Membrane protein self-assembly microbubble for targeting thrombus and preparation method of membrane protein self-assembly microbubble

Abstract

The invention provides a membrane protein self-assembly microbubble for targeting thrombus and a preparation method thereof, and belongs to the field of biomedical engineering.The preparation method combines freezing fusion and ultrasonic treatment and comprises the steps that firstly, a Langmuir Blodgett membrane balance is used for testing and determining a formula of phospholipid and a formula of membrane protein; secondly, non-covalent coupling of the phospholipid and the membrane protein is achieved in the repeated freezing and thawing process, and in the process, the phospholipid and the membrane protein are self-assembled to form a compound with a stable structure at normal temperature; then, SF6 bubbles are introduced under the action of external ultrasound, and membrane protein self-assembled microbubbles are formed. The prepared membrane protein self-assembled microbubble has good targeting performance, can accurately recognize and act on a thrombus part, and can be used as an excellent contrast agent in the aspect of ultrasonic application, so that a doctor can clearly observe information such as the position, the size and the form of thrombus, and an important basis is provided for diagnosis of thrombus-related diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering, and in particular relates to a membrane protein self-assembled microbubble for targeting thrombus and a preparation method thereof. Background Art

[0002] As a key tool in medical imaging, microbubble contrast agents significantly enhance the accuracy and sensitivity of ultrasound diagnosis through their unique physical properties. It can significantly improve image contrast, make the microvascular structure and lesion details clearly visible, and provide more detailed and accurate diagnostic information for clinicians. Through specific molecular design, microbubbles can selectively bind to specific components in thrombi, thereby enhancing the ultrasound imaging effect on thrombi. For example, by chemically modifying the RGD-type peptide segment to the surface of microbubbles, this targeted microbubble can successfully bind to the IIb / IIIa receptor on the surface of activated platelets in thrombi, achieving thrombus-specific contrast enhancement. However, the targeting recognition efficiency of chemically modified microbubbles is limited by many factors such as the surface density and molecular conformation of the modified ligand. In addition, chemical modification will also affect the functionality of molecular recognition.

[0003] In order to improve the molecular recognition function of microbubbles, the present invention non-covalently self-assembles cell membrane proteins on the surface of microbubbles. Cell membrane proteins, because of their transmembrane domains, are embedded in the surface of the phospholipid bilayer of cells through non-covalent self-assembly, maintaining the optimal surface density and natural active conformation, and have efficient molecular recognition function, making them an excellent choice for microbubble targeting ligands. However, how to non-covalently self-assemble membrane proteins on the surface of microbubbles, that is, in the phospholipid monolayer at the gas-liquid interface, is still extremely challenging. Summary of the invention

[0004] Purpose of the invention: In order to solve the problem of how to non-covalently self-assemble membrane proteins on the surface of microbubbles, the present invention provides a membrane protein self-assembled microbubbles for targeting thrombus and a preparation method thereof, by repeatedly freezing and fusing phospholipids and membrane proteins, and using ultrasonic technology to make phospholipids and proteins self-assemble at the gas-liquid interface, to form membrane protein self-assembled microbubbles for targeting thrombus.

[0005] The present invention discloses a method for preparing membrane protein self-assembled microbubbles for targeting thrombus, comprising the following steps:

[0006] Step 1, selecting a phospholipid composition and a membrane protein for assembling microbubbles, and determining the ratio of each component in the phospholipid composition and the ratio of the phospholipid composition to the membrane protein when the surface tension of the gas-liquid interface is the lowest;

[0007] Step 2: according to the result determined in step 1, a mixture of the phospholipid composition and the membrane protein is prepared, and the phospholipid composition and the membrane protein in the mixture are fully fused by a freeze-thaw method to form a phospholipid-protein freeze-thaw conjugate;

[0008] Step 3: Bubble sulfur hexafluoride into the phospholipid-protein freeze-thaw conjugate by means of probe ultrasonic treatment to form membrane protein self-assembled microbubbles.

[0009] Furthermore, in the step 1, the phospholipids in the phospholipid composition are artificial phospholipids; the phospholipid composition is DPPC (dipalmitoylphosphatidylcholine), DPPG (distearate phosphatidylglycerol) and DSPE-PEG 2000 (distearate phosphatidylethylamine alcohol-polyethylene glycol 2000) or a combination of several thereof;

[0010] The membrane protein (membrane protein is the abbreviation of platelet membrane protein) is αⅡbβ3 membrane protein.

[0011] Furthermore, in step 1, the ratio of each component in the phospholipid composition and the ratio of the phospholipid composition to the membrane protein are determined by surface tension control method, and the specific steps are as follows:

[0012] Step 1.1, determining the ratio of each component in the phospholipid composition;

[0013] Using chloroform as solvent, phospholipid solutions with the same total phospholipid mass but different mass ratios of each component were prepared, and the surface tension of the phospholipid solutions with different ratios at the gas-liquid interface was measured using a Langmuir-Blodgett multifunctional membrane balance to obtain the mass ratio of the components when the surface tension of the gas-liquid interface was the lowest.

[0014] Step 1.2, determining the ratio of phospholipid composition to membrane protein;

[0015] The phospholipid composition determined in step 1.1 was used to prepare mixed solutions of phospholipid compositions and membrane proteins with different mass ratios. The surface tension of the mixed solutions of phospholipid compositions and membrane proteins with different ratios at the gas-liquid interface was measured using a Langmuir-Blodgett multifunctional membrane balance to obtain the mass ratio of the components when the surface tension at the gas-liquid interface was the lowest.

[0016] Further, the phospholipid composition is DPPC, DPPG and DSPE-PEG (2000) The composition of DPPC, DPPG and DSPE-PEG (2000) The mass ratio is 8:1:1;

[0017] The mass ratio of the αⅡbβ3 membrane protein to the phospholipid composition is 0.02.

[0018] Furthermore, in step 2, the freeze-thaw method is: freezing with liquid nitrogen and then thawing at room temperature, and the number of freeze-thaw cycles is no less than three times.

[0019] Furthermore, the average particle size of the membrane protein self-assembled microbubbles is 1.2 μm to 12 μm.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The present invention tests and optimizes the phospholipid-protein ratio by Langmuir Blodgett membrane balance, so that its surface tension at the gas-liquid interface is significantly reduced, and the αⅡbβ3 membrane protein is fused with phospholipids by repeated freezing-thawing at room temperature to obtain a freeze-thaw conjugate, and then sulfur hexafluoride gas is blown into the phospholipid-protein freeze-thaw conjugate by ultrasound assistance to form membrane protein self-assembled microbubbles at the gas-liquid interface. The present invention reduces the surface tension of the cell membrane by regulating the ratio of protein to phospholipid in the freeze-thaw conjugate, and the freeze-thaw complex after the surface tension is reduced can be spontaneously adsorbed at the gas-liquid interface under the action of high shear force to form bubbles coated with the freeze-thaw conjugate.

[0022] 2) The membrane protein self-assembled microbubbles prepared by the present invention have good targeting performance and can accurately identify and act on the site of thrombus. In terms of ultrasound application, the microbubbles can serve as excellent contrast agents, allowing doctors to clearly observe the location, size, morphology and other information of thrombus, providing an important basis for the diagnosis of thrombus-related diseases.

[0023] 3) In the present invention, the αⅡbβ3 membrane protein was located by fluorescent labeling and successfully traced in the membrane protein self-assembled microbubbles. By quantifying the number of microbubbles and protein concentration, it was calculated that there were about 23,000 proteins on each microbubble. Through the microbubble targeting blood clot experiment, it was proved that the synthesized membrane protein self-assembled microbubbles can specifically identify clots and have targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The surface pressure and area isotherm curves of the regulating effect of liposomes with different phospholipid formulas on the surface tension of the air-liquid interface;

[0025] Figure 2 The surface pressure and area isotherm curves of the regulating effect of liposomes with different phospholipid protein formulas on the surface tension of the air-liquid interface;

[0026] Figure 3 This is the surface pressure and area isotherm curve of liposomes repeatedly pulling the membrane when the protein-phospholipid ratio is 0.02;

[0027] Figure 4 This is the particle size distribution diagram of the synthesized membrane protein self-assembled microbubbles;

[0028] Figure 5 This is a fluorescence confocal schematic diagram of the fusion of αⅡbβ3 membrane protein and phospholipid;

[0029] Figure 6 Flow cytometric diagram to verify the fusion of αⅡbβ3 membrane protein and phospholipid;

[0030] Figure 7 Small animal fluorescence imaging of αⅡbβ3 membrane protein self-assembled microbubbles targeting blood clots;

[0031] Figure 8 This is a quantitative graph of the small animal fluorescence signal of αⅡbβ3 membrane protein self-assembled microbubbles targeting blood clots;

[0032] Fig. 9 Ultrasound contrast imaging of rat liver after intravenous administration of αⅡbβ3 membrane protein self-assembled microbubbles. DETAILED DESCRIPTION

[0033] In order to make the purpose, technology and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should also be noted that, in order to avoid blurring the present invention due to unnecessary details, only structures and / or processing steps closely related to the scheme of the present invention are shown in the accompanying drawings, and other details that are not closely related to the present invention are omitted.

[0034] The present invention provides a method for preparing membrane protein self-assembled microbubbles for targeting thrombus, and the preparation method of the present invention combines freezing fusion with ultrasonic treatment. First, the formula of phospholipids and membrane proteins is determined by Langmuir Blodgett membrane balance test. Secondly, phospholipids and membrane proteins are non-covalently coupled during repeated freezing and thawing, during which phospholipids and membrane proteins self-assemble to form a complex with a stable structure at room temperature; then, SF6 bubbles are introduced under external ultrasound to form membrane protein self-assembled microbubbles.

[0035] In the embodiment of the present invention, DPPC, DPPG, DSPE-PEG (2000) All of them were from Avituo Pharmaceutical Technology Co., Ltd., PBS, DiD, and PE were purchased from Jiangsu Keygen Biotechnology Co., Ltd., and blood clots were extracted from mouse raw blood; chloroform was purchased from Nanjing Wanqing Chemical Glass Instrument Co., Ltd., and mice were purchased from Qinglongshan Animal Farm.

[0036] Example 1: Regulation of gas-liquid interfacial tension of protein and phospholipid compositions

[0037] Step 1, preparing solutions with different phospholipid ratios in chloroform solvent, and measuring the surface tension of phospholipids with different ratios at the gas-liquid interface using a Langmuir-Blodgett multifunctional membrane balance;

[0038] First, prepare DPPC:DPPG=9:1, DPPC:DPPG:DSPE-PEG 2000=8:1:1, DPPC:DPPG:DSPE-PEG 2000 =16:2:3,DPPC:DPPG:DSPE-PEG 2000 =16:2:1, DPPC:DPPG:DSPE-PEG 2000 =9:1:2 and pure DSPE-PEG 2000 Six solutions with different ratios. 20 μl of phospholipid solutions with different ratios (total phospholipid concentration 1 mg / ml) were evenly dripped onto the Langmuir-Blodgett membrane balance, and the chloroform was evaporated for 10 minutes. After the initial surface pressure stabilized, the initial surface area was set to 282 cm 2 , clear the surface pressure, compress the film at a speed of 0.2 mm / s at 20°C, and record the surface tension-surface area curve. Figure 1 As shown, DPPC:DPPG:DSPE-PEG 2000 When the ratio is 8:1:1, the surface tension of the gas-liquid interface is the lowest (the greater the surface pressure, the lower the surface tension), which is 69mN / m.

[0039] Step 2, regulating the air-liquid interfacial tension of the mixture of the phospholipid composition and the membrane protein;

[0040] After optimizing the phospholipid ratio, the phospholipid composition used was DPPC:DPPG:DSPE-PEG 2000 The mass ratio of the membrane protein to the phospholipid composition is 8:1:1; solutions with a mass ratio of αⅡbβ3 membrane protein to the phospholipid composition of 1:50, 1:20, and 1:3.3 (chloroform is used as the solvent, and the total phospholipid concentration is 1 mg / ml) and an αⅡbβ3 membrane protein solution without the phospholipid composition (chloroform is used as the solvent);

[0041] 20 μl of the above solution was evenly dripped onto the Langmuir-Blodgett film balance and the chloroform was allowed to evaporate for 10 min. After the initial surface pressure stabilized, the initial surface area was set to 282 cm 2 , clear the surface pressure, compress the film at a speed of 0.2 mm / s at 20°C, and record the surface tension-surface area curve. Figure 2 As shown, when the mass ratio of αⅡbβ3 membrane protein to phospholipid is 0.02, the surface tension of the complex is the lowest.

[0042] like Figure 3 As shown, the surface circulation performance of phospholipid compositions and membrane proteins was determined. 2000 The ratio of 8:1:1 and the ratio of protein to phospholipids of 0.02 were tested for 5 film drawing tests, and the surface pressure did not collapse and was able to maintain good performance.

[0043] Example 2: Preparation of αⅡbβ3 membrane protein solution

[0044] αⅡb protein and β3 protein were synthesized by GenScript and combined at 4° to form αⅡbβ3 membrane protein; the concentration of αⅡb protein was 0.19 mg / ml, the concentration of β3 protein was 1.17 mg / ml, and the final protein concentration of the lipoprotein obtained by the ratio was 0.34 mg / ml.

[0045] Example 3: Preparation of phospholipid composition solution

[0046] The selected phospholipid composition is dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000 ), a combination of distearate phosphatidylglycerol (DPPG);

[0047] Afterwards, according to DPPC:DPPG:DSPE-PEG 2000 The liposome solution was dissolved in chloroform at a mass ratio of 8:1:1, and rotary evaporated at 50°C and 100 rpm until a uniform film was formed at the bottom of the round-bottom flask; the flask was removed and dried at room temperature for 1 hour. Then, phosphate buffer (PBS) was added to the dried flask, and water bath ultrasound was performed at room temperature, 100 W, 53 kHz to obtain a liposome solution, and the liposome pH was adjusted to 7.4, with a final concentration of 10 mg / ml.

[0048] Example 4: Synthesis of membrane protein self-assembled microvesicles

[0049] The αⅡbβ3 membrane protein solution prepared in Example 2 and the phospholipid composition solution prepared in Example 3 were mixed evenly at a mass ratio of 50:1 between the phospholipid composition and the αⅡbβ3 protein. The mixture was repeatedly frozen and thawed three times in liquid nitrogen and room temperature to obtain a phospholipid-protein freeze-thaw conjugate. The phospholipid-protein freeze-thaw conjugate was treated with probe ultrasound (300W, 5s on, 5s off), sulfur hexafluoride gas was blown into the system, and membrane protein self-assembled microbubbles were formed by ultrasound. The phospholipid composition and membrane protein self-assembled at the gas-liquid interface to form a large number of stable micron-sized bubbles.

[0050] 1) Microbubble particle size determination:

[0051] Microbubbles were photographed using a microscope, and the microbubble size was counted using image J software. Figure 4 As shown, 472 microbubbles were counted, and the particle size was 1.20±0.57 μm.

[0052] 2) Quantitative statistics of microvesicle numbers and αⅡbβ3 membrane protein numbers:

[0053] Take 2 μl of membrane protein self-assembled microbubble solution, dilute it five times, count it three times using a cell counting plate and take the average number, which is 2.16*10 7 microbubbles, that is, the number of self-assembled microbubbles of membrane protein without dilution is 5.4*10 7 / μl; the concentration of microvesicle protein measured by microplate reader was 0.184mg / ml; the molar mass of αⅡbβ3 membrane protein was 2.23*10 5 g / mol; the calculated molar number of αⅡbβ3 membrane protein in 1μl microbubble is 8.25*10 7 / μmol, each microbubble contains 3.82*10 -14 / μmol, that is, each microbubble contains 8.52*10 -9 / μg protein, i.e. 2.30*10 4 A protein molecule.

[0054] 3) Verification of membrane protein self-assembled microbubble fusion:

[0055] The phospholipid composition and αⅡbβ3 protein were stained with excess DiD (cell membrane red fluorescent probe) and PE (phycoerythrin) respectively (αⅡbβ3 membrane protein solution prepared in Example 2 and phospholipid composition solution prepared in Example 3), and then the un-membrane-embedded dye was removed by centrifugation at 12000g, 10min, and 4°C, and incubated in a constant temperature box for 30min. After being taken out, they were fused in a mass ratio of 50:1 between the phospholipid composition and the αⅡbβ3 protein, and repeatedly frozen and thawed three times in liquid nitrogen and room temperature to obtain a phospholipid-protein freeze-thaw conjugate. The phospholipid-protein freeze-thaw conjugate was treated with probe ultrasound (300W, 5s on, 5s off), and sulfur hexafluoride gas was blown into the system to form αⅡbβ3 membrane protein self-assembled microbubbles by ultrasound. After that, the microbubbles were diluted 20 times and dropped onto a glass slide with a groove, and then covered with a coverslip, and confocal microscopy was used to take pictures with excitation light sources of 635nm and 535nm, respectively. The results are as follows Figure 5 As shown, the phospholipid-protein microbubbles contain blue fluorescence from phospholipids and orange-red fluorescence from αⅡbβ3 protein, and the fluorescence superposition proves that the phospholipid protein has been successfully fused.

[0056] 4) Flow cytometry detection of membrane protein self-assembled microvesicle fusion:

[0057] like Figure 6 As shown, PE fluorescent antibody dye was used to stain phospholipid microvesicles, platelet membrane microvesicles, and αⅡbβ3 membrane protein self-assembled microvesicles, respectively, and flow cytometry was used for detection. Among them:

[0058] Preparation method of phospholipid microbubbles: according to DPPC:DPPG:DSPE-PEG 2000=8:1:1 mass ratio to prepare a phospholipid solution, and ultrasonically bubble sulfur hexafluoride into it to obtain phospholipid microbubbles.

[0059] Preparation method of platelet membrane microbubbles: extract whole blood from mice, centrifuge the whole blood with a centrifugal force of 100g for 10 minutes and take the supernatant; put the supernatant into the centrifuge, set the parameters to 1000g for 10 minutes, and take the bottom precipitate to obtain platelets. Dissolve the platelets in PBS buffer and extract the platelet membrane by freezing and thawing with liquid nitrogen; repeatedly freeze and thaw the platelet membrane and phospholipid solution at a mass ratio of 1:50, and inflate with ultrasound to obtain platelet membrane microbubbles.

[0060] The results showed that both the αⅡbβ3 membrane protein self-assembled microbubbles and the platelet membrane microbubbles had fluorescent antibody binding proteins, proving that the phospholipid proteins were successfully fused.

[0061] 5) Blood clot targeting experiment:

[0062] Blood clots were pre-added to a 96-well plate, and DiD (cell membrane far-red fluorescent probe) was used to stain phospholipid microbubbles, platelet membrane microbubbles, and αⅡbβ3 membrane protein self-assembled microbubbles. PBS buffer, phospholipid microbubbles, platelet membrane microbubbles, and αⅡbβ3 membrane protein self-assembled microbubbles were co-incubated with blood clots for 1 hour, and then the microbubbles were removed with a pipette, and washed with PBS three times. The targeting situation was observed using small animal imaging. The results are shown in Figure 2. Figure 7 and Figure 8 shown.

[0063] Figure 7 The right side of the figure is the standard bar; Figure 7 In the middle left picture, PBS buffer was added in the first row, stained phospholipid microbubbles were added in the second row, stained platelet membrane microbubbles were added in the third row, and stained αⅡbβ3 membrane protein self-assembled microbubbles were added in the fourth row. Since no protein was added to the second row, there is no targeting and the signal intensity is low. Platelet membrane microbubbles and αⅡbβ3 membrane protein microbubbles have the ability to target blood clots and have high signal intensity. Figure 8 The fluorescence signal intensity is quantified. Figure 7 and Figure 8 The results show that both αⅡbβ3 membrane protein self-assembled microbubbles and platelet membrane microbubbles have targeting effects, and the effects are similar, proving that the synthesized membrane protein microbubbles have good targeting properties. Before the synthesis of microbubbles, αⅡbβ3 lipoprotein was not in an activated state. Through the blood clot targeting experiment, it can be proved that the αⅡbβ3 membrane protein on the surface of the αⅡbβ3 membrane protein self-assembled microbubbles can maintain an activated conformation.

[0064] To evaluate the in vivo stability of αⅡbβ3 membrane protein self-assembled microbubbles as blood pool contrast agents, Doppler ultrasound examinations were performed on the livers of SD rats to assess blood flow and tissue integrity. Representative ultrasound contrast images of rat livers were obtained after intravenous administration of αⅡbβ3 membrane protein microbubbles, as shown in Figure 2. Fig. 9 As shown, it indicates that αⅡbβ3 membrane protein microbubbles have a good perfusion imaging effect on blood flow.

[0065] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.

Claims

1. A method for preparing membrane protein self-assembled microbubbles for targeting thrombus, characterized in that: The following steps are involved: Step 1, selecting a phospholipid composition and a membrane protein for assembling microbubbles, and determining the ratio of each component in the phospholipid composition and the ratio of the phospholipid composition to the membrane protein when the surface tension of the gas-liquid interface is the lowest; Step 2: according to the result determined in step 1, a mixture of the phospholipid composition and the membrane protein is prepared, and the phospholipid composition and the membrane protein in the mixture are fully fused by a freeze-thaw method to form a phospholipid-protein freeze-thaw conjugate; Step 3: Bubble sulfur hexafluoride into the phospholipid-protein freeze-thaw conjugate by means of probe ultrasonic treatment to form membrane protein self-assembled microbubbles.

2. The preparation method according to claim 1, characterized in that: In the step 1, the phospholipids in the phospholipid composition are artificial phospholipids; the phospholipid composition is DPPC, DPPG and DSPE-PEG 2000 One or a combination of the following: The membrane protein is αⅡbβ3 membrane protein.

3. The preparation method according to claim 1, characterized in that: In the step 1, the method for determining the ratio of each component in the phospholipid composition and the ratio of the phospholipid composition to the membrane protein is a surface tension control method, and the specific steps are as follows: Step 1.1, determining the ratio of each component in the phospholipid composition; Using chloroform as solvent, phospholipid solutions with the same total phospholipid mass but different mass ratios of each component were prepared, and the surface tension of the phospholipid solutions with different ratios at the gas-liquid interface was measured using a Langmuir-Blodgett multifunctional membrane balance to obtain the mass ratio of the components when the surface tension of the gas-liquid interface was the lowest. Step 1.2, determining the ratio of phospholipid composition to membrane protein; The phospholipid composition determined in step 1.1 was used to prepare mixed solutions of phospholipid compositions and membrane proteins with different mass ratios. The surface tension of the mixed solutions of phospholipid compositions and membrane proteins with different ratios at the gas-liquid interface was measured using a Langmuir-Blodgett multifunctional membrane balance to obtain the mass ratio of the components when the surface tension at the gas-liquid interface was the lowest.

4. The preparation method according to claim 2, characterized in that: The phospholipid composition is DPPC, DPPG and DSPE-PEG (2000) The composition of DPPC, DPPG and DSPE-PEG (2000) The mass ratio is 8:1:1; The mass ratio of the αⅡbβ3 membrane protein to the phospholipid composition is 0.

02.

5. The preparation method according to claim 2, characterized in that: In step 2, the freeze-thaw method is: freezing with liquid nitrogen and then thawing at room temperature, and the number of freeze-thaw cycles is no less than three times.

6. The preparation method according to claim 1, characterized in that: The average particle size of the membrane protein self-assembled microbubbles is 1.2 μm to 12 μm.

7. A membrane protein self-assembled microbubble for targeting thrombus, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6.

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