CN115944752A - Engineered fused membrane bubble, preparation method and application thereof - Google Patents
Engineered fused membrane bubble, preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a preparation method of an engineered fused membrane bubble, the fused membrane bubble obtained by the preparation method and application of the fused membrane bubble in targeted ultrasound contrast of atherosclerotic plaques. The method comprises treating cells with Bafilomycin A1 (BAF), and extracting cell membrane with cell membrane protein extraction kit; preparing liposome by rotary evaporation; preparing a fusion membrane by repeatedly freezing and thawing the extracted cell membrane and liposome by using liquid nitrogen; and preparing the fused membrane bubbles by using an ultrasonic-assisted method for the prepared fused membrane. The invention adds BAF to up-regulate the expression of APOA1 in the process of in vitro culturing HepG2 cells, then extracts the cell membranes of the cells to fuse with artificially synthesized liposome, finally fills sulfur hexafluoride gas in the molecular rearrangement process of gas-liquid interface ultrasound to form fused membrane bubbles with uniform particle size and stable structure, and applies the fused membrane bubbles in the ultrasound molecular image of atherosclerotic plaques.
Description
Technical Field
The invention relates to a preparation method of an engineered fusion membrane bubble and application thereof in targeted ultrasound contrast of atherosclerotic plaques.
Background
Ultrasonic imaging is the most common method for detecting atherosclerosis in clinic except blood examination, and the influence of lesion on blood flow can be observed while the blockage situation of plaque in blood vessel is observed. Compared with common ultrasound, contrast enhancement ultrasound can obviously enhance the contrast effect and clearly display the fine vascular structures of normal and pathological tissues. Since the general contrast agent lacks specificity or has poor specificity and is easily cleared by the immune system in vivo, a contrast agent with strong specificity and long circulation time in vivo is urgently needed to realize accurate detection of atherosclerotic plaques.
APOA1 is a major functional protein of High Density Lipoprotein (HDL), and not only can bind to B-type scavenger receptor SR-BI, which is highly expressed by foam cells, but also can form a new high density lipoprotein with phospholipids to take up cholesterol, activate Lecithin Cholesterol Acyltransferase (LCAT) to convert cholesterol into cholesterol ester and transfer it from peripheral tissues to the liver, and thus APOA1 can be used to target atherosclerotic plaques. The literature reports that APOA1 is contained in the membrane fraction of HepG2 cells. Under conditions of nutrient enrichment, APOA1 levels in HepG2 cells are maintained in dynamic equilibrium by autophagy clearance and protein synthesis dependent on the mammalian rapamycin target protein complex 1 (mTORC 1) pathway. Therefore, inhibiting autophagy is a potential method for improving the expression level of APOA1. CD47 is a transmembrane glycoprotein widely expressed on cancer cell membranes, has an immune escape function, and can remarkably prolong the circulation time of a contrast agent in a body.
Disclosure of Invention
The invention mainly aims to design an engineered fused membrane bubble preparation method, which comprises the steps of adding BAF to up-regulate APOA1 expression in the process of in vitro culture of HepG2 cells, extracting cell membranes of the cells to fuse with artificially synthesized liposomes, finally filling sulfur hexafluoride gas in the molecular rearrangement process of gas-liquid interface ultrasound to form fused membrane bubbles with uniform particle size and stable structure, and applying the fused membrane bubbles in the ultrasound molecular image of atherosclerotic plaques.
In order to achieve the above purpose, the invention provides a preparation method of an engineered fused membrane bubble, which comprises the following steps:
step S1, treating cells by Bafilomycin A1 (BAF), and extracting cell membranes by using a cell membrane protein extraction kit;
s2, preparing a liposome by a rotary evaporation method;
s3, preparing a fusion membrane by repeatedly freezing and thawing the cell membrane extracted in the step S1 and the liposome prepared in the step S2 by using liquid nitrogen; and
and S4, preparing the fused membrane bubbles by using an ultrasonic-assisted method for the fused membrane prepared in the step S3.
A further development of the invention is that step S1 comprises the following steps:
s11, culturing human liver cancer cells (HepG 2) in a T75 cell culture bottle containing 10% serum RMPI-1640 culture medium, adding Phosphate Buffer Solution (PBS) for cleaning for 2-3 times, adding 4mL of trypsin solution containing Ethylene Diamine Tetraacetic Acid (EDTA) for digestion for 2-3min, adding 6mL of 10% serum-containing RMPI-1640 culture medium to stop digestion after 80% of cells are detached under microscope observation, transferring the cells into a 15mL centrifuge tube for centrifugation at 900rpm for 3min, sucking supernatant, then resuspending the cells with 1mL of culture medium, adding 10 mu L of cell suspension into a cell counting plate, counting by using a cell counter, adding 4 multiplied by 106 cells into a round dish with the diameter of 10cm for culture for 24 hours;
step S12, taking out the culture medium in the round dish in the step S11, adding PBS (phosphate buffer solution) for washing for 2-3 times, adding an RMPI-1640 incomplete culture medium containing BAF (basic organic fertilizer) for incubation for 6 hours in an incubator, adding the RMPI-1640 incomplete culture medium into a control group, sucking out the culture medium after incubation is finished, adding 4mL of PBS after PBS is washed for 2-3 times, scraping cells by using a cell scraper and transferring the cells to a centrifuge tube;
and S13, centrifuging the cells obtained in the step S12 at 900rpm for 3 minutes to absorb the supernatant, adding a cell membrane protein extraction reagent A for resuspension, repeatedly freezing and thawing the cells for 2-3 times after uniformly mixing the cells, centrifuging the lysed cells for 10 minutes at 700g and 4 ℃ to remove cell nucleus precipitates, collecting the supernatant, centrifuging the supernatant for 30 minutes at 14000g and 4 ℃ to obtain precipitates, namely cell membrane fragments which comprise cell membranes and organelle membranes, and resuspending the cell membrane fragments in a PBS solution for 3-5 minutes by water bath ultrasound to obtain a cell membrane suspension.
A further development of the invention is that step S2 comprises the following steps:
and in the step S21 and the step S2, the mass ratio of the phospholipid to the phosphatidylcholine Dipalmitate (DPPC), the phosphatidylethanolamine distearate-polyethylene glycol 2000 (DSPE-PEG 2000), the stearic acid to the phospholipid glycerol Distearate (DPPG) is 10:10:5:4 is dissolved in chloroform and methanol with the volume ratio of 5:1;
and S22, carrying out rotary evaporation on the liposome organic solution prepared in the step S21 at 50 ℃ and 100rpm until a layer of uniform thin film appears at the bottom of the round-bottom flask, taking off the round-bottom flask, carrying out vacuum drying for 15 minutes, adding 2mL of PBS (phosphate buffer solution) into each 29mg of liposome in the dried flask, and hydrating for 5-10 minutes in 50 ℃,100w and 53kHz water bath under ultrasonic conditions to obtain a liposome suspension.
A further development of the invention is that step S3 comprises the following steps:
step S31, mixing the cell membrane suspension obtained in the step S1 and the liposome solution obtained in the step S2 according to the mass ratio of cell membrane suspension protein to liposome of 1:0,1:12.5,1:25,1:50,0:1, preferably, the ratio of the cell membrane suspension protein to the liposome is 1;
and step S32, freezing the mixed solution obtained in the step S31 in liquid nitrogen for 2 minutes, then thawing the mixed solution under water bath ultrasound at 37 ℃, and repeating the cycle for 3-5 times to obtain a fusion membrane solution.
A further development of the invention is that step S4 comprises the following steps:
s41, placing 1mL of the fusion membrane solution prepared in the step S3 in a 10mL penicillin bottle, placing the penicillin bottle on ice, opening a gas bottle to introduce sulfur hexafluoride into the penicillin bottle, simultaneously placing an ultrasonic probe on a gas-liquid interface of the sulfur hexafluoride and the fusion membrane solution, controlling the ultrasonic power to be 300w, the frequency to be 25KHZ, the opening time to be 5 seconds, the closing time to be 5 seconds and the total time to be 1 minute, and preparing a fusion membrane bubble emulsion;
s42, adding 5mL of the fusion membrane bubble emulsion obtained in the step S42 into a 20mL penicillin bottle, placing the penicillin bottle in liquid nitrogen for freezing for 5-10 minutes, taking out the penicillin bottle, and performing freeze drying in a freeze dryer at the temperature of-40 ℃ to obtain freeze-dried powder;
and S43, filling 60mg of sulfur hexafluoride gas into the freeze-dried powder bottle obtained in the step S43, sealing the bottle by using a rubber plug, keeping the bottle in an aluminum cover jaw, storing the bottle in a 4-degree refrigerator, adding ultrapure water with the same volume for redissolving when in use, ultrasonically mixing the bottle in a water bath with the room temperature of 53kHZ, and then inflating the bottle in the step S41.
In order to achieve the above object, the present invention also provides an engineered fused membrane bubble prepared according to the preparation method.
In order to achieve the above object, the present invention also provides an engineered fused membrane bubble, comprising an inert gas core and an engineered fused membrane formed by fusing a cell membrane and a liposome, which is coated outside the gas core.
The invention is further improved in that the particle size of the fusion membrane bubble is 1.518 +/-0.056 mu m, and the zeta potential is-18.94 +/-9.07 mV.
The invention further improves that the shell layer of the fused membrane bubble is composed of a cell membrane and liposome, and the shell layer of the fused membrane bubble contains CD47 and APOA1.
In order to achieve the aim, the invention also provides application of the engineered fused membrane bubble in targeted ultrasound imaging of atherosclerotic plaques.
The invention has the beneficial effects that: the method comprises the steps of stimulating liver cancer cells cultured in vitro to express APOA1 by using BAF, performing differential centrifugation to extract cell membranes after cell thawing and cell lysis, promoting full fusion between phospholipid bilayers in a repeated freezing and thawing mode to obtain a fusion membrane, and finally filling sulfur hexafluoride gas in a gas-liquid interface ultrasonic molecular rearrangement process to form fusion membrane bubbles with uniform particle size and stable structure. In the experimental verification process, CD47 and APOA1 on the cell membrane are detected on the surface of the fusion membrane bubble, and then compared with the liposome bubble specific targeting foam cell in an in-vitro foam cell model experiment, the fusion membrane bubble disclosed by the invention has the potential feasibility of targeting the atherosclerotic plaque in vitro.
Drawings
FIG. 1 is a diagram representing the content of APOA1 protein in fused membrane vesicle of the present invention: 1) cell membrane of HepG2 cell after BAF treatment, 2) HepG2 cell membrane, 3) APOA1 protein content on the fusion membrane bubble.
FIG. 2 is a representation of the cell membrane, liposome, fused membrane bubble of the present invention: 1) cell membrane, 2) fusion membrane, 3) particle size and Zeta potential characterization diagram of fusion membrane bubble.
FIG. 3 is a representation of a fused membrane bubble of the present invention: 1) Cell membrane transmission electron microscopy characterization, ruler =100nm; 2) Transmission electron microscopy characterization of liposomes, scale =200nm; 3) Fusing a film transmission electron microscope characterization image, and enabling a ruler to be =500nm; 3) Transmission electron microscopy characterization of fused membrane bubbles, scale =2 μm.
FIG. 4 is a characterization diagram of fused membrane bubbles, which is obtained by labeling cell membrane and liposome with membrane dyes DiO and DiI, respectively, repeatedly freezing and thawing, fusing, exciting with 460nm laser, and receiving fluorescence emission spectrogram in 480-680nm wavelength range.
FIG. 5 is a representation of a fused membrane bubble of the present invention, and a confocal representation of the fused membrane bubble: 1) fused membrane bubble bright field plot, scale =5 μm, 2) green fluorescence from DiO-labeled cell membranes, 3) red fluorescence from DiI-labeled liposomes, 4) fluorescent colocalization of DiO and DiI dyes.
FIG. 6 is a representation of the fusion membrane vesicle protein of the invention, the flow representation of the fluorescent antibody label of the vesicle, the red curve representing the liposome vesicle and the blue curve representing the fusion membrane vesicle labeled with the fluorescent antibody.
FIG. 7 is a representation of fusion membrane vesicle proteins of the invention: 1) fusion membrane bubble, 2) fusion membrane, 3) SDS-PAGE characterization of cell membrane.
FIG. 8 is a representation of the fusion membrane vesicle protein of the invention: 1) fusion membrane bubbles, 2) cell membranes, and 3) Western Blot characterization results of the HepG2 cell total protein.
FIG. 9 is a representation of the confocal mapping of foam cell targeting in vitro of the present invention 1) PBS group with scale =10 μm; 2) DiO-labeled liposome vesicles L-MB; 3) DiO-labeled fused membrane bubbles CL-MB.
FIG. 10 is a flow chart of a method for producing engineered fused membrane bubbles according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
It should be emphasized that in describing the present invention, various formulas and constraints are identified with consistent reference numerals, but the use of different reference numerals to identify the same formula and/or constraint is not precluded and is provided for the purpose of more clearly illustrating the features of the present invention.
The invention provides a preparation method of an engineering fusion membrane bubble. The method comprises the steps of stimulating hepatic cells cultured in vitro to express apolipoprotein A1 by using baverromycin A1, performing differential centrifugation to extract cell membranes after cell lysis by freeze thawing, promoting full fusion among phospholipid bilayers in a repeated freeze thawing manner to obtain a fusion membrane, and finally filling sulfur hexafluoride gas in the molecular rearrangement process of gas-liquid interface ultrasound to form fusion membrane bubbles with uniform particle size and stable structure. The engineered fusion membrane bubble comprises an inert gas core and an engineered fusion membrane which is coated outside the gas core and formed by fusing a cell membrane and a liposome. The particle diameter of the fusion membrane bubble is 1.518 +/-0.056 mu m, and the zeta potential is-18.94 +/-9.07 mV. The cell membrane and the liposome of the fused membrane bubble form a shell layer, and the shell layer contains CD47 and APOA1. The engineered fusion membrane bubble can be applied to targeted ultrasound imaging of atherosclerotic plaques.
The preparation method of the engineering fusion membrane bubble mainly comprises the following steps:
step S1, extracting cell membranes by using a cell membrane protein extraction kit after treating cells by BAF;
s2, preparing a liposome by a rotary evaporation method;
s3, preparing a fusion membrane by repeatedly freezing and thawing the cell membrane extracted in the step S1 and the liposome prepared in the step S2 by using liquid nitrogen;
and S4, preparing the fused membrane bubbles by using an ultrasonic-assisted method for the fused membrane prepared in the step S3.
Wherein, step S1 includes the following steps:
s11, culturing human liver cancer cells (HepG 2) in a T75 cell culture bottle containing 10% serum RMPI-1640 culture medium, adding Phosphate Buffer Solution (PBS) for cleaning for 2-3 times, adding 4mL of trypsin solution containing Ethylene Diamine Tetraacetic Acid (EDTA) for digestion for 2-3min, adding 6mL of 10% serum-containing RMPI-1640 culture medium to stop digestion after 80% of cells are detached under microscope observation, transferring the cells into a 15mL centrifuge tube for centrifugation at 900rpm for 3min, sucking supernatant, then resuspending the cells with 1mL of culture medium, adding 10 mu L of cell suspension into a cell counting plate, counting by using a cell counter, adding 4 multiplied by 106 cells into a round dish with the diameter of 10cm for culture for 24 hours;
step S12, taking out the culture medium in the round dish in the step S11, adding PBS (phosphate buffer solution) for washing for 2-3 times, adding an RMPI-1640 incomplete culture medium containing BAF (basic organic fertilizer) for incubation for 6 hours in an incubator, adding the RMPI-1640 incomplete culture medium into a control group, sucking out the culture medium after incubation is finished, adding 4mL of PBS after PBS is washed for 2-3 times, scraping cells by using a cell scraper and transferring the cells to a centrifuge tube;
and S13, centrifuging the cells obtained in the step S12 at 900rpm for 3 minutes to suck out the supernatant, adding a cell membrane protein extraction reagent A for resuspension, mixing uniformly, repeatedly freezing and thawing the cells for 2-3 times by using liquid nitrogen to lyse the cells, centrifuging the lysed cells for 10 minutes at 700g and 4 ℃ to remove cell nucleus precipitates, collecting the supernatant, centrifuging the supernatant for 30 minutes at 14000g and 4 ℃ to take precipitates, namely cell membrane fragments, wherein the cell membrane fragments comprise cell membranes and organelle membranes, and resuspending the cell membrane fragments in a PBS solution for 3-5 minutes by water bath ultrasound to obtain a cell membrane suspension.
Wherein, step S2 includes the following steps:
and in the step S21 and the step S2, the mass ratio of the phospholipid to the phosphatidylcholine Dipalmitate (DPPC), the phosphatidylethanolamine distearate-polyethylene glycol 2000 (DSPE-PEG 2000), the stearic acid to the phospholipid glycerol Distearate (DPPG) is 10:10:5:4 is dissolved in chloroform and methanol with the volume ratio of 5:1;
and S22, carrying out rotary evaporation on the liposome organic solution prepared in the step S21 at 50 ℃ and 100rpm until a layer of uniform thin film appears at the bottom of the round-bottom flask, taking off the round-bottom flask, carrying out vacuum drying for 15 minutes, adding 2mL of PBS (phosphate buffer solution) into each 29mg of liposome in the dried flask, and hydrating for 5-10 minutes in 50 ℃,100w and 53kHz water bath under ultrasonic conditions to obtain a liposome suspension.
Wherein, step S3 includes the following steps:
step S31, mixing the cell membrane suspension obtained in the step S1 and the liposome solution obtained in the step S2 according to the mass ratio of cell membrane suspension protein to liposome of 1:0,1:12.5,1:25,1:50,0:1, preferably, the ratio of the cell membrane suspension protein to the liposome is 1;
and step S32, freezing the mixed solution obtained in the step S31 in liquid nitrogen for 2 minutes, then thawing the mixed solution under water bath ultrasound at 37 ℃, and repeating the cycle for 3-5 times to obtain a fusion membrane solution.
Wherein, step S4 comprises the following steps:
s41, placing 1mL of the fusion membrane solution prepared in the step S3 in a 10mL penicillin bottle, placing the penicillin bottle on ice, opening a gas bottle to introduce sulfur hexafluoride into the penicillin bottle, simultaneously placing an ultrasonic probe on a gas-liquid interface of the sulfur hexafluoride and the fusion membrane solution, controlling the ultrasonic power to be 300w, the frequency to be 25KHZ, the opening time to be 5 seconds, the closing time to be 5 seconds and the total time to be 1 minute, and preparing a fusion membrane bubble emulsion;
s42, adding 5mL of the fusion membrane bubble emulsion obtained in the step S42 into a 20mL penicillin bottle, placing the penicillin bottle in liquid nitrogen for freezing for 5-10 minutes, taking out the penicillin bottle, and performing freeze drying in a freeze dryer at the temperature of-40 ℃ to obtain freeze-dried powder;
and S43, filling 60mg of sulfur hexafluoride gas into the freeze-dried powder bottle obtained in the step S43, sealing the bottle by using a rubber plug, clamping a jaw by using an aluminum cover, storing in a refrigerator with 4 degrees, adding ultrapure water with the same volume when in use, carrying out ultrasonic mixing in a water bath with the room temperature of 53kHZ, and then carrying out gas filling in the step S41.
The present invention is described in detail below with reference to the drawings and examples.
1. Engineering cells, and preparing cell membrane suspension. Culturing human hepatocyte HepG2 in a T75 cell culture bottle containing 10% serum RMPI 1640 culture medium, washing with PBS for 2-3 times, adding 4mL of EDTA-containing trypsin solution for digestion for 2-3min, adding 6mL of 10% serum-containing RMPI 1640 culture medium after 80% of cells are detached under microscope observation, terminating digestion, transferring to a centrifuge tube 15mL of 900rpm for 3min, sucking out supernatant, suspending with 1mL of culture medium, adding 10 μ L of cell suspension to a cell counting plate, counting with a cell counter, adding 4 × 106 cells into a 10 cm-diameter round dish for culture for 24 hours, taking out the culture medium in the round dish, washing with PBS for 2-3 times, adding BAF-containing RMPI 1640 incomplete culture medium, incubating in an incubator for 6 hours, adding RMPI 1640 incomplete culture medium into a control group, sucking out the culture medium after incubation is completed, washing the PBS for 2-3 times, adding 4mL of PBS, scraping the cells by using a cell scraper, transferring the cells into a centrifuge tube, centrifuging the obtained cells at 900rpm for 3 minutes, sucking out supernatant, adding a cell membrane protein extraction reagent A for heavy suspension, repeatedly freezing and thawing for 2-3 times after uniform mixing to lyse the cells, centrifuging the lysed cells for 10 minutes under the conditions of 700g and 4 ℃ to remove cell nucleus precipitates, collecting the supernatant, centrifuging the supernatant for 30 minutes under the conditions of 14000g and 4 ℃ to obtain precipitates, namely cell membrane fragments, adding water for heavy suspension, quantifying the total protein concentration of cell membranes by using a BCA method, and storing the cell membrane suspension in a refrigerator at-80 ℃ for later use.
2. A liposome membrane solution was prepared. The phospholipid is prepared from the following components in a mass ratio of 10:10:5:4 in chloroform and methanol with the volume ratio of 5:2, carrying out rotary evaporation on the obtained liposome organic solution at the temperature of 50 ℃ and the speed of 100rpm until a layer of uniform thin film appears at the bottom of the round-bottom flask, taking off the round-bottom flask, carrying out vacuum drying for 15 minutes, adding 2mL of 0.01M phosphate buffer solution into each 29mg of liposome in the dried flask, and hydrating in a 50 ℃,100w and 53kHZ water bath for 5-10 minutes to obtain a liposome suspension.
3. A fusion membrane solution was prepared. And (3) carrying out ultrasonic full mixing on the mixed solution obtained in the step at room temperature of 53kHZ water bath, then placing the mixed solution in liquid nitrogen for freezing for 2 minutes, taking out the mixed solution, then unfreezing the mixed solution in the room temperature of 53kHZ water bath, and repeatedly freezing and thawing for 3 times to obtain a fusion membrane solution.
4. Preparing the fused membrane bubble. And (3) placing 1mL of the fusion membrane solution obtained in the step into a 10mL penicillin bottle, placing the penicillin bottle on ice, opening the gas bottle, introducing sulfur hexafluoride into the penicillin bottle, simultaneously placing an ultrasonic probe on a gas-liquid interface of the sulfur hexafluoride and the fusion membrane solution, controlling the ultrasonic power to be 300w, the frequency to be 25KHZ, the opening time to be 5 seconds, the closing time to be 5 seconds and the total time to be 1 minute, and thus obtaining the fusion membrane bubble emulsion. Freezing the prepared bubble suspension in liquid nitrogen for 5-10 min, taking out, and freeze-drying in a freeze dryer; after drying, filling sufficient sulfur hexafluoride into the bottle, covering the bottle cap, and storing at normal temperature.
5. And (3) characterizing the particle size, zeta potential and morphology of different components in the preparation process of the fused membrane bubble. mu.L of each of the cell membrane, the confluent membrane, and the confluent membrane was taken and added to 2mL of a 10% (w/v) glucose solution, and the particle size was measured by a ZetaPALS laser particle size analyzer, and 15. Mu.L of each of the components was diluted in 1.5mL of ultrapure water and inserted into an electrode to measure the Zeta potential. Diluting the prepared bubble suspension by 100 times, dripping 10 mu L of the diluted bubble suspension, settling the diluted bubble suspension on a 200-mesh copper net, standing and drying. Diluting cell membrane, liposome and fusion membrane by 100 times, dripping 10 μ L of the diluted solution to settle on a 200-mesh copper net, drying, negative staining with 1% phosphotungstic acid for 2min, removing staining solution with filter paper, dripping a drop of pure water on the copper net, removing with filter paper, repeating for two or three times, standing, and drying. Characterization was performed using Hitachi HT7700 transmission electron microscopy at an acceleration voltage of 100 kv.
6. Protein characterization of the fused membrane vesicles. And (3) performing APOA1Elisa characterization, namely taking BAF treated cells and control group untreated HepG2 cells to extract cell membranes of the BAF treated cells through a cell membrane protein extraction reagent A, fusing a part of cell membranes extracted in the previous step with liposome, ultrasonically inflating to obtain bubbles, centrifuging for 1 minute at 50g, taking down the supernatant, re-inflating, repeating the operation for 3 times, adding PBS to re-suspend the remaining bubbles, quantitatively diluting the cell membranes, the cell membranes and the bubbles of the BAF treated cells to 1 mu g/mu L by using a BCA protein quantitative kit, and taking the cell membranes, the cell membranes and the bubbles containing 100 mu L of the BAF treated cells to detect the content of APOA1 through a human APOA1Elisa quantitative kit. Flow characterization, 100ul of fused membrane bubbles and liposome bubbles were incubated with 10ul of AF488-labeled Rat anti-APOA1 antibody for flow cytometry characterization, and fluorescence signals of 3X 105 bubbles were collected and analyzed for fluorescence intensity using FlowJ software. And (3) SDS-PAGE characterization, preparing SDS-PAGE electrophoresis liquid, diluting total cell protein, cell membranes and fusion membrane bubbles to a protein concentration of 1 mu g/ul, adding 5x protein loading buffer solution containing DTT, boiling for 5min, and cooling at room temperature for later use. And (3) adding 10 mu L of pre-dyeing Marker into the pre-prepared glue, wherein each 20 mu L of the cell total protein sample liquid, the cell membrane sample liquid and the fusion membrane bubble sample liquid. The voltage of the electrophoresis apparatus is set to be 100v, the time is 100 minutes, and the electrophoresis is finished after the last color strip appears on the pre-dyed Marker. And cutting a target strip by using a cutting rubber plate, soaking the cut target strip in Coomassie brilliant blue ultrafast dyeing liquid for 3h, and decoloring the dyed strip in decoloring liquid for one night to take out and photograph so as to obtain a target image. And (3) WB characterization, taking out the prefabricated glue, adding a PVDF membrane with a proper size into methanol for activation for 1-2 minutes, sequentially paving the fiber pad, whatman3MM filter paper, the PVDF membrane, the gel, the Whatman3MM filter paper and the fiber pad into a membrane transferring clamp in the membrane transferring liquid, removing bubbles among all layers by using a roller in the paving process, putting the layers into a transferring groove, and transferring the membrane by using a Trans-Blot Turbo all-round protein transferring system. And taking out the PVDF membrane and marking the PVDF membrane after the membrane conversion is finished. Putting the PVDF membrane into a box, adding 5% of skimmed milk powder, shaking for 2h, and sealing. After blocking, the PVDF membrane was removed and washed with 1 XTBST buffer for 10 minutes each 3 times with slow shaking. And (3) taking the primary antibody of the membrane, slowly shaking and incubating for 2h at room temperature, recovering the primary antibody, adding TBST buffer solution to repeatedly wash the membrane for 3 times, incubating for 2h at room temperature, repeatedly washing the membrane for 3 times, taking out the membrane, and placing the membrane in the TBST buffer solution. Preparing ECL chemiluminescence working solution. The gel was imaged with the membrane side facing up and a suitable amount of working solution added, using G: BOX ChemiXR 5.
7. Fused membrane bubbles target foam cells in vitro. Blowing and beating RAW264.7 cells cultured in a 10cm round dish, re-suspending with 1mL of DMEM high-sugar complete culture medium after centrifugation, adding 10 microliters of DMEM into a cell counting plate for counting, preparing the concentration of the cell suspension into 100000 cells/mL, adding 1mL of the cell suspension into a confocal dish for culturing for 24 hours, sucking the culture medium, washing with PBS for 2-3 times, adding 1mL of prepared DMEM incomplete culture medium with the concentration of Ox-LDL of 20 mu g/mL for incubation for 24h, washing with PBS for 3 times, adding 400 mu LPBS, diO-labeled liposome bubbles and DiO-labeled fusion membrane bubbles into a control group and an experimental group respectively for incubation for 15-30min, and washing with PBS for 2 times and observing by using a confocal microscope.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the present invention.
Claims (10)
1. A preparation method of an engineered fused membrane bubble is characterized by comprising the following steps:
s1, treating cells by Bafilomycin A1 (BAF), and extracting cell membranes by using a cell membrane protein extraction kit;
s2, preparing a liposome by a rotary evaporation method;
s3, preparing a fusion membrane by repeatedly freezing and thawing the cell membrane extracted in the step S1 and the liposome prepared in the step S2 by using liquid nitrogen; and
and S4, preparing the fused membrane bubbles by using an ultrasonic-assisted method for the fused membrane prepared in the step S3.
2. The method for preparing an engineered fused membrane bubble according to claim 1, wherein: the step S1 includes the steps of:
s11, culturing human liver cancer cells (HepG 2) in a T75 cell culture bottle containing a RMPI-1640 culture medium containing 10% serum, adding Phosphate Buffer Solution (PBS) for washing for 2-3 times, adding 4mL of trypsin solution containing Ethylene Diamine Tetraacetic Acid (EDTA) for digesting for 2-3min, adding 6mL of RMPI-1640 culture medium containing 10% serum after observing 80% of cells to stop digesting after observing by a microscope, transferring the cells into a 15mL centrifuge tube for centrifuging at 900rpm for 3min, sucking supernatant, resuspending by using 1mL of culture medium, adding 10 mu L of cell suspension into a cell counting plate, counting by using a cell counter, and adding 4 multiplied by 106 cells into a round dish with the diameter of 10cm for culturing for 24 hours;
s12, taking out the culture medium in the round dish in the step S11, adding PBS (phosphate buffer solution) for washing for 2-3 times, adding an RMPI-1640 incomplete culture medium containing BAF (basic fibroblast growth factor) for incubation for 6 hours in an incubator, adding an RMPI-1640 incomplete culture medium in a control group, sucking the culture medium after the incubation is finished, adding 4mL of PBS after washing for 2-3 times by the PBS, scraping the cells by using a cell scraper and transferring the cells to a centrifuge tube;
and S13, centrifuging the cells obtained in the step S12 at 900rpm for 3 minutes to absorb the supernatant, adding a cell membrane protein extraction reagent A for resuspension, repeatedly freezing and thawing the cells for 2-3 times after uniformly mixing the cells, centrifuging the lysed cells for 10 minutes at 700g and 4 ℃ to remove cell nucleus precipitates, collecting the supernatant, centrifuging the supernatant for 30 minutes at 14000g and 4 ℃ to obtain precipitates, namely cell membrane fragments which comprise cell membranes and organelle membranes, and resuspending the cell membrane fragments in a PBS solution for 3-5 minutes by water bath ultrasound to obtain a cell membrane suspension.
3. The method for preparing an engineered fused membrane bubble according to claim 2, wherein: the step S2 includes the steps of:
and in the step S21 and the step S2, the mass ratio of the phospholipid to the phosphatidylcholine Dipalmitate (DPPC), the phosphatidylethanolamine distearate-polyethylene glycol 2000 (DSPE-PEG 2000), the stearic acid to the phospholipid glycerol Distearate (DPPG) is 10:10:5:4 is dissolved in chloroform and methanol with the volume ratio of 5:1;
and S22, carrying out rotary evaporation on the liposome organic solution prepared in the step S21 at 50 ℃ and 100rpm until a layer of uniform thin film appears at the bottom of the round-bottom flask, taking off the round-bottom flask, carrying out vacuum drying for 15 minutes, adding 2mL of PBS (phosphate buffer solution) into each 29mg of liposome in the dried flask, and hydrating for 5-10 minutes in 50 ℃,100w and 53kHz water bath under ultrasonic conditions to obtain a liposome suspension.
4. The method for preparing an engineered fused membrane bubble according to claim 3, wherein: step S3 includes the following steps:
step S31, mixing the cell membrane suspension obtained in the step S1 and the liposome solution obtained in the step S2 according to the mass ratio of cell membrane suspension protein to liposome of 1:0,1:12.5,1:25,1:50,0:1, preferably, the ratio of the cell membrane suspension protein to the liposome is 1;
and step S32, freezing the mixed solution obtained in the step S31 in liquid nitrogen for 2 minutes, then thawing the mixed solution under water bath ultrasound at 37 ℃, and repeating the cycle for 3-5 times to obtain a fusion membrane solution.
5. The method for preparing an engineered fused membrane bubble according to claim 4, wherein: step S4 includes the following steps:
s41, placing 1mL of the fusion membrane solution prepared in the step S3 in a 10mL penicillin bottle, placing the penicillin bottle on ice, opening a gas cylinder to introduce sulfur hexafluoride into the penicillin bottle, simultaneously placing an ultrasonic probe on a gas-liquid interface of the sulfur hexafluoride and the fusion membrane solution, controlling the ultrasonic power to be 300w, the frequency to be 25KHZ, the opening time to be 5 seconds, the closing time to be 5 seconds and the total time to be 1 minute, and preparing a fusion membrane bubble emulsion;
step S42, adding 5mL of the fusion membrane bubble emulsion obtained in the step S42 into a 20mL penicillin bottle, placing the penicillin bottle in liquid nitrogen for freezing for 5-10 minutes, taking out the penicillin bottle, and performing freeze drying in a freeze dryer at the temperature of-40 ℃ to obtain freeze-dried powder;
and S43, filling 60mg of sulfur hexafluoride gas into the freeze-dried powder bottle obtained in the step S43, sealing the bottle by using a rubber plug, clamping a jaw by using an aluminum cover, storing in a refrigerator with 4 degrees, adding ultrapure water with the same volume when in use, carrying out ultrasonic mixing in a water bath with the room temperature of 53kHZ, and then carrying out gas filling in the step S41.
6. An engineered fused membrane bubble characterized by: the production method according to any one of claims 1 to 5.
7. An engineered fused membrane bubble characterized by: comprises an inert gas core and an engineered fusion membrane coated outside the gas core and formed by fusing a cell membrane and a liposome.
8. The engineered fused membrane bubble of claim 7, wherein: the particle diameter of the fusion membrane bubble is 1.518 +/-0.056 mu m, and the zeta potential is-18.94 +/-9.07 mV.
9. The engineered fused membrane bubble of claim 7, wherein: the shell layer of the fused membrane bubble is composed of a cell membrane and a liposome, and the shell layer of the fused membrane bubble contains CD47 and APOA1.
10. Use of an engineered fused membrane vesicle according to any one of claims 1-5, 7-9 in targeted ultrasound imaging of atherosclerotic plaques.
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