CN115998706A - Preparation method and application of liposome microbubble complex for targeted loading of AIE and vancomycin - Google Patents

Abstract

The invention discloses a preparation method of a liposome microbubble compound of targeted loading AIE and vancomycin, which is used for preparing liposome and biotinylated microbubble of biotinylated AIE and vancomycin; and after the biotinylated microbubbles washed by the buffer saline are incubated at room temperature, adding the biotinylated AIE and vancomycin-loaded liposome, and then incubating to obtain the liposome microbubble complex. The liposome has a slow release function, the AIE and vancomycin in the liposome are released from the inside of the liposome to the periphery of the liposome, the AIE targets and identifies bacteria and is combined, ROS (reactive oxygen species) reaction is exerted to destroy bacterial membranes, bacterial death is promoted, and meanwhile, the vancomycin also exerts sterilization efficacy, so that the killing capacity to bacteria is further improved. After the liposome microbubble compound is formed by connecting the liposome microbubble compound with the microbubble through biotin, the substance is subjected to ultrasonic radiation, and the microbubble is contracted, expanded and ruptured to exert cavitation effect and acoustic pore effect, so that the permeability of peripheral blood vessels and cells is enhanced, the local medicine concentration is improved, and the medicine efficacy is improved.

Description

Preparation method and application of liposome microbubble complex for targeted loading of AIE and vancomycin

Technical Field

The invention relates to the technical field of medical treatment, in particular to a preparation method of a liposome microbubble compound for targeted loading of AIE and vancomycin.

Background

With the development of drug-resistant bacteria due to the large amount of antibiotics, vancomycin has been recommended by the expert consensus as a first-line drug for the treatment of MRSA as a narrow-spectrum antibacterial agent that exerts a strong killing effect only on gram-positive bacteria. However, the aqueous solution of vancomycin has unstable properties, so that the effective concentration of the vancomycin is reduced, and the bioavailability is lower through oral administration; if administered intravenously, the required concentration of the drug administered to achieve therapeutic concentrations is exceeded by the limits that the human body can withstand, and can increase otonephrotoxicity and the occurrence of "red syndrome". In order to improve the stability of vancomycin and the effective concentration of the drug, formulation of vancomycin has been studied by researchers, fan et al proposed to prepare gelatin/oxidized Salecan hydrogel as a carrier for delivery of vancomycin, and as a result, it was found that the resulting hydrogel exhibited excellent antibacterial ability and variable properties. In addition, the hydrogel shows ideal drug release kinetics and mechanism, and is successfully applied to the controlled release of vancomycin, so that the stable maintenance of the concentration of the vancomycin is realized. However, the substance is greatly influenced by the pH value, and when the substance is applied to the body, the property and the action of the substance can be influenced because the pH value in the body is in a dynamic change process, and the pore size and the structure of the hydrogel are also key factors for determining the efficacy of the hydrogel in tissues, and the pore size can influence the adhesive force of the hydrogel to surrounding cells or tissues, so that the gelatin/oxidized Salecan hydrogel is used as a carrier to load vancomycin to act in the body, and the uncontrollable factors are too many. There are many researchers who propose the use of liposomes as carriers, with vancomycin loading being a likely good choice. The existing liposome is used for delivering medicines for treating diseases, the treatment effect on the diseases is obvious on the premise of ensuring safety, and the invention patent with the application number of 201811105558 discloses a vancomycin nano liposome composition and a preparation method thereof. However, the effect of the substance is not remarkable when the substance is applied to the treatment of peri-prosthetic infections (Periprosthetic joint infection, PJI) after clinical artificial joint replacement, mainly because pathogenic bacteria can form a biological film in the occurrence process of the PJI, and the biological film mainly comprises polysaccharide, protein, nucleic acid and lipid, and the polysaccharide, the protein, the nucleic acid and the lipid are combined with each other to form a compact three-dimensional polymer network structure and are connected with bacteria in the biological film, so that the substance has good mechanical stability. The physical characteristics of the biological membrane prevent the vancomycin nano liposome from entering, so that the vancomycin cannot reach the deep layer of the biological membrane, the sterilization effect is exerted, and the treatment effect is reduced. Under the mediation of ultrasound, cavitation effect and sound pore effect can occur, so that the permeability of surrounding cells or blood vessels is enhanced, the permeation of medicines is promoted, and the therapeutic effect is better exerted. Tang Benzhong institutions and organizations develop a focusing induction luminescent material (AIE) which can well identify the types of pathogenic bacteria and has targeting effect on bacteria, and the substance gathers under the irradiation of near infrared light to exert oxidation stress (ROS) reaction, destroy the cell membrane of the bacteria identified in a targeting manner, lead the bacteria to crack and die, and exert the effect of targeted killing of the bacteria.

Based on the above, the liposome microbubble compound with the target loading AIE and vancomycin is proposed to be prepared, and the drug delivery mode is already applied to the treatment of diseases such as cancers and the like, and has a good application prospect.

Disclosure of Invention

The invention aims to provide a preparation method and application of a liposome microbubble compound for targeted loading of AIE and vancomycin.

In order to achieve the above purpose, the invention adopts the following technical scheme: the preparation method of the liposome microbubble compound for targeting and loading AIE and vancomycin comprises the following steps:

A. preparation of liposomes of biotinylated AIE and vancomycin: weighing proper amounts of DPPC, cholesterol, DSPE-PEG 2000-biotin, aggregation-induced emission material and vancomycin respectively, fully dissolving in acetone, fully dissolving, mixing, and extruding for several times through a polycarbonate lipid membrane filter to obtain a liposome of the biotinylation loaded AIE and vancomycin;

B. preparing biotinylated microbubbles;

C. washing and incubation: the obtained microbubbles were washed three times with phosphate buffered saline in a bucket centrifuge, incubated for 30min at room temperature with gentle shaking, and then biotinylated AIE and vancomycin-loaded liposomes were added and incubated for 30min at room temperature to obtain AIE and vancomycin-loaded liposome microbubble complexes.

Further, the preparation method of the biotinylated microbubbles comprises the following steps:

b1 weighing distearoyl phosphatidylcholine (DSPC) and biotin dipalmitoyl phosphatidylcholine (DSPE-PEG 2000-biotin) according to a proportion, respectively dissolving in chloroform, and respectively putting the two solutions in warm water to enable the two solutions to be fully dissolved;

b2, fully and uniformly mixing the two solutions in a test tube and placing the test tube on a test tube rack, introducing inert gas flow into the mixed solution to evaporate the mixed solution and form a layer of film on the wall of the test tube, and then placing the test tube in a vacuum pump for vacuum drying to obtain a lipid film;

adding the lipid membrane into Tris liquid for resuspension, then placing the solution into an ultrasonic vibration instrument for vibration to enable the lipid membrane to be completely dissolved in the Tris liquid, and then sub-packaging the solution into small penicillin bottles according to 1 mL/bottle;

b4, after the cover is sealed, the cover is opened, the sealing film is used for sealing, the sealed small penicillin bottle is connected with the injector, two ends of an iron frame for placing the injector are communicated, and one end of the iron frame is perfluoropropane (C ₃ F ₈ ) One end of the vacuum pump is connected, the valve connected with the vacuum pump is firstly closed, the valve connected with the vacuum pump is opened, after vacuumizing for 45min, the valve is closed, the valve connected with the gas is opened, the gas valve is kept for 10s, the vacuum valve is opened again, the operation is repeated for 6-8 times, finally the valve connected with the vacuum pump is closed, the valve connected with the gas is opened, the gas is kept to be filled for 10s, the penicillin bottle is removed from the injector, and finally the sealing film is used for sealing;

and B5, oscillating the prepared solution for 30s by using a silver mercury capsule blender to obtain the biotinylation microbubbles.

Further, in the step A, the molar ratio of the DPPC, the cholesterol, the DSPE-PEG 2000-biotin and the aggregation-induced emission material to the vancomycin is 60-70:25-35:3.5-4.5:0.8-1.2:0.8-1.2; the preferred molar ratio to mass ratio is 65:30:4:1:1.

further, in the step A, the extrusion times of the filter are more than or equal to 11.

Further, in the step A, the aperture of the polycarbonate lipid membrane is 200nm.

Further, in the step B1, the amounts of DSPC and DSPE-PEG2000 are as follows: 12 parts, 3.6 parts.

Further, in step B3, the Tris solution is prepared from glycerol: propylene glycol: 0.1M triple buffered saline solution at 1:1: 8.

The liposome microbubble compound of the targeted load AIE and vancomycin prepared by the method has the encapsulation rate of 83.22 percent, the average particle size of (165.07 +/-4.01) nm and the dispersity index of 0.33+/-0.04.

The liposome prepared by the invention has a slow release function, the AIE and vancomycin in the liposome are released from the inside of the liposome to the periphery of the liposome, the AIE targets and identifies bacteria and is combined, after near infrared light irradiation, the AIE gathers to exert oxidative stress (ROS) reaction to destroy bacterial cell membranes and promote bacterial death, and meanwhile, the released vancomycin also exerts sterilization effect to further increase the killing effect on the bacteria. After the liposome microbubble compound is formed by connecting the liposome microbubble compound with the microbubble through a biomembrane, the substance is subjected to shrinkage, expansion and rupture after ultrasonic radiation, and cavitation effect and acoustic pore effect are exerted, so that the permeability of peripheral blood vessels and cells is enhanced, the penetration of local medicine into the inside of the cells is promoted, the local medicine concentration is improved, and the medicine efficacy is improved.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is a flow chart of the preparation process of the invention;

FIG. 2 is a graph showing the results of the targeted loading of AIE and vancomycin liposomes according to example 4 of the present invention;

FIG. 3 is a graph showing the results of a scanning electron microscope before and after treatment of bacteria with AIE-loaded vancomycin liposome microbubble complex according to example 5 of the present invention;

FIG. 4 is a graph showing the results of confocal laser microscopy of the positional relationship between the biofilm and the complex in example 6 of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

The preparation method of the liposome microbubble complex (shown in figure 1) for targeting and loading AIE and vancomycin comprises the following steps:

A. preparation of liposomes of biotinylated AIE and vancomycin: weighing a proper amount of DPPC, cholesterol, DSPE-PEG 2000-biotin and an aggregation-induced emission material (DPPC, cholesterol, DSPE-PEG 2000-biotin, the molar ratio of the aggregation-induced emission material to vancomycin is 60:25:3.5:0.8), respectively and fully dissolving vancomycin (0.8 mg) in acetone, fully dissolving, mixing, extruding through a 200nm polycarbonate lipid film filter for 11 times, and obtaining a liposome of the biotin-loaded AIE and vancomycin by using a micro extruder;

B. preparation of biotinylated microbubbles

B1, weighing 12mg of distearoyl phosphatidylcholine (DSPC) and 3.6mg of biotin dipalmitoyl phosphatidylcholine (DSPE-PEG 2000-biotin) according to a proportion, respectively dissolving in 600 mu L of chloroform and 200 mu L of chloroform, and respectively putting the two solutions into warm water to fully dissolve the two solutions;

b2, fully and uniformly mixing the two solutions in a test tube and placing the test tube on a test tube rack, introducing inert gas flow into the mixed solution to evaporate the mixed solution and form a layer of film on the wall of the test tube, and then placing the test tube in a vacuum pump for vacuum drying to obtain a lipid film;

adding the lipid membrane into Tris liquid (glycerol: propylene glycol: 0.1M triple buffer saline (pH 7.4) solution in a ratio of 1:1:8) for resuspension, then placing into an ultrasonic vibration instrument for vibration to enable the lipid membrane to be completely dissolved in the Tris liquid, and then sub-packaging into small penicillin bottles according to 1 mL/bottle;

b4, after the cover is sealed, the cover is opened, the sealing film is used for sealing, the packaged small penicillin bottle is connected with the injector, two ends of an iron frame for placing the injector are communicated, and one end of the iron frame is perfluoropropane (C ₃ F ₈ ) One end of the vacuum pump is connected, the valve of the gas is firstly closed, the valve connected with the vacuum pump is opened, after the vacuum pump is vacuumized for 45min, the valve is closed, the valve connected with the gas is opened, the vacuum pump is kept for 10s, the valve of the gas is closed again, the vacuum valve is opened, the operation is repeated for 6 times, and finally the vacuum pump and the valve are closedA valve connected with a pump is opened, the gas is kept filled for 10 seconds, the small penicillin bottle is removed from the injector, and finally, a sealing film is used for sealing;

and B5, oscillating the prepared solution for 30s by using a silver mercury capsule blender to obtain the biotinylation microbubbles.

C. Washing and incubation: the resulting microbubbles were washed three times with phosphate buffered saline in a 10mL syringe, washed with a 400g bucket centrifuge for 3min to remove excess non-pooled lipids, and after incubation of the washed biotinylated microbubbles with gentle shaking at room temperature for 30min, 200 μl of biotinylated AIE-and vancomycin-loaded liposomes were added and incubated at room temperature for a further 30min to obtain AIE-and vancomycin-loaded liposome microbubble complexes.

Example 2

The preparation method of the liposome microbubble complex (shown in figure 1) for targeting and loading AIE and vancomycin comprises the following steps:

A. preparation of liposomes of biotinylated AIE and vancomycin: weighing a proper amount of DPPC, cholesterol, DSPE-PEG 2000-biotin and an aggregation-induced emission material (the molar mass ratio of the DPPC, the cholesterol, the DSPE-PEG 2000-biotin and the aggregation-induced emission material is 65:30:4:1), respectively and fully dissolving 1mg of vancomycin in acetone, fully dissolving and then mixing to finally obtain a liposome with the lipid concentration of 20mg/mL, extruding the liposome for 11 times through a 200nm polycarbonate lipid membrane filter, and obtaining the liposome loaded with AIE and vancomycin by using a micro extruder;

B. preparation of biotinylated microbubbles

B1, respectively weighing 11.8mg of DSPC and 3.5mg of DSPE-PEG2000, dissolving in 600 mu L of chloroform and 200 mu L of chloroform, respectively placing the three solutions in a beaker filled with warm water, and fully dissolving the three solutions;

b2, after dissolution, mixing the three materials and placing the mixture into a test tube, placing the test tube containing the mixed solution on a test tube rack, enabling the mixed solution in the test tube to act on the test tube by using stable nitrogen flow to accelerate the evaporation of the mixed solution, forming a layer of film on the wall of the test tube after the evaporation, and placing the test tube in a vacuum pump for vacuum drying for 3 hours;

b3, re-suspending the obtained lipid membrane in 5mL of Tris (glycerol: propylene glycol: 0.1M triple buffer saline (pH 7.4) solution in a ratio of 1:1:8), placing a test tube in an ultrasonic vibration instrument at 65 ℃ for vibrating for 5min to completely dissolve the lipid membrane in the Tris, packaging the obtained solution in small penicillin bottles, and uniformly packaging 1mL of the solution in each small bottle;

b4, after sealing the cover, opening the cover, sealing with sealing film, connecting the sealed small penicillin bottle with the injector (two ends of the iron frame for placing the injector are communicated, one end of the iron frame is perfluoropropane (C) ₃ F ₈ ) One end of the vacuum pump is connected, the valve connected with the vacuum pump is firstly closed, the valve connected with the vacuum pump is opened, after vacuumizing for 45min, the valve is closed, the valve connected with the gas is opened, the gas valve is kept for 10s, the vacuum valve is opened again, the operation is repeated for 7 times, finally the valve connected with the vacuum pump is closed, the valve connected with the gas is opened, after keeping the gas filling for 10s, the penicillin bottle is removed from the injector, and finally the sealing film is used for sealing;

and B5, finally, oscillating the prepared solution for 30s by using a silver mercury capsule blender to obtain Vm-MBs suspension C, washing and incubating: the resulting microbubbles were washed three times with phosphate buffered saline in a 10mL syringe, washed with a 400g bucket centrifuge for 3min to remove excess non-pooled lipids, and after incubation of the washed biotinylated microbubbles with gentle shaking at room temperature for 30min, 200 μl of biotinylated AIE-and vancomycin-loaded liposomes were added and incubated at room temperature for a further 30min to obtain AIE-and vancomycin-loaded liposome microbubble complexes.

Example 3

The preparation method of the liposome microbubble complex (shown in figure 1) for targeting and loading AIE and vancomycin comprises the following steps:

A. preparation of liposomes of biotinylated AIE and vancomycin: weighing a proper amount of DPPC, cholesterol, DSPE-PEG 2000-biotin and an aggregation-induced emission material (the molar mass ratio of the DPPC, the cholesterol, the DSPE-PEG 2000-biotin and the aggregation-induced emission material to vancomycin is 70:35:4.5:1.2), respectively fully dissolving vancomycin (1.2 mg) in acetone, fully dissolving, mixing, extruding through a 200nm polycarbonate lipid film filter for 11 times, and obtaining a liposome of the biotin-loaded AIE and the vancomycin by using a micro extruder;

B. preparation of biotinylated microbubbles

B1, respectively weighing 12.4mg of DSPC and 3.8mg of DSPE-PEG2000, dissolving in 600 mu L and 200 mu L of chloroform, dissolving 1mg of vancomycin in 20 mu L of physiological saline, and respectively placing the three solutions in a beaker filled with warm water to fully dissolve the three solutions;

b2, after dissolution, mixing the three materials and placing the mixture into a test tube, placing the test tube containing the mixed solution on a test tube rack, enabling the mixed solution in the test tube to act on the test tube by using stable nitrogen flow to accelerate the evaporation of the mixed solution, forming a layer of film on the wall of the test tube after the evaporation, and placing the test tube in a vacuum pump for vacuum drying for 3 hours;

b3, re-suspending the obtained lipid membrane in 5mL of Tris (glycerol: propylene glycol: 0.1M triple buffer saline (pH 7.4) solution in a ratio of 1:1:8), placing a test tube in an ultrasonic vibration instrument at 65 ℃ for vibrating for 5min to completely dissolve the lipid membrane in the Tris, packaging the obtained solution in small penicillin bottles, and uniformly packaging 1mL of the solution in each small bottle;

b4, after sealing the cover, opening the cover, sealing with sealing film, connecting the sealed small penicillin bottle with the injector (two ends of the iron frame for placing the injector are communicated, one end of the iron frame is perfluoropropane (C) ₃ F ₈ ) One end of the vacuum pump is connected, the valve connected with the vacuum pump is firstly closed, the valve connected with the vacuum pump is opened, after vacuumizing for 45min, the valve is closed, the valve connected with the gas is opened, the gas valve is kept for 10s, the vacuum valve is opened again, the operation is repeated for 8 times, finally the valve connected with the vacuum pump is closed, the valve connected with the gas is opened, after keeping the gas filling for 10s, the penicillin bottle is removed from the injector, and finally the sealing film is used for sealing;

b5, finally oscillating the prepared solution for 30s by using a silver mercury capsule blender to obtain Vm-MBs suspension

C. Washing and incubation: the resulting microbubbles were washed three times with phosphate buffered saline in a 10mL syringe, washed with a 400g bucket centrifuge for 3min to remove excess non-pooled lipids, and after incubation of the washed biotinylated microbubbles with gentle shaking at room temperature for 30min, 200 μl of biotinylated AIE-and vancomycin-loaded liposomes were added and incubated at room temperature for a further 30min to obtain AIE-and vancomycin-loaded liposome microbubble complexes.

Example 4

Targeted AIE-loaded vancomycin liposome slow release performance investigation

(1) Measuring the slow release efficiency of the targeted loading AIE and vancomycin liposome by adopting a high performance liquid chromatograph;

(2) The liquid phase was applied to an Agilent TC-C18 column (250.0mm. Times.4.6mm, 5 μm);

(3) The mobile phase is acetonitrile/0.0125 mol/L potassium dihydrogen phosphate buffer (pH value is 3.2) (10:90);

(4) The flow rate is 1ml/min, the column temperature is 35 ℃, the detection wavelength is 236nm, and the sample injection amount is 10 mu L;

(5) The results are shown in figure 2, and the targeted AIE and vancomycin-loaded lipids can release the vancomycin in the lipid at different times, so that the liposome has a slow release effect.

Example 5

The sterilization efficacy of the targeting AIE and vancomycin loaded liposome microbubble complex prepared in example 1 is examined

(1) Methicillin-resistant staphylococcus aureus (MRSA) was selected from 2-3 single colonies from platelets and placed in tryptone soy broth medium, which was incubated in a 160rpm/min incubator at 37℃for 24h.

(2) After the bacterial liquid is uniformly mixed, the wavelength is set to 570nm by an enzyme-labeled instrument, and the absorbance of the bacterial liquid is measured, so that the absorbance value of the final bacterial liquid is 0.5, and the bacterial liquid concentration is 10 ⁶ CFU/mL。

(3) 2mL of the prepared concentrated stock solution is taken and placed in a culture dish with a sterile cover slip, after incubation is carried out for 24 hours, the cover slip with bacteria attached is placed in glutaraldehyde and stored in a refrigerator at 4 ℃ for 4 hours, then the cover slip is taken out and washed 3 times with distilled water, tertiary butanol with concentration gradient of 50%, 70%, 90% and 100% (3 times) is respectively used for each operation, 15 minutes is kept, finally, metal spraying operation is carried out, and the density degree and the bacterial morphology of the bacteria attached on the cover slip are observed through a scanning electron microscope.

(4) Taking 2mL of the stock solution prepared in the step (2), placing the stock solution in a culture dish with a sterile cover slip, incubating for 24 hours, removing the stock solution, washing 3 times with distilled water, and adding 2mL of the stock solution with the concentration of 10 ⁷ After the vancomycin-carrying nano drug delivery system is continuously incubated for 24 hours, taking out a cover glass, placing the cover glass in glutaraldehyde, storing the cover glass in a refrigerator at the temperature of 4 ℃ for 4 hours, taking out the cover glass, cleaning the cover glass with distilled water for 3 times, respectively carrying out tertiary butanol with the concentration gradient of 50%, 70%, 90% and 100% (3 times), keeping the cover glass for 15 minutes each time, finally carrying out metal spraying operation, and observing the bacterial density and bacterial morphology attached to the cover glass by a scanning electron microscope.

(5) Comparing the morphological differences of bacteria attached to the cover glass before and after treatment by the targeting AIE and vancomycin liposome microbubble complex, and the result is shown in figure 3, wherein A. The cover glass is observed by a scanning electron microscope to be attached with a large amount of aggregated MRSA without any treatment, the bacteria have uniform morphology and better bacterial activity; B. after the targeting AIE and vancomycin liposome microbubble complex is processed, the scanning electron microscope observes that bacteria on the cover glass are dispersed, the number is obviously reduced, and the bacteria are uneven in morphology and size.

(6) The scanning electron microscope results show that compared with the control group, the target-loaded AIE and vancomycin liposome microbubble complex is obviously reduced in bacteria, and the activity of residual bacteria is obviously affected, so that the target-loaded AIE and vancomycin liposome microbubble complex has obvious sterilization efficacy (shown in figure 3).

Example 6

Investigation of the property of Targeted AIE-loaded and vancomycin-loaded Liposome microbubble Complex that can penetrate into biological Membrane

(1) Methicillin-resistant staphylococcus aureus (MRSA) was selected from 2-3 single colonies from platelets and placed in tryptone soy broth medium, which was incubated in a 160rpm/min incubator at 37℃for 24h.

(2) After the bacterial liquid is uniformly mixed, the wavelength is set to 570nm by an enzyme-labeled instrument, and the absorbance of the bacterial liquid is measured, so that the final product is obtainedThe absorbance value of the bacterial liquid is 0.5, and the bacterial liquid concentration is 10 ⁶ CFU/mL。

(3) And 2mL of the prepared concentrated stock solution is taken and placed in a confocal culture dish, after incubation is carried out for 24 hours, the culture medium is removed, after washing for 3 times by distilled water, SYTO59 fluorescent dye is added in a dark place, and after incubation is carried out for 15 minutes, the redundant liquid is removed, so that the biological membrane is marked by SYTO59 fluorescence.

(4) And incubating the prepared targeting AIE-loaded vancomycin liposome microbubble complex and DIL fluorescent staining agent for 15min in a dark place, and marking the complex with DIL red fluorescence.

(5) After the biological film and the compound which are respectively and fluorescently marked in the step (3) and the step (4) are incubated for 5min together, the liquid on the surface of the biological film is removed, the biological film is lightly washed for 3 times by double distilled water, and finally the positional relationship between the biological film and the compound is observed by a laser confocal microscope.

(6) The complex with red fluorescence was observed by laser confocal microscopy to be located in the deep, middle and surface layers of the biofilm with green fluorescence, and this result indicated that the complex material was able to penetrate into the deep layers of the biofilm, as shown in fig. 4.

Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be encompassed by the claims.

Claims (10)

1. The preparation method of the liposome microbubble complex for targeting and loading AIE and vancomycin is characterized by comprising the following steps:

A. preparation of liposomes of biotinylated AIE and vancomycin: weighing proper amounts of DPPC, cholesterol, DSPE-PEG 2000-biotin, aggregation-induced emission material and vancomycin respectively, fully dissolving in acetone, fully dissolving, mixing, and extruding for several times through a polycarbonate lipid membrane filter to obtain a liposome of the biotinylation loaded AIE and vancomycin;

B. preparing biotinylated microbubbles;

C. washing and incubation: the obtained microbubbles were washed three times with phosphate buffered saline in a bucket centrifuge, incubated for 30min with gentle shaking at room temperature, biotinylated AIE and vancomycin loaded liposomes were added and incubated for another 30min at room temperature to obtain AIE and vancomycin loaded liposome microbubble complexes.

2. The method of claim 1, wherein in step B, the method of preparing biotinylated microbubbles comprises:

b1 weighing distearoyl phosphatidylcholine (DSPC) and biotin dipalmitoyl phosphatidylcholine (DSPE-PEG 2000-biotin) according to a proportion, respectively dissolving in chloroform, and respectively putting the two solutions in warm water to enable the two solutions to be fully dissolved;

b2, fully and uniformly mixing the two solutions in a test tube and placing the test tube on a test tube rack, introducing inert gas flow into the mixed solution to evaporate the mixed solution and form a layer of film on the wall of the test tube, and then placing the test tube in a vacuum pump for drying to obtain a lipid film;

adding the lipid membrane into Tris liquid for resuspension, then placing the solution into an ultrasonic vibration instrument for vibration to enable the lipid membrane to be completely dissolved in the Tris liquid, and then sub-packaging the solution into small penicillin bottles according to 1 mL/bottle;

b4, after the cover is sealed, the cover is opened, the sealing film is used for sealing, the sealed small penicillin bottle is connected with the injector, two ends of an iron frame for placing the injector are communicated, and one end of the iron frame is perfluoropropane (C ₃ F ₈ ) One end of the vacuum pump is connected, the valve connected with the vacuum pump is firstly closed, the valve connected with the vacuum pump is opened, after vacuumizing for 45min, the valve is closed, the valve connected with the gas is opened, the gas valve is kept for 10s, the vacuum valve is opened again, the operation is repeated for 6-8 times, finally the valve connected with the vacuum pump is closed, the valve connected with the gas is opened, the gas is kept to be filled for 10s, the penicillin bottle is removed from the injector, and finally the sealing film is used for sealing;

and B5, oscillating the prepared solution for 30s by using a silver mercury capsule blender to obtain the biotinylation microbubbles.

3. The method according to claim 1, wherein in step a, the molar ratio of DPPC, cholesterol, DSPE-PEG 2000-biotin, aggregation-inducing luminescent material to vancomycin is 60-70:25-35:3.5-4.5:0.8-1.2:0.8-1.2.

4. The method according to claim 1, wherein in step a, the molar ratio to mass ratio of DPPC, cholesterol, DSPE-PEG 2000-biotin, aggregation-inducing luminescent material to vancomycin is 65:30:4:1:1.

5. the method according to claim 1, wherein in step A, the number of times the filter is pressed is not less than 11.

6. The method according to claim 1, wherein in step a, the polycarbonate lipid membrane has a pore size of 200nm.

7. The method of claim 2, wherein in step B1, the DSPC and DSPE-PEG2000 are used in an amount of: 12mg, 3.6mg.

8. The method of claim 2, wherein in step B3, the Tris solution consists of glycerol: propylene glycol: 0.1M triple buffered saline solution at 1:1: 8.

9. A targeted AIE and vancomycin loaded liposome microbubble complex prepared according to the method of any one of claims 1-8.

10. Use of the liposome microbubble complex targeted to support AIE and vancomycin according to claim 9 in the treatment of periprosthetic infections.

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