Alg takes place rapidly in a short time to form a network structure, and due to the presence of MMT, Alg does not adhere completely to the core material wall after cross-linking, which makes the surface of the produced microparticles non-smooth (see FIG. 5 b).

According to the statistical result of the scanning electron microscope photos, the particle size of the dried particles is mostly 1-3 μm, the particle size distribution is uniform, but a small amount of agglomeration occurs. This may be due to the inability to disperse the wet granules uniformly in water during drying.

Example 7

(1) Determination of minimum inhibitory concentration

FIG. 6 is a graph of MIC determination comparison test of carvacrol microparticles, which is 850, 700, 550, 400, 250, 125, 62.5. mu.g/mL, blank from left to right.

As shown in fig. 6, under all test conditions, the minimum concentration of undeveloped carvacrol in a 96-well plate is 250 μ g/mL, which indicates that the minimum inhibitory concentration of carvacrol loaded by the montmorillonite-based Pickering emulsion particles is 250 μ g/mL, which is much lower than the minimum inhibitory concentration measured by carvacrol alone, but compared with carvacrol loaded by emulsion, the minimum inhibitory concentration is not changed, and the concentration of carvacrol which is too low to inhibit bacterial growth may not be reached.

(2) Determination of pyocyanin content

Pyocin can reflect the virulence of the strain and is one of the important virulence factors of pseudomonas aeruginosa. It is involved in a variety of cellular functions, and excessive amounts of pyocin directly result in cell death.

The inhibition result of the montmorillonite-based Pickering emulsion particles on the yield of the pyocin of the pseudomonas aeruginosa is shown in figure 7, and under the sub-inhibitory concentration, the yield of the pyocin is gradually reduced along with the increase of the concentration of the carvacrol. In fig. 7, the control group is a blank without any substance added, and the blank particles are the group of composite microparticles without carvacrol added.

When the mass concentration of the carvacrol is 31.25 mu g/mL (1/8 MIC), the inhibition rate of the carvacrol-loaded particles on pyocin reaches 28.64%, while the inhibition rate of the corresponding pure carvacrol essential oil is 3.39%, and under the same concentration, the inhibition rate of the carvacrol Pickering emulsion is 15.92%. When the mass concentration is 125 mug/mL, namely 1/2MIC, the inhibition rate of the carvacrol-loaded particles on pyocin is as high as 56.92%, the inhibition rate of corresponding pure carvacrol essential oil is 28.36%, and the inhibition rate of the carvacrol Pickering emulsion is 45.6% under the same concentration.

The result shows that under the action of carvacrol with the same mass concentration, the effect of inhibiting the bacteria from generating pyocyanin is as follows: the particle is larger than the emulsion and larger than the pure essential oil, and the inhibition rate of the particle to the pyocin is at least nine percent higher than that of the emulsion under each sub-bacteriostatic concentration. At these concentrations, there was no effect on the growth of pseudomonas aeruginosa, indicating that drug-loaded microparticles inhibited the quorum-sensing regulated production of pyocin.

(3) Determination of biofilm content

The biofilm of pseudomonas aeruginosa is the most important cause of bacterial resistance. The bacterial biofilm is in a highly structured sheet shape, most bacteria can generate the biofilm under a proper condition, but the formation of the biofilm can obviously increase the drug resistance of the bacteria in the membrane to drugs, which can cause serious damage to the putrefaction of goods and the health of human bodies. After the culture, a circle of film is formed on the inner wall of the 96-well plate, and the formed amount of the biological film is reflected by the absorbance after dyeing.

As shown in fig. 8, the amount of biofilm formation decreased significantly as the mass concentration of carvacrol increased. When the mass concentration is 125 mug/mL, namely 1/2MIC, the inhibition rate of the drug-loaded particles on the biological membrane reaches the maximum, namely 74.03%. Compared with the drug-loaded Pickering emulsion and pure carvacrol essential oil with the same concentration, the concentration of the drug-loaded Pickering emulsion and the pure carvacrol essential oil are respectively 8.75 percent and 23.67 percent higher, because the Alg on the surface of the particle is more easily adhered to a cell membrane for action after solidification. The blank particles have little inhibition on the biological membrane because bacteria are adhered to the surfaces of the particles, so that partial bacteria can not reach enough concentration for quorum sensing.

Example 8

(1) Inhibition of exercise capacity

The motility of bacteria is regulated by flagella, and can move through the contraction and extension processes of the bacteria. The motility of bacteria affects the formation and structural morphology of biofilms and involves the initial adhesion of bacteria. Therefore, the research on the movement capability of the pseudomonas aeruginosa has important significance for controlling the formation of the biofilm. The experiment researches the inhibition effect of carvacrol-loaded particles on the motility of pseudomonas aeruginosa. The experimental result shows that the carvacrol particles have the weakening effect on the swimming motility and the clustering motility of the pseudomonas aeruginosa under the condition that the bacterial growth is not inhibited, and have good dose dependence relationship. The control group without carvacrol has better swimming motility of pseudomonas aeruginosa. The bacteria in the experiment group added with the carvacrol cannot be fully diffused and form a colony around the inoculation position, and the inhibition on the bacteria migration ability is correspondingly enhanced along with the increase of the concentration of the carvacrol. The results are shown in FIG. 9, and the inhibitory effect of samples of different concentrations on Pseudomonas aeruginosa (a) swimming and (b) colonization.

As can be seen from FIG. 9a, the inhibition rate of the migration ability of Pseudomonas aeruginosa is 84.3% when the concentration of the drug-loaded particles is 1/2 MIC. The mean diameter of the colonies at concentrations 1/4MIC and 1/8MIC were 5.73mm and 9.71mm, respectively, while the mean diameter of the colonies under the inhibition of the drug-loaded emulsion was 9.24mm and 13.63mm, respectively, and the mean diameter of the colonies under the inhibition of carvacrol alone was 11.78mm and 14 mm. The average colony diameter under the action of carvacrol particles is obviously smaller than that of the other two control groups through comparison.

Similar to the above experimental results, carvacrol microparticles were also able to significantly inhibit colonization of pseudomonas aeruginosa and also had a good dose-dependent relationship, with the results shown in fig. 9b, where the carvacrol concentration in the drug-loaded microparticles increased from 1/8MIC to 1/2MIC, the inhibition of colonization of pseudomonas aeruginosa increased from 60.17% to 74.64%.

In conclusion, from the inhibition data of carvacrol particles on the swimming motility and the cluster movement of pseudomonas aeruginosa, the particles have better inhibition effect on the quorum sensing of the pseudomonas aeruginosa than the emulsion and the carvacrol essential oil alone, and are potential carriers for transporting quorum sensing inhibitors.

(2) Observation of bacterial adhesion

Micrographs by scanning electron microscopy are shown in FIG. 10, where (a-c) bacteria treated with carvacrol microparticles; (d-f) blank particle treated bacteria; (g) untreated bacteria; (h) adsorbing the bacteria by the particles; (i) a damaged bacterium.

It was found that the bacteria treated with the carvacrol microparticles had largely failed to see intact cell structures, and that bacterial colonies were progressively smaller and dispersed as the concentration of carvacrol increased. Similar to the bacteria treated with the carvacrol Pickering emulsion, the bacteria showed significant shrinkage, cracking, denting, etc. Furthermore, destruction of bacteria after carvacrol microparticle treatment was more severe (fig. 10 i). This is probably because the particle size of the particles is smaller and it is easier to penetrate the cell membrane to reach the interior of the bacterial cells, and the micrograph 10h shows that the surface of the particles can adsorb bacteria, so that the active substance can better act on the bacterial biofilm, and a better quorum sensing inhibition effect can be achieved.

(3) Bacterial biofilm observation

In order to explain the effect of the carvacrol-loaded montmorillonite-based particles on the pseudomonas aeruginosa biofilm more intuitively, the experiment is observed by using an ultra-depth-of-field microscope. The formation of the biofilm mainly comprises the stages of initial adhesion of bacteria, formation of early biofilm and mature diffusion. The biofilms of pseudomonas aeruginosa provide enhanced resistance to the environment, immune system and antibacterial drugs by establishing a physical barrier to the spread of bacteria to other environments.

FIG. 11 is a bacterial biofilm map wherein (a-c) bacterial biofilms (1/2MIC, 1/4MIC, 1/8MIC) were treated with carvacrol microparticles; (d) untreated biofilms.

The study elected to observe the initial biofilm formation stage when the bacteria adhered (24 h). After 24h incubation, it was observed that pseudomonas aeruginosa had begun to be in an initial adherent state, that bacteria not treated with carvacrol particles began to clump together in large numbers, and that the mucus layer surrounded a large number of bacteria. Moreover, untreated pseudomonas aeruginosa formed a complete, dense biofilm. However, the biofilm treated with carvacrol particles gradually decreased in membrane area and increased in structure with increasing concentration (see fig. 11).

The cross-linking agent is selected from calcium chloride, the dosage is 0.05-5 mol/L, the optimal dosage is 2.5mol/L, when the dosage exceeds the range, the cross-linking is incomplete and the particles are easy to break when the dosage is too low; when the concentration is too high, a large amount of particles are agglomerated together, the dispersibility is poor, and the particles at the agglomerated center cannot be crosslinked to a poor degree, and at this time, the salt particles cannot be sufficiently washed off.

In conclusion, through the research on the content of pyocin generated by pseudomonas aeruginosa, the carvacrol-loaded montmorillonite-based particles have the strongest inhibition on the toxicity of pseudomonas aeruginosa. Probably because the surface of the particles can adsorb bacteria and directly transmit active substances to the surface of the bacteria, the generation of virulence factors of the bacteria is inhibited. Scanning microscope shows that the surface of the thallus is rough, the cell wall is damaged, and intracellular substances leak out. The biomembrane content and morphological characterization show that the effect of the carvacrol particles is far better than that of pure carvacrol essential oil. Several studies simultaneously show that the carvacrol particles prepared by taking the montmorillonite-based Pickering emulsion as the template have the best anti-quorum sensing activity, and the action mechanism is as follows: firstly, after the solid oil and the carvacrol essential oil are compounded, the carvacrol is not easy to leak, and the available concentration is guaranteed. And the carvacrol can be better stored in the shell layer after the Alg of the shell layer is gelled. Secondly, because the particle size of the particles is smaller, the transportation resistance of the active substances can be efficiently reduced, and the passive absorption of bacteria is enhanced. In addition, the smaller size can also increase the adhesion of particles on the surface of bacteria, and better block signal communication between bacteria, thereby achieving the anti-quorum sensing property.

The solid lipid is used as an oil phase to encapsulate carvacrol, and the solid lipid-encapsulated quorum sensing inhibitor used in the prior art is only lipid, so that polycrystalline transformation is easy to cause, the drug loading rate is low, and an unpredictable gelation trend can occur. In addition, in the scheme of taking the existing solid lipid particle as an entrapment system, an organic solvent and an active substance are required to be compounded firstly, and the problem of incomplete removal of the organic solvent and increased toxicity possibly exists during preparation of the drug-loaded particle.

The whole preparation process of the invention avoids the use of all organic reagents or toxic substances. Generally, the preparation of the particles adopts a liquid oil phase, and oily liquid active substances such as carvacrol have over-high fluidity during entrapment, so that the phenomena of instability or content outflow and the like are caused. In addition, the system outer wall material is not single sodium alginate, and montmorillonite is added, so that the rigidity of the prepared particles is enhanced, the loaded drug can be better protected, and the montmorillonite has adsorption performance, so that the montmorillonite can be synergized with carvacrol in application, and the quorum sensing resistance effect is greatly improved.

The invention introduces the montmorillonite-based Pickering emulsion as a template to prepare particles, has low biological toxicity, obviously improves the encapsulation rate and stability of the medicament, and greatly reduces the leakage of active ingredients in the storage process. Meanwhile, the study on the anti-pseudomonas aeruginosa quorum sensing activity of the carvacrol particles, the study on the contents of pyocin, the bacterial motility, the influence on bacterial cell biomembranes and the like is carried out, and the thought is widened for the practical application of the carvacrol particles.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A preparation method of coated quorum sensing inhibitor composite particles is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

preparing an oil phase containing a quorum sensing inhibitor;

2. The method of preparing the encapsulated quorum sensing inhibitor composite microparticle of claim 1, wherein: the montmorillonite and sodium alginate mixed solution is prepared by the following steps,

3. The method of preparing the encapsulated quorum sensing inhibitor composite microparticles of claim 1 or 2, wherein: the montmorillonite and sodium alginate mixed solution comprises 1-2.5 wt% of montmorillonite and 0.25-2.5 wt% of sodium alginate.

4. The method of preparing the encapsulated quorum sensing inhibitor composite microparticle of claim 3, wherein: the mass fraction of the montmorillonite is 2 wt%, and the mass fraction of the sodium alginate is 2 wt%.

5. The method of preparing the encapsulated quorum sensing inhibitor composite microparticle of claim 1, wherein: the formulation comprises an oil phase containing a quorum sensing inhibitor, including,

6. The method of preparing the encapsulated quorum sensing inhibitor composite microparticle of claim 5, wherein: the solid lipid comprises beeswax and monoglyceryl stearate, and the quorum sensing inhibitor comprises carvacrol; the mass ratio of the quorum sensing inhibitor to the oil phase is 1: 1-2.

7. The method of preparing the encapsulated quorum sensing inhibitor composite microparticles of claim 1 or 2, wherein: adding the oil phase into the water phase, and shearing and mixing to obtain the microemulsion, wherein the oil-water ratio is 1: 1-5, the shearing and mixing speed is 8000-25000 rpm, and the shearing and mixing time is 2-4 min.

8. The method of preparing the encapsulated quorum sensing inhibitor composite microparticles of claim 1 or 2, wherein: cooling and diluting the microemulsion, wherein the cooling temperature of the microemulsion is 1-10 ℃, and the dilution multiple is 5-100 times; the ionic crosslinking agent is any one or a mixture of two of zinc chloride, barium chloride, calcium dihydrogen phosphate, calcium sulfate, calcium hydrogen hydrochloride and calcium lactate aqueous solution, the concentration of the ionic crosslinking agent is 0.05-5 mol/L, and the volume ratio of the ionic crosslinking agent to the emulsion aqueous dispersion is 1: 3.

9. The method of preparing the encapsulated quorum sensing inhibitor composite microparticles of claim 1 or 2, wherein: and stirring and crosslinking, wherein the stirring and crosslinking time is 30-60 min, and the stirring rotating speed is 100 rpm.

10. The coated quorum sensing inhibitor composite particles prepared by the preparation method of the coated quorum sensing inhibitor composite particles as claimed in any one of claims 1 to 9.