CN117568255B - Cell membrane surface tension regulating and controlling method - Google Patents
Cell membrane surface tension regulating and controlling method Download PDFInfo
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- CN117568255B CN117568255B CN202410063289.5A CN202410063289A CN117568255B CN 117568255 B CN117568255 B CN 117568255B CN 202410063289 A CN202410063289 A CN 202410063289A CN 117568255 B CN117568255 B CN 117568255B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N13/00—Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects
- G01N13/02—Investigating surface tension of liquids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0644—Platelets; Megakaryocytes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N25/00—Investigating or analyzing materials by the use of thermal means
- G01N25/20—Investigating or analyzing materials by the use of thermal means by investigating the development of heat, i.e. calorimetry, e.g. by measuring specific heat, by measuring thermal conductivity
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Abstract
The invention discloses a cell membrane surface tension regulating method, which comprises the steps of doping artificial phospholipid with high surface activity into a cell membrane in a liquid nitrogen freezing and thawing mode to form a cell membrane freezing and thawing compound, and assembling phospholipid and protein components of the cell membrane freezing and thawing compound on the surface of microbubbles of the cell membrane freezing and thawing compound in a gas-liquid interface assembling mode. The method provided by the invention can improve the surface activity of the cell membrane, can stably and orderly assemble the cell membrane into a monolayer membrane at a gas-liquid interface, effectively reduces the surface tension of the gas-liquid interface, and simultaneously effectively regulates the surface tension of the cell and effectively regulates and controls the physical properties such as the size potential and the like of the formed cell membrane freeze-thawing compound microbubbles. The method provided by the invention can also be applied to acute thrombosis diagnosis, and has good effect.
Description
Technical Field
The invention belongs to the technical fields of biomedical engineering and ultrasonic molecular imaging, and particularly relates to a cell membrane surface tension regulation and control method.
Background
Cell membranes are typically bilayer structures composed of molecules such as proteins and cholesterol embedded in phospholipids. Cell membrane biomimetic technology has been successful in achieving surface modification assembly of nanoparticles and nanodroplets, and in achieving long circulation in vivo and good biocompatibility. Typical cell membrane biomimetic techniques are to coat the peeled cell membrane directly onto the surface of nanoparticles or droplets in the form of a phospholipid bilayer. However, due to surface tension, the phospholipid bilayer of the cell membrane cannot be directly coated on the surface of the micron-sized bubbles.
In view of the above, there is a need to provide a method for regulating the surface tension of cell membranes to solve the above-mentioned problems.
Disclosure of Invention
The invention aims to provide a method for regulating and controlling the surface tension of a cell membrane so as to reduce the surface tension of the cell membrane.
In order to achieve the above purpose, the present invention adopts the following technical scheme, including:
step 1: doping artificial phospholipid into the cell membrane in a freeze thawing mode to obtain a cell membrane freeze thawing compound;
Step 2: the phospholipid and protein components of the cell membrane freeze-thawing complex are assembled on the microbubble surface of the cell membrane freeze-thawing complex in a gas-liquid interface assembly mode.
As a further improvement of the invention, the cell membrane freeze-thawing complex is fused by liquid nitrogen freezing at room temperature,
As a further improvement of the invention, the liquid nitrogen is thawed at room temperature for 5 times.
As a further improvement of the present invention, the cell membrane is a platelet membrane in the cell membrane freeze-thaw complex and cell membrane freeze-thaw complex microbubbles.
As a further improvement of the present invention, the classes of artificial phospholipids in the cell membrane freeze-thaw complex include DPPC, DPPG, DSPE-PEG (2000) and combinations of the DPPC, DPPG and DSPE-PEG (2000).
As a further improvement of the present invention, the platelet membrane freeze-thaw complex microbubbles have a minimum glass transition temperature at a protein to phospholipid ratio of 0.02.
As a further improvement of the invention, the platelet membrane freeze-thawing complex microbubbles have the lowest gas-liquid interfacial surface tension at a protein to phospholipid ratio of 0.02.
As a further improvement of the present invention, the platelet membrane freeze-thaw complex microbubbles have an average particle size of 1000nm to 10 μm.
As a further improvement of the present invention, the platelet membrane freeze-thaw complex microbubbles have integrin alpha IIbβ3 on the surface of the original platelet membrane and maintain an activated conformation.
The beneficial effects are that:
The invention adopts a mode of repeated liquid nitrogen freezing-room temperature thawing to dope phospholipid into the platelet membrane and obtain the platelet membrane freeze-thawing compound vesicle. The surface tension of phospholipid protein molecules in the freeze-thawing compound at a gas-liquid interface is obviously reduced by a Langmuir-Blodgett membrane balance test, and the glass transition temperature of the doped platelet membrane is reduced by a differential and moderate thermal scanning technology test. And blowing sulfur hexafluoride gas into the platelet membrane freeze-thawing compound vesicles in an ultrasonic auxiliary mode to form the platelet membrane freeze-thawing compound vesicles at a gas-liquid interface. Platelet membrane proteins were located by fluorescent labeling and successfully spiked into platelet membrane freeze-thaw complex microbubbles. By proteomic analysis of platelet membrane freeze-thaw complex vesicles and platelet membrane freeze-thaw complex microbubbles, it was found that the platelet membrane freeze-thaw complex microbubbles inherited 61.4% of the protein species of the platelet membrane freeze-thaw complex vesicles and maintained the conformation of integrin αiiβ3 activation on the platelet membrane surface. In the experiment of the acute and chronic thrombus model of the inferior vena cava of a rat, the platelet membrane freeze-thawing complex microbubbles can specifically identify the acute thrombus, the average signal-to-noise ratio for diagnosing the acute thrombus is 12.47 dB, and the chronic thrombus is 0.1dB.
Drawings
FIG. 1 is a schematic diagram of a method for regulating and controlling cell membrane surface tension according to the present invention;
FIG. 2 is a graph showing the isothermal surface pressure and area of liposomes of different phospholipid formulations according to the present invention for the gas-liquid interfacial surface tension modulating effect;
FIG. 3 is a graph showing the effect of mass ratios of different proteins to phospholipids on gas-liquid interfacial surface tension: a is a schematic diagram of the assembly of bovine serum albumin and phospholipid at a gas-liquid interface; b is an isothermal graph of gas-liquid interface surface pressure and area with different protein to phospholipid mass ratios; c is the surface tension value of different protein to phospholipid mass ratio;
FIG. 4 is a graph depicting the fusion efficiency of platelet membrane and liposome according to the present invention: a is a schematic diagram of mutual fusion of platelet membrane vesicles and liposomes; b is a fluorescence spectrum chart of lipophilic dyes DiO and DiI after being respectively fused with each other in platelet membrane vesicles and liposomes; c is a flow chart of the fluorescent-labeled platelet membrane vesicles and liposomes after being mutually fused;
FIG. 5 is a graph of differential thermal scan after cell membrane liposome fusion;
FIG. 6 is a graph showing the ratio of protein to lipid in platelet membrane vesicles modulated by fusion with liposomes using a freeze-thawing method;
FIG. 7 is a graph of gas-liquid interface surface pressure versus area isotherms for different protein-lipid ratios for platelet membrane freeze-thaw complexes;
FIG. 8 is a chart of a fixed-bit flow analysis of the microbubble composition of the platelet membrane freeze-thaw compound formed in different preparation modes;
FIG. 9 is a graph of a positioning fluorescence imaging analysis of the microbubble components of the platelet membrane freeze-thawing complex formed by different preparation methods;
FIG. 10 is a graph representing the morphology of the platelet membrane freeze-thawing complex of the present invention: a is a platelet membrane transmission electron microscope characterization diagram with a1 μm scale; b is a platelet membrane nano bubble transmission electron microscope characterization diagram with a1 μm scale; c is a platelet membrane freeze-thawing complex diagram with a1 μm scale; d is a platelet membrane freeze-thawing compound bubble transmission electron microscope characterization diagram with a1 μm scale;
FIG. 11 is a graph of platelet membrane, platelet membrane nanovesicles, platelet membrane freeze-thaw complex vesicles, and particle size and potential statistics of platelet membrane freeze-thaw complex vesicles;
FIG. 12 is a graph of a proteomic analysis characterization of a platelet membrane freeze-thaw complex of the present invention;
FIG. 13 is a confocal image of the characterization of the microbubble protein of the platelet membrane freeze-thaw complex of the present invention;
FIG. 14 is a graph depicting the conformational state of the platelet membrane freeze-thaw complex microbubble alpha IIb beta 3 protein of the present invention;
FIG. 15 is an ultrasonic diagnostic chart of acute thrombus of microbubbles of the platelet membrane freeze-thaw compound of the present invention;
FIG. 16 is a diagnostic ultrasound of a platelet membrane freeze-thaw complex microbubble chronic thrombus of the present invention;
FIG. 17 is a graph showing the diagnostic effect of the platelet membrane freeze-thawing complex microbubbles of the present invention on acute and chronic thrombosis.
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 noted that, in order to avoid obscuring the present invention due to unnecessary details, only structures and/or processing steps closely related to aspects of the present invention are shown in the drawings, and other details not greatly related to the present invention are omitted.
The invention provides a method for regulating and controlling the surface tension of a cell membrane, which is shown in a figure 1, wherein phospholipids with different surface activities are doped into a phospholipid bilayer of the cell membrane in a freeze-thawing fusion mode to form a freeze-thawing complex, and the surface tension of the cell membrane is reduced by regulating and controlling the ratio of protein and phospholipid of the freeze-thawing complex. The freeze-thawing compound with reduced surface tension can be spontaneously adsorbed at the gas-liquid interface under the action of high shearing force to form bubbles coated by the freeze-thawing compound.
Examples:
1. extraction of platelet membrane:
Firstly, selecting platelets from whole blood of human, rat, mouse, rabbit, cow and pig, wherein the selected phospholipids are the combination of dipalmitin phosphatidylcholine, distearate phosphatidylethylamine alcohol-polyethylene glycol 2000, stearic acid and distearate phosphatidylglycerol; then, to the platelets extracted from whole blood, mannose at a final concentration of 42 mM, prostaglandin E1 at a final concentration of 1. Mu.M, and a phosphatase protease inhibitor mixture were added to give platelet suspensions at a concentration of 2X 10 8/mL. Freezing the platelet suspension in a-80 ℃ refrigerator for 1 hour, then thawing in a 37 ℃ constant temperature water bath, repeating the steps three times at 4 ℃ and 12000 And (3) centrifuging for 15 minutes under the condition g to obtain platelet membrane suspension, quantifying the protein concentration by using a BCA protein quantification kit, and quantifying the phospholipid concentration by using a phospholipid kit.
2. Preparation of phospholipids:
The phospholipids are dissolved in a volume ratio of 1 according to a series of mass ratios of dipalmitin phosphatidylcholine, distearate phosphatidylethylamine alcohol-polyethylene glycol 2000, stearic acid and distearate phosphatidylglycerol: 1 in chloroform and methanol at 50 ℃ and 100 rpm until a uniform film appears at the bottom of the round bottom flask, the flask is removed and dried in vacuo for 8 hours. To the dried flask was added a phosphate buffer and sonicated in a water bath at 50℃and 100 w, 53 and kHZ to obtain a liposome membrane solution, and the pH of the liposome membrane solution was adjusted to 7.4. Platelet membranes were mixed with liposomes at different mass ratios of protein phospholipids. And (3) repeatedly freezing and thawing the mixture for 6 times under the conditions of liquid nitrogen and room temperature to obtain the platelet membrane freeze-thawing compound. Transferring the platelet membrane or the platelet membrane freeze-thawing compound into a 10 mL glass bottle placed on ice, blowing sulfur hexafluoride gas into the system, and dispersing the sulfur hexafluoride gas into an aqueous medium by using probe ultrasonic treatment (300W, 5s on/5 s off) for 4 cycles to respectively obtain the platelet membrane nanobubbles and the platelet membrane freeze-thawing compound microbubbles.
3. Regulating and controlling the gas-liquid interfacial tension of the protein phospholipid complex:
The surface tension of the prepared phospholipid complex at the gas-liquid interface was measured using a Langmuir-Blodgett multifunctional membrane balance. 20 μl of a solution of phospholipid (1 mg/mL lipid concentration) in chloroform was applied with a microinjector to a Langmuir-Blodgett multifunctional membrane balance and the chloroform was evaporated for 10 minutes. The initial area of the surface was set to 282 cm 2 and the film was compressed at 20 ℃ at a rate of 10 mm/min, recording the surface tension-surface area curve. As shown in FIG. 2, in a series of phospholipid formulations, DSPE-PEG 2000 had the lowest surface tension (3 mN/m) at the gas-liquid interface at a mole percentage of 10%, DPPC: DPPG: the ratio of DSPE-PEG 2000 is 8:1: the surface pressure of the gas-liquid interface at 1 reached 69mN/m, and thus had the lowest gas-liquid interface surface tension (2 mN/m). In order to explore the rule of assembling proteins and phospholipids at a gas-liquid interface, bovine Serum Albumin (BSA) is selected as a standard protein as shown in A in FIG. 3, a series of phospholipid protein complexes with different mass ratios of proteins to phospholipids are prepared, 20 mu L of chloroform solution of the phospholipid protein complexes (1 mg/mL lipid concentration) is coated on a Langmuir-Blodgett multifunctional membrane balance by a microinjector, and chloroform is evaporated for 10 minutes. The initial area of the surface was set to 282 cm 2 and the film was compressed at 20 ℃ at a rate of 10 mm/min, recording the surface tension-surface area curve. As shown in B and C in fig. 3, there is the lowest gas-liquid interfacial surface tension in a series of phospholipid protein complexes with different mass ratios when the mass ratio of protein to phospholipid is 0.02.
4. Platelet membrane-phospholipid fusion validation:
Platelet membrane vesicles and liposomes were stained with excess DiO and DiI, respectively, followed by removal of the dye without membrane embedded with 12000 g,15 min,4 ℃. According to the different proteins: the two membranes were added in mass ratio and incubated in an incubator at 37℃for 30 minutes, removed and placed in liquid nitrogen for 1 minute, then melted at 37℃and the cycle repeated 5 times. And placing the freeze-thawing compound solution after freeze-thawing cycle on ice, and performing ultrasonic treatment by using a probe with the power of 10w and the frequency of 25 Khz, controlling the ultrasonic treatment to be on for 5 seconds, and controlling the ultrasonic treatment to be off for 5 seconds, wherein the total time is 2 minutes, so as to obtain the fluorescent-labeled platelet membrane freeze-thawing compound vesicle. 2 ml water and 20 ul samples were added to a 3 ml quartz cuvette, and 480-680 nm emission was received using a 460 nm wavelength laser as excitation light. As shown in FIG. 4A, because of Foster Fluorescence Resonance Energy Transfer (FRET) phenomenon of DiI and DiO molecules at a distance of less than 10nm, molecules embedded at the hydrophobic end of the phospholipid bilayer are close to each other and less than 10nm, it can be demonstrated that platelet membrane and phospholipid of liposome fuse with each other. As shown in B in fig. 4, the fluorescence resonance transfer phenomenon becomes more pronounced as the mass ratio of protein to phospholipid decreases, indicating an increase in the degree of membrane fusion. Platelet membrane vesicles and liposomes were stained with excess DiO and DiD, respectively, followed by removal of the dye without membrane embedded with 12000 g,15 min,4 ℃. The stained platelet vesicles and the liposomes are fused by mixing incubation and freeze thawing respectively when the mass ratio of the protein to the phospholipid is 0.02, and the fusion efficiency of the platelet vesicles and the liposomes is observed by flow cytometry, as shown in C in FIG. 4, when the platelet vesicles and the liposomes form a freeze thawing complex, the two components can be effectively fused. Taking different membrane proteins: the platelet membrane freeze-thawing compound microbubbles with the mass ratio of the liposome are freeze-dried, and the freeze-dried powder is subjected to differential calorimetric scanning analysis, wherein the scanning speed is 5 ℃/min. As shown in FIG. 5, as the proportion of doped phospholipid increases, the glass transition temperature of the platelet membrane freeze-thaw complex decreases, with the lowest glass transition temperature at a protein to phospholipid mass ratio of 0.02. And regulating and controlling the gas-liquid interfacial tension of the platelet membrane. The composition of the freeze-thawing complex formed by doping different proportions of phospholipids into platelet membranes using the freeze-thawing method is shown in figure 6. The platelet membrane is doped with phospholipids in different proportions, so that the surface tension of the formed platelet membrane freeze-thawing compound at the gas-liquid interface is reduced along with the reduction of the mass ratio of protein to phospholipid, and the platelet membrane freeze-thawing compound has the lowest gas-liquid interface surface tension when the mass ratio of protein to phospholipid is 0.02 as shown in fig. 7.
5. Positioning of platelet membrane gas-liquid interface assembly components:
Platelet membranes were protein labeled with cy5.5 for component localization during gas-liquid interface assembly. The platelet membrane, the platelet membrane freeze-thaw complex or the mixture of platelet membrane and liposome is ultrasonically emulsified in an SF 6 atmosphere. The resulting bubbles were used for flow cytometry analysis, the resulting bubbles were isolated in FSC/SSC gating, and cy5.5 fluorescence was quantified in the Q2 and Q4 regions, as shown in fig. 8, which was effective in assembling platelet membrane components onto microbubbles using platelet membrane freeze-thaw complexes relative to a mixture of platelet membranes and liposomes. For fluorescence localization analysis, the platelet membrane fraction was targeted with cy5.5 label. All vesicle systems were centrifuged at 12000g for 10 min before sonication, at 50g for 10 min after sonication, and imaged by an in vivo small animal imaging system (IVIS) to detect cy5.5 signals. After sonication, the particle size of the raw mixture and the top or down portion of the centrifuge tube was measured by a Zetasizer. As shown in fig. 9, fluorescent molecule cy5.5 labeling the platelet membrane was detected in microbubbles prepared using the platelet membrane freeze-thaw complex.
6. And (3) morphology and potential characterization of a product in the preparation process:
And adopting a transmission electron microscope to characterize the shapes of the platelet membrane, the platelet membrane freeze-thawing compound, the platelet membrane nanobubble and the platelet membrane freeze-thawing compound microbubble. All samples were diluted to 0.1 mg/mL (lipid concentration). And (3) dripping 10 mu L of the platelet membrane and the platelet membrane freeze-thawing compound onto the carbon-coated copper mesh. After drying in vacuo for 15 minutes, the samples were counterstained with 1% uranium acetate. 10 μl of PNBs and PMBs were dropped onto the carbon coated copper mesh at room temperature for 2h to be adsorbed onto the carbon film surface. All samples were examined using a transmission electron microscope at an accelerating voltage of 100 kV. As shown in fig. 10, the platelet membrane (a) and the platelet membrane (C) have a typical phospholipid bilayer structure, and the platelet membrane nanobubble (B) and the platelet membrane freeze-thawing complex microbubbles (D) have a typical monolayer bubble structure. Platelet membrane, platelet membrane freeze-thaw complex, platelet membrane nanobubble, and platelet membrane freeze-thaw complex microbubble were measured by Zetasizer, and the hydrated particle size and zeta potential of the platelet membrane and platelet membrane nanobubble were similar to those of the platelet membrane nanobubble, as shown in fig. 11, with a significant decrease of-47 mV in the zeta potential of the platelet membrane freeze-thaw complex and platelet membrane freeze-thaw complex microbubble after phospholipid doping.
7. Platelet membrane freeze-thawing complex microvesicle protein characterization:
Proteomic analysis was performed on the prepared platelet membrane freeze-thaw complexes and platelet membrane freeze-thaw complex microbubbles, which inherited 61.7% of the protein species of the platelet membrane freeze-thaw complexes, as shown in fig. 12. The prepared platelet membrane freeze-thaw complex microbubbles were vortexed with FITC-labeled CD41 antibody for 2 minutes and then incubated in 37 ℃ with slow agitation for 30 minutes. The parallel groups were then nonspecifically stained with membrane dye DiI with phospholipid and removed at 200 G, centrifugation conditions for 2 minutes separate microbubbles from unbound antibody. The microbubbles were diluted 20-fold and dropped onto grooved slides, which were covered with coverslips and confocal microscopes were imaged with 484nm and 559nm excitation light sources, respectively. As shown in fig. 13, green fluorescence from the platelet membrane was effectively co-localized with red fluorescence from the liposomes in the platelet membrane freeze-thaw complex microbubbles.
8. Platelet membrane freeze-thawing complex microglobulin αiibβ3 protein conformational analysis:
The collected platelets were allowed to stand in Tyrode buffer (5×10 5). The platelet or platelet membrane freeze-thaw complex microbubbles were incubated with normal saline or thrombin (0.5U/mL) in a final volume of Tyrode buffer of 200 μl for 15 minutes. Then stained 20 min with 10 μl PE-JON/a antibody in the dark at room temperature and flow analyzed. In the co-immunoprecipitation experiments, the platelets of the mice were stimulated with 0.5U/mL thrombin, and the reaction was stopped by adding lysis buffer. Activated platelet lysate, platelet membrane freeze-thaw complex lysate and platelet membrane freeze-thaw complex microbubble lysate are then incubated with anti-beta 3 antibody (50 μg/mL) at 4 ℃ overnight. The next day rProtein A/G magnetic beads were added to absorb the immune complex. The samples were separated on a magnetic rack and washed 6 times with lysis buffer. Immunoprecipitation was analyzed using SDS-polyacrylamide gel electrophoresis and Western blotting with β3 and talin-1 antibodies. As shown in fig. 14, glycoprotein αiibβ3 on the surface of the platelet membrane freeze-thaw complex microbubbles has an activated conformation and activation thereof is independent of the tanin-1 protein within the platelets.
9. Construction of acute and chronic thrombus models:
The rat was anesthetized with isoflurane, the inferior vena cava was found after the abdominal opening, and a section of filter paper strip with a length of 4 mm and a width of 2 mm was soaked in 20% ferric trichloride, and after removal, the filter paper strip was stuck to the surface of the inferior vena cava, after 1 minute, the filter paper strip was removed, and the place was rewashed with PBS to construct an acute thrombus model of the rat. In the chronic thrombus model, rats were kept under sterile conditions for 10 days after wound suturing, and a chronic thrombus model was constructed.
10. Ultrasound molecular image diagnosis of acute and chronic thrombus models:
The ultrasonic contrast device adopts LOGIQ E whole body application ultra-high-end color ultrasound produced by GE company, firstly, 18 MHz probe mechanical index MI is selected to be 1.0, the inferior vena cava is contrast in B mode, the size and the position of thrombus are judged, and the blood flow at the thrombus is contrast in Doppler mode. Extracting 5ml normal saline by a 5ml syringe, adding the normal saline into the platelet membrane freeze-thawing compound micro-bubble freeze-dried powder, and forcibly shaking and re-dissolving to form the platelet membrane freeze-thawing compound micro-bubble. The platelet membrane freeze-thawing complex microbubbles were injected into the tail vein of rats via the tail vein, respectively. And selecting a probe of 9 MHz, adjusting the mechanical index MI to 0.14, and carrying out contrast enhancement ultrasonic image analysis on inferior vena cava in a contrast enhancement mode (CEUS), and carrying out vascular perfusion and thrombus targeting. Images were continuously acquired for 5 minutes, and signal intensities of the region of interest of the images were analyzed using MATLAB software, and ultrasound signal values of the region of interest were determined from a scale of ultrasound intensities. As shown in fig. 15, in the acute thrombus model, the platelet membrane freeze-thaw complex microbubbles can effectively target the acute thrombus, thus effectively improving the ultrasonic signal-to-noise ratio of the acute thrombus diagnosis. As shown in fig. 16, in the chronic thrombus model, the platelet membrane freeze-thaw complex microbubbles were not enriched at the chronic thrombus, and the ultrasonic signal-to-noise ratio was not significantly changed. The hematoxylin-eosin and masson staining analysis of acute thrombus and chronic thrombus prove that the components of the platelet membrane freeze-thawing compound microbubble have obvious differences, and the average ultrasonic signal-to-noise ratio of the platelet membrane freeze-thawing compound microbubble in the diagnosis of the acute thrombus and the chronic thrombus is mainly based on fibrin.
In conclusion, the invention adopts a mode of repeatedly freezing with liquid nitrogen and thawing at room temperature to dope phospholipid into the platelet membrane and obtain the platelet membrane freeze-thawing complex vesicle. The surface tension of phospholipid protein molecules in the freeze-thawing compound at a gas-liquid interface is obviously reduced by a Langmuir-Blodgett membrane balance test, and the glass transition temperature of the doped platelet membrane is reduced by a differential and moderate thermal scanning technology test. And blowing sulfur hexafluoride gas into the platelet membrane freeze-thawing compound vesicles in an ultrasonic auxiliary mode to form the platelet membrane freeze-thawing compound vesicles at a gas-liquid interface. Platelet membrane proteins were located by fluorescent labeling and successfully spiked into platelet membrane freeze-thaw complex microbubbles. By proteomic analysis of platelet membrane freeze-thaw complex vesicles and platelet membrane freeze-thaw complex microbubbles, it was found that the platelet membrane freeze-thaw complex microbubbles inherited 61.4% of the protein species of the platelet membrane freeze-thaw complex vesicles and maintained the conformation of integrin αiiβ3 activation on the platelet membrane surface. In the experiment of the acute and chronic thrombus model of the inferior vena cava of a rat, the platelet membrane freeze-thawing complex microbubbles can specifically identify the acute thrombus, the average signal-to-noise ratio for diagnosing the acute thrombus is 12.47 dB, and the chronic thrombus is 0.1dB.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, 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 and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention.
Claims (8)
1. A method for regulating and controlling the surface tension of a platelet membrane, which is characterized by comprising the following steps:
step 1: using a freeze thawing mode to dope liposome into the platelet membrane to obtain a platelet membrane freeze thawing compound;
Step 2: the liposomes and platelet membrane protein components of the platelet membrane freeze-thawing complex are assembled to the microbubble surface of the platelet membrane freeze-thawing complex by means of gas-liquid interface assembly.
2. The method for regulating and controlling the surface tension of a platelet membrane according to claim 1, wherein: the platelet membrane freeze-thawing compound is thawed in a liquid nitrogen freezing room temperature manner.
3. A method for regulating and controlling the surface tension of a platelet membrane according to claim 2, wherein: the liquid nitrogen was thawed 5 times at room temperature.
4. The method for regulating and controlling the surface tension of a platelet membrane according to claim 1, wherein: in the platelet membrane freeze-thawing complex, the liposome-derived phospholipid is any one of DPPC, DPPG, DSPE-PEG 2000, a DPPC and DPPG combination, a DPPC and DSPE-PEG 2000 combination, a DPPG and DSPE-PEG 2000 combination and a DPPC, DPPG and DSPE-PEG 2000 combination.
5. The method for regulating and controlling the surface tension of a platelet membrane according to claim 1, wherein: in platelet membrane freeze-thaw complex microbubbles, the protein to liposome mass ratio of 0.02 has the lowest glass transition temperature.
6. The method for regulating and controlling the surface tension of a platelet membrane according to claim 1, wherein: in the platelet membrane freeze-thawing complex microbubbles, the protein to liposome mass ratio was 0.02 with the lowest gas-liquid interfacial surface tension.
7. The method for regulating and controlling the surface tension of a platelet membrane according to claim 1, wherein: the average particle size of the platelet membrane freeze-thawing complex microbubbles is 1000nm to 10 μm.
8. The method for regulating and controlling the surface tension of a platelet membrane according to claim 7, wherein: the surface of the platelet membrane freeze-thawing complex microbubbles has integrin alpha IIbβ3 on the surface of the original platelet membrane and maintains the activated conformation.
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