Preparation method and application of ultrasound contrast microbubbles targeting tumor vascular endothelial CD93 molecules

Abstract

This invention belongs to the field of ultrasound contrast microbubble synthesis technology, and relates to a method for preparing and applying ultrasound contrast microbubbles targeting CD93 molecules in tumor vascular endothelium. The method includes the following steps: constructing a recombinant expression vector capable of expressing the CD93 ligand MMRN2 protein, transfecting cells, and extracting the cell membrane using differential centrifugation; mixing dipalmitoylphosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol 2000, and chloroform, adding DiI, and preparing a liposome membrane using a thin-film hydration method; mixing the cell membrane and the liposome membrane, performing ice-bath sonication, and mixing and shaking with perfluoropropane to obtain an ultrasound contrast agent targeting CD93 molecules in tumor vascular endothelium. The ultrasound contrast microbubbles prepared by the method of this invention have good biocompatibility compared to conventional antibody-targeting strategies, and can also improve stability and targeting, thereby enabling the assessment of CD93 molecule expression in tumors.

Description

Technical Field

[0001] This invention belongs to the field of ultrasound contrast microbubble synthesis technology, specifically relating to a method for preparing and applying ultrasound contrast microbubbles targeting tumor vascular endothelial CD93 molecules. Background Technology

[0002] In recent years, immunotherapy has become one of the most popular areas of cancer research. However, due to tumor heterogeneity and the infeasibility of repeated biopsies, predicting and monitoring efficacy remains a significant challenge for immunotherapy. PET and SPECT are existing molecular imaging tools for assessing the efficacy of tumor immunotherapy, based on labeling and tracking individual immune cells or signaling molecules in the tumor parenchyma. However, their applications are limited due to high imaging costs, long cycles, and high radiation doses. Ultrasound molecular imaging utilizes targeted contrast microbubbles as molecular probes for imaging in contrast-enhanced ultrasound mode. It offers advantages such as real-time, non-invasive, safe, and repeatable ultrasound imaging, allowing for the assessment of tumor blood perfusion and the targeting of specific receptors to achieve microscopic molecular-level imaging. However, existing microbubble preparation methods and techniques for attaching ligands to microbubbles are still immature. Targeted contrast agents suffer from poor stability, low connectivity and target-finding capabilities, and the use of exogenous materials raises biosafety concerns. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing ultrasound contrast microbubbles targeting tumor vascular endothelial CD93 molecules and their application. The ultrasound contrast microbubbles prepared by the method of this invention exhibit good biocompatibility, and can also improve stability and targeting, thereby enabling the evaluation of tumor CD93 molecule expression.

[0004] This invention provides a method for preparing ultrasound contrast microbubbles targeting tumor vascular endothelial CD93 molecules, comprising the following steps:

[0005] A recombinant expression vector capable of expressing MMRN2 protein was constructed, and the recombinant expression vector was transfected into HEK293T cells to obtain HEK293T cells expressing MMRN2 protein;

[0006] The cell membrane of HEK293T cells expressing MMRN2 protein was extracted using differential centrifugation to obtain the HEK293T cell membrane expressing MMRN2 protein.

[0007] Liposome membranes were prepared by mixing dipalmitoylphosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol 2000 and chloroform, adding DiI, and using a membrane hydration method.

[0008] The HEK293T cell membrane expressing MMRN2 protein and the liposome membrane were mixed and subjected to ice bath sonication to obtain a mixture. The mixture was then mixed with perfluoropropane in a closed device and mechanically vibrated to obtain ultrasound imaging microbubbles targeting tumor vascular endothelial CD93 molecules.

[0009] Preferably, the method for constructing the recombinant expression vector capable of expressing the MMRN2 protein includes the following steps:

[0010] A recombinant gene capable of expressing the MMRN2 protein was constructed into a basic plasmid to obtain a recombinant expression vector expressing the MMRN2 protein. The recombinant gene includes, from the 3' end to the 5' end, a Hind III restriction site, a Kozak fragment, a signal peptide, an MMRN2 fragment, a PTGFRN fragment, a FLAG tag, and a Kpn I restriction site connected in sequence. The basic plasmid includes pcDNA3.1(+).

[0011] Preferably, the nucleotide sequence of the recombinant gene is as shown in SEQ ID NO.1.

[0012] Preferably, the differential centrifugation method for extracting cell membranes includes the following steps: 24 h after transfection, HEK293T cells expressing MMRN2 protein are digested with trypsin, centrifuged, the precipitate is resuspended in hypotonic lysis buffer, lysed, homogenized and centrifuged to remove cell nuclei and unbroken cells, the supernatant is then centrifuged to separate mitochondria, and the resulting supernatant is subjected to ultracentrifugation to obtain the HEK293T cell membrane expressing MMRN2 protein.

[0013] Preferably, the mass ratio of dipalmitoylphosphatidylcholine, the mass of distearate phosphatidylethanolamine-polyethylene glycol 2000, the volume of chloroform, and the volume of DiI is (5-15) mg:(3-5) mg:(1-3) mL:(8-12) μL; and the concentration of DiI is 930 μg / mL.

[0014] Preferably, the steps for preparing liposome films using the thin-film hydration method include:

[0015] Dipalmitoylphosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol 2000 and chloroform were mixed, and DiI was added under light-protected conditions. After dissolution, the mixture was rotary evaporated to obtain a dry lipid film. A hydration solution was added to the dry lipid film and the mixture was shaken to obtain a liposome film.

[0016] Preferably, the amount of HEK293T cell membrane expressing MMRN2 protein added is based on the amount of dipalmitoylphosphatidylcholine and distearate phosphatidylethanolamine-polyethylene glycol 2000 added, and the mass ratio of dipalmitoylphosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol 2000 and the mass of HEK293T cell membrane expressing MMRN2 protein calculated according to protein quantification is (5-15):(3-5):(0.25-0.35).

[0017] Preferably, the ultrasonic power is 100W, the ultrasonic frequency is 40-45kHz, and the ultrasonic duration is 2-4min.

[0018] The present invention also provides targeted tumor vascular endothelial CD93 molecular ultrasound contrast microbubbles prepared by the preparation method described in the above technical solution.

[0019] This invention also provides the application of the targeted tumor vascular endothelial CD93 molecule ultrasound contrast microbubbles prepared by the preparation method described above, or the ultrasound contrast microbubbles described above, in the preparation of products for evaluating the tumor immune microenvironment; the evaluation of the tumor immune microenvironment includes monitoring immune-related hemodynamic changes or reflecting the tumor's immune infiltration status through targeted signals.

[0020] This invention provides a method for preparing CD93-targeting ultrasound imaging microbubbles for tumor vascular endothelial cells. The invention designs a simple and feasible ultrasound imaging microbubble, namely a CD93-targeting microbubble, for intravascular molecular assessment, evaluating CD93 expression within tumors. The microbubble is prepared using phospholipids and HEK293T cell membranes transfected with recombinant genes as shell materials, exhibiting good biocompatibility and ensuring the stability and targeting of the contrast agent. CD93 is a transmembrane protein expressed on vascular endothelial cells. Its binding to its ligand MMRN2 promotes pathological angiogenesis in tumors, and CD93 expression is also correlated with tumor immune infiltration. Ultrasound molecular imaging based on CD93-targeting microbubbles can assess tumor immune status by monitoring CD93 expression. The ultrasound imaging microbubbles prepared by the method described in this invention can improve the stability and targeting of ultrasound molecular imaging, thereby assessing the tumor immune microenvironment. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1This is a schematic diagram of a recombinant gene provided by the present invention;

[0023] Figure 2 This is a diagram showing the results of high MMRN2 expression in the cell membrane after transfection with recombinant genes, as provided by the present invention.

[0024] Figure 3 This is a schematic diagram of targeted imaging microbubble synthesis provided by the present invention;

[0025] Figure 4 The targeted microbubble scanning electron microscope image provided by this invention;

[0026] Figure 5 This is a schematic diagram of the targeted microbubble particle size distribution provided by the present invention;

[0027] Figure 6 This is a schematic diagram of the targeted microbubble potential distribution provided by the present invention;

[0028] Figure 7 The targeted microbubble fluorescence microscope image provided by this invention;

[0029] Figure 8 The image shows the results of confocal microscopy observation of the in vitro target-finding capability of targeted microbubbles provided by this invention.

[0030] Figure 9 The result image shows the in vitro ultrasound contrast imaging capability of the targeted microbubbles provided by this invention. Detailed Implementation

[0031] This invention provides a method for preparing ultrasound contrast microbubbles targeting tumor vascular endothelial CD93 molecules, comprising the following steps:

[0032] A recombinant expression vector capable of expressing MMRN2 protein was constructed, and the recombinant expression vector was transfected into HEK293T cells to obtain HEK293T cells expressing MMRN2 protein;

[0033] The cell membrane of HEK293T cells expressing MMRN2 protein was extracted using differential centrifugation to obtain the HEK293T cell membrane expressing MMRN2 protein.

[0034] Liposome membranes were prepared by mixing dipalmitoylphosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol 2000 and chloroform, adding DiI, and using a membrane hydration method.

[0035] The HEK293T cell membrane expressing MMRN2 protein and the liposome membrane were mixed and subjected to ice bath sonication to obtain a mixture. The mixture was then mixed with perfluoropropane in a closed device and mechanically vibrated to obtain ultrasound imaging microbubbles targeting tumor vascular endothelial CD93 molecules.

[0036] In this invention, the targeted tumor vascular endothelial CD93 molecular ultrasound imaging microbubble is a CD93 targeted microbubble, the shell of which is composed of phospholipids and HEK293T cell membranes transfected with recombinant genes, and the interior is filled with inert gas perfluoropropane (C3F8).

[0037] This invention constructs a recombinant expression vector capable of expressing the MMRN2 protein, and transfects the recombinant expression vector into HEK293T cells to obtain HEK293T cells expressing the MMRN2 protein. In this invention, the construction method of the recombinant expression vector capable of expressing the MMRN2 protein preferably includes the following steps: constructing a recombinant gene capable of expressing the MMRN2 protein into a basic plasmid to obtain a recombinant expression vector expressing the MMRN2 protein; the recombinant gene includes, from the 3' end to the 5' end, a Hind III restriction enzyme site, a Kozak fragment, a signal peptide, an MMRN2 fragment, a PTGFRN fragment, a FLAG tag, and a Kpn I restriction enzyme site connected sequentially; the basic plasmid includes pcDNA3.1(+). In this invention, the nucleotide sequence of the recombinant gene is shown in SEQ ID NO. 1. The recombinant gene capable of expressing the MMRN2 protein of this invention can achieve the expression of the CD93 ligand MMRN2 to the extracellular space. The recombinant expression vector of this invention is preferably constructed by Nanjing Genscript Biotech Co., Ltd., and the recombinant expression vector is preferably transfected into *E. coli* for subsequent plasmid extraction. This invention preferably uses the alkaline cleavage method to extract plasmids (recombinant expression vectors capable of expressing MMRN2 protein). After obtaining the plasmids, HEK293T cells are preferably transfected with HighGene transfection reagent. In this invention, the mass ratio of the recombinant expression vector to the biological volume of the transfection reagent during transfection is preferably (2-4) μg:(4-8) μL. The transfected HEK293T cells are cultured in complete medium containing the transfection reagent (DMEM medium (Gibco, USA) + 10% fetal bovine serum (Gibco, USA) + 1% penicillin-streptomycin (Hyclone, USA)). In this invention, 6 hours after transfection, the medium is preferably changed to complete medium without the transfection reagent. This invention preferably extracts total RNA and total protein at 12 hours and 24 hours after transfection for identification. This invention preferably uses Western blot for protein identification (e.g.,...). Figure 2 The results confirmed that the plasmid transfection was successful, and cell membranes expressing MMRN2 protein were extracted, which were then used to synthesize targeted microvesicles.

[0038] After obtaining HEK293T cells expressing MMRN2 protein, this invention uses differential centrifugation to extract the cell membrane of the HEK293T cells expressing MMRN2 protein, thus obtaining the HEK293T cell membrane expressing MMRN2 protein. In this invention, the differential centrifugation method for cell membrane extraction preferably includes the following steps: 24 h after transfection, trypsin digestion of HEK293T cells expressing MMRN2 protein, centrifugation, resuspending the pellet in hypotonic lysis buffer, lysis, homogenization, centrifugation, removal of cell nuclei and unbroken cells, followed by centrifugation of the supernatant to separate mitochondria, and ultracentrifugation of the resulting supernatant to obtain the HEK293T cell membrane expressing MMRN2 protein. In this invention, the centrifugation conditions after trypsin digestion are preferably 1000 rpm for 5 min. In this invention, the lysis preferably includes the following steps: lysis on ice, followed by grinding and squeezing to obtain a lysed homogenate. In this invention, the lysis time on ice is preferably 25–35 min, more preferably 30 min. In this invention, the hypotonic lysis buffer preferably comprises 20 mM Tris-HCl, pH 7.5, 10 mM KCl, and 2 mM MgCl2, with a protease inhibitor added (one EDTA-free protease inhibitor tablet (Merck, USA) added per 10 ml of hypotonic lysis buffer). In this invention, the grinding and squeezing are preferably performed using a homogenizer. The commercially available homogenizer is preferably Kimble 885300-0002, Mexico. The number of grinding and squeezing cycles is preferably 20-30 times, more preferably 25 times. The centrifugation conditions for the homogenate are preferably 4°C, 500 g, 10 min, to remove cell nuclei and unbroken cells. The supernatant is then centrifuged at 4°C, 10000 g, 10 min, a step that enables mitochondrial separation. Subsequently, the supernatant is ultracentrifuged, preferably at 4°C, 100000 g, 30 min. The present invention preferably uses the BSA protein assay to determine the protein content in the cell membrane, and then prepares microbubbles.

[0039] After obtaining the HEK293T cell membrane expressing MMRN2 protein, this invention mixes dipalmitoylphosphatidylcholine (DPPC), distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and chloroform, adds DiI, and prepares liposome membranes using a thin-film hydration method. In this invention, the preferred mass ratio of dipalmitoylphosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol 2000, chloroform, and DiI is (5-15) mg:(3-5) mg:(1-3) mL:(8-12) μL, more preferably 10 mg:4 mg:2 mL:10 μL. In this invention, the preferred steps for preparing liposome films using the thin-film hydration method include: mixing dipalmitoylphosphatidylcholine, distearylphosphatidylethanolamine-polyethylene glycol 2000, and chloroform; adding DiI under light-protected conditions; dissolving and then rotary evaporating to obtain a dry lipid film; adding hydration solution to the dry lipid film; and shaking to obtain the liposome film. Specifically, this invention preferably weighs 0.01 g of dipalmitoylphosphatidylcholine and 0.004 g of distearylphosphatidylethanolamine-polyethylene glycol 2000, i.e., the mass ratio of DPPC to DSPE-PEG2000 is 10:4, mixes them in a 25 mL rotary evaporator, adds 2 mL of chloroform to the rotary evaporator, adds 10 μL of DiI under light-protected conditions, and gently shakes until fully dissolved. In this invention, the preferred mass concentration of DiI in the DiI solution is 930 μg / mL. In this invention, the preferred rotary evaporation speed is 120 rpm. In this invention, the rotary evaporation time is preferably 25-35 min, more preferably 30 min. Preferably, the rotary evaporation is performed until a light pink, uniformly thick dry lipid film forms on the flask wall. Preferably, the hydration solution is prepared and preheated with 10 min remaining in the rotary evaporation. Preferably, the hydration solution consists of 900 μl PBS + 100 μl glycerol + 20 μl Pronek F-68. Preferably, the preheating temperature is 37°C. When adding the hydration solution to the dry lipid film, the rotary evaporation flask is shaken (i.e., the rotary evaporation flask is wrapped in aluminum foil and placed on a constant temperature shaker at 37°C, 130 rpm, and shaken for 1 hour) to allow the liposome film to detach from the flask wall.

[0040] After obtaining the liposome membrane, the present invention mixes the HEK293T cell membrane expressing MMRN2 protein with the liposome membrane and performs ice bath sonication to obtain a mixture. The mixture is then mixed with perfluoropropane in a closed device and mechanically vibrated to obtain ultrasound imaging microbubbles targeting tumor vascular endothelial CD93 molecules. In this invention, the amount of HEK293T cell membrane expressing MMRN2 protein added is based on the amount of dipalmitoylphosphatidylcholine and distearate-phosphatidylethanolamine-polyethylene glycol 2000 added. The preferred mass ratio of dipalmitoylphosphatidylcholine, distearate-phosphatidylethanolamine-polyethylene glycol 2000, and the mass of HEK293T cell membrane expressing MMRN2 protein (calculated according to protein quantification) is (5-15):(3-5):(0.25-0.35), preferably 10:4:0.3, that is, the mass ratio of DPPC, DSPE-PEG2000, and cell membrane (calculated according to protein quantification) is 10:4:0.3. Specifically, based on the calculation of adding 10 mg of DPPC and 4 mg of DSPE-PEG2000 for the preparation of liposome membrane, it is preferable to mix the cell membrane with a protein quantification of 0.3 mg with the liposome membrane for subsequent operations. When the phospholipid content is determined, the higher the cell membrane content, the worse the gastric bubble stability. The above-mentioned added settings of this invention can balance the stability and targeting of the bubble. In this invention, the ultrasonic power is preferably 100W, the ultrasonic frequency is preferably 40-45kHz, more preferably 42kHz, and the ultrasonic time is preferably 2-4min, more preferably 2min. In this invention, the sealing device preferably includes a bottle sealed with a rubber cap. In this invention, the mixture is preferably placed in the bottle first, then the air in the bottle is exchanged with perfluoropropyne (C3F8), and then the rubber cap is closed and vibration is performed. In this invention, the vibration is preferably performed in a mechanical vibrator, and the number of vibrations is preferably 1-3 times, more preferably 2 times. In this invention, the total vibration time is preferably 90s.

[0041] This invention also provides targeted tumor vascular endothelial CD93 molecular ultrasound contrast microbubbles prepared by the preparation method described in the above technical solution. This invention designs a simple and feasible ultrasound contrast microbubble, namely a CD93-targeting microbubble, for intravascular molecular assessment of the tumor immune microenvironment. The recombinant gene described in this invention can express the CD93 ligand MMRN2 on the outer side of the cell membrane. After transfecting HEK293T cells with the above recombinant gene, a cell membrane with high-efficiency MMRN2 expression can be obtained. Using this cell membrane to prepare targeted ultrasound microbubbles breaks through the traditional method of attaching target molecule-specific antibodies or ligands to the surface of contrast agents, ensuring the stability and targeting of the contrast agent. The ultrasound contrast microbubbles described in this invention can improve the stability and targeting of ultrasound molecular imaging, thereby assessing the tumor immune microenvironment.

[0042] This invention also provides the application of the targeted tumor vascular endothelial CD93 molecule ultrasound contrast microbubbles prepared by the preparation method described above, or the ultrasound contrast microbubbles described above, in the preparation of products for evaluating the tumor immune microenvironment; the evaluation of the tumor immune microenvironment includes monitoring immune-related hemodynamic changes or reflecting the tumor's immune infiltration status through targeted signals.

[0043] To further illustrate the present invention, the preparation method and application of a targeted tumor vascular endothelial CD93 molecular ultrasound contrast microbubble provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Constructing recombinant expression vectors:

[0046] 1) This invention links MMRN2 to the N-terminus of the transmembrane protein PTGFRN via a linker, allowing MMRN2 to be transported to the outer side of the cell membrane via PTGFRN. (See...) Figure 1 .

[0047] 2) Using pcDNA3.1(+) as the vector, Kozak, signal peptide, MMRN2, Linker, PTGFRN, and FLAG fragments were sequentially inserted, with HindIII restriction enzyme site added upstream and KpnI restriction enzyme site added downstream. Plasmid synthesis was performed by Nanjing Genscript Biotech Co., Ltd. The nucleotide sequence is as follows:

[0048] The 3' to 5' ends of the recombinant gene consist of the HindIII restriction site, the Kozak fragment, the signal peptide, MMRN2, the linker, PTGFRN, the FLAG tag, and the KpnI restriction site. The nucleotide sequence of the recombinant gene (SEQ ID NO.1) is as follows:

[0049] Figure 1 As shown.

[0050] The base plasmid of the recombinant expression vector was pcDNA3.1(+). The recombinant expression vector was constructed by Nanjing Genscript Biotech Co., Ltd., and then transferred into *E. coli* for preservation.

[0051] 3) Bacterial culture:

[0052] ①LB formula: Weigh 1.5g tryptone + 1.0g yeast powder + 0.5g NaCl, add 100mL double-distilled water, autoclave for 30min and store at 4℃. Before use, add ampicillin antibiotic at a ratio of 1:1000, shake well, and the bacterial culture medium is ready.

[0053] ② Add 15 mL of bacterial culture medium to the shake tube, add 200 μL of bacterial strain (Escherichia coli containing recombinant expression vector obtained by Nanjing Genscript Biotech Co., Ltd.), mix well, place in a shaker at 37℃ and 286 r / min for 16 h to obtain the culture solution.

[0054] Plasmid extraction and transfection:

[0055] Plasmids were extracted from the culture medium using the alkaline lysis method, and HEK293T cells were transfected with the plasmids using HighGene transfection reagent. 3 μg of the recombinant expression vector was mixed with 7.2 μL of transfection reagent, incubated at 37°C for 20 min, and then slowly added to HEK293 T cell culture medium (DMEM complete medium: DMEM medium (Gibco, USA) + 10% fetal bovine serum (Gibco, USA) + 1% penicillin-streptomycin (Hyclone, USA)). After incubation for 6 h (i.e., 6 h after transfection), the medium was changed to complete medium without the transfection reagent. Total RNA and total protein were extracted at 12 h and 24 h post-transfection for identification. 24 h after transfection, the cell membrane (CM) of HEK293 T cells was extracted using differential centrifugation.

[0056] Extraction of MMRN2 protein-expressing HEK293T cell membranes: Cell membranes were extracted 24 h after recombinant gene transfection using differential centrifugation. HEK293T cells transfected with the recombinant expression vector were digested with trypsin, centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in hypotonic lysis buffer and lysed on ice for 30 min. The hypotonic lysis buffer was prepared as follows: 20 mM Tris-HCl, pH 7.5, 10 mM KCl and 2 mM MgCl2, with the addition of a protease inhibitor (1 tablet of EDTA-free protease inhibitor (Merck, USA) per 10 ml of hypotonic lysis buffer). The cells were then homogenized and squeezed 25 times using a Kimble 885300-0002 (Mexico) to fully lyse the cells. The cell homogenate was centrifuged (4℃, 500 g, 10 min) to remove the nuclei and undisturbed cells, and then the supernatant was centrifuged (4℃, 10000 g, 10 min) to separate the mitochondria. The resulting supernatant was subjected to ultrafiltration (4℃, 100,000g, 30 min) to obtain the cell membrane. The protein content in the purified cell membrane was determined using the BSA protein assay.

[0057] HEK293T cells (Cell) and cell membranes (CM) without transfection with the recombinant gene were collected simultaneously as controls (Ctrl).

[0058] Example 2

[0059] The obtained cells and cell membranes were analyzed by Western blot. FLAG tag

[0060] The extracted cells and cell membranes were added to 100 μL of RIPA lysis buffer (Beyotime, China) and incubated on ice for 30 min to fully lyse the proteins. Protein concentration was determined using a BCA protein assay kit (Thermo, USA). Equal volumes of protein samples (15 μg) were subjected to 5% and 12% SDS-PAGE electrophoresis (concentration gel voltage 120 V, separation gel voltage 160 V) and then transferred to a PVDF membrane. After blocking with 5% skim milk for 1 h at room temperature, the membrane was incubated overnight at 4°C with primary antibody. The primary antibody selected was β-actin (81115-1-RR, Proteintech). + / K + ATPase (14418-1-AP, Proteintech) and FLAG (ab205606, Abcam) antibodies were used. After washing three times in TBST, the mixture was incubated at room temperature for 1 hour with the corresponding anti-rabbit (7074, CST) and anti-mouse (7076, CST) secondary antibodies. Finally, luminescence was performed (ECL luminescent solution, GE, USA).

[0061] The results are as follows Figure 2As shown, the cells and cell membranes transfected with the plasmid express the MMRN2 protein.

[0062] Example 3

[0063] Synthesis of CD93-targeted microbubbles

[0064] Cell membranes were extracted using differential centrifugation according to the method in Example 1. Liposome membranes were prepared using a membrane hydration method. MMRN2-expressing cell membranes and liposome membranes were mixed using ultrasound, and then filled with an inert gas to obtain CD93-targeting microbubbles. The process is as follows: Figure 3 As shown.

[0065] Specifically, liposome membranes were prepared using the membrane hydration method: 0.01 g of dipalmitoylphosphatidylcholine (DPPC) and 0.004 g of distearate-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, i.e., the mass ratio of DPPC to DSPE-PEG2000 was 10:4. These were mixed in a 25 ml rotary evaporator flask. 2 ml of chloroform was added to the flask, and 10 μl of DiI (930 μg / ml) was added in the dark. The mixture was gently shaken until fully dissolved. The flask was then connected to a rotary evaporator at 120 rpm. The flask was then placed in a water bath at 55 °C and rotary evaporated for 30 min until a light pink, uniformly thick, dry lipid membrane formed on the flask wall. With 10 min remaining in the rotary evaporation, the hydration solution was preheated in a 37 °C constant temperature bed. The hydration solution consisted of 900 μl PBS + 100 μl glycerol + 20 μl Pluronic F-68. Remove the rotary evaporator flask, add 2 ml of preheated hydration solution, and shake thoroughly (place the rotary evaporator flask on a constant temperature shaker at 37°C, 130 rpm, for 1 hour) to allow the lipid membrane to detach from the flask wall.

[0066] Synthesis of CD93-targeted microbubbles: The extracted cell membranes and liposome membranes were mixed using an ice bath sonication method (100W power, 42kHz frequency, 2min). The mixture was placed in a small vial with a rubber cap, and the air in the vial was exchanged with perfluoropropane (C3F8). The solution was then placed in a mechanical vibrator and shaken twice for 90s to obtain a targeted microbubble suspension.

[0067] Example 4

[0068] CD93-targeted microbubble characteristics identification

[0069] To observe the morphology of microbubbles (non-targeted microbubbles were prepared using DPPC and DSPE-PEG2000 via a thin-film hydration method without cell membranes; the synthesis process was identical to that of targeted microbubbles, resulting in a microbubble stock solution) and targeted microbubbles, 200 μL of the microbubble stock solution was diluted in 2 mL of 1×PBS and thoroughly mixed by pipetting. One drop each of the microbubble diluent, targeted microbubble diluent (obtained by diluting the microbubble suspension from Example 3 10-fold with PBS), and SonoVue microbubble diluent (SonoVue contrast agent (Bracco, Italy)) were placed on clean lead foil, dried in a desiccator, and sputtered with gold for 5 min after complete drying. The morphology was observed using a scanning electron microscope (HITACHI S-4800, Japan). Figure 4 As shown, the CD93-targeting microbubbles are approximately spherical in shape and disperse well in solution, similar to commercially available SonoVe microbubbles. This invention successfully synthesizes contrast microbubbles.

[0070] Take 1 mL of each of the following microbubble diluents: targeted microbubble diluent, and SonoVe microbubble diluent. Place them in the sample cell of a Beckman Coulter particle size analyzer. Detect the particle size distribution of each group of microbubbles at 25℃, an excitation wavelength of 660 nm, and an excitation angle of 90°. The results are as follows: Figure 5 As shown, the average particle sizes of CD93-targeted microbubbles and microbubbles are 960.5±143nm and 834.3±109nm, respectively, which are smaller than the particle size of SonoVue (1256.3±212nm).

[0071] To assess the fusion of cell membrane and liposome membrane, a zeta potential analyzer was used to analyze the zeta potential distribution of microvesicles, HEK293T cell membranes transfected with recombinant genes, and targeted microvesicles. Figure 6 As shown, the average potential of the targeted microbubbles is -28.07mV, which is between the cell membrane potential of -32.06mV and the potential of the simple liposome microbubbles of -22.65mV, indicating that the targeted microbubbles integrate cell membrane and liposome membrane components.

[0072] The fusion of cell membrane and liposomes was observed using a fluorescence microscope (OLYMPUS IX71, Japan). The synthesized liposome membrane was labeled with DiI (3 μM) using a mixture of DPPC and DSPE-PEG2000. The 293T cell membrane solution extracted after transfection with MMRN2 plasmid was labeled with DiO (3 μM). Microvesicles were synthesized following the above steps in the dark. Figure 7 As shown, DiI-labeled liposome membranes are red, DiO-labeled cell membranes are green, and the fusion of the two presents a yellow color.

[0073] Example 5

[0074] CD93 targeting microbubble target acquisition ability identification

[0075] To induce CD93 expression in human umbilical vein endothelial cells (HUVECs), cells were seeded in confocal culture dishes and 500 ng / mL lipopolysaccharide (LPS, 916374, Sigma-Aldrich, USA) was added. The cells were then cultured for 8 hours. The culture medium was then changed, and either DiI-labeled microbubbles (control group, non-targeted microbubbles, i.e., microbubbles prepared solely with DPPC and DSPE-PEG2000 without cell membrane addition) or targeted microbubbles (5.0 × 10⁻⁶) were added. 6 Incubate for 40 min with 4% paraformaldehyde for 10 min. Detect CD93 expression in HUVEC cells using primary antibody (AF2379, R&D Systems) and secondary antibody (SA00003-3, Proteintech). Stain cell nuclei with Hoechst 33342 (1:1000, Beyotime Biotechnology) for 10 min. Protect from light throughout the process. Observe using a laser confocal microscope. Figure 8 As shown, compared with the control group, the targeted microvesicles have a higher affinity for CD93 molecules in endothelial cells.

[0076] Example 6

[0077] To determine the ratio of cell membrane to phospholipids during CD93-targeted microbubble construction and the ultrasound contrast imaging capability of the microbubbles, microbubbles were prepared by mixing phospholipids and cell membranes in different proportions, specifically as follows: 10 mg DPPC + 4 mg DSPE-PEG2000 were used to prepare liposome membranes using the aforementioned thin-film hydration method. Cell membranes with protein quantifications of 0 mg, 0.15 mg, 0.30 mg, 0.60 mg, 1.20 mg, and 2.40 mg were mixed with the above liposome membranes to prepare microbubbles. The microbubble suspensions were added to the wells of an agarose gel, and ultrasound contrast imaging was performed from the side of the agarose gel using a Vevo 2100 imaging system. The imaging parameters were as follows: frequency 18 MHz; contrast gain 40 dB; depth 11.56 mm; dynamic range 35 dB. Image retention times were 0 min, 5 min, 10 min, 15 min, and 30 min.

[0078] like Figure 9As shown, when microbubbles were prepared using 10 mg DPPC, 2000 4 mg DSPE-PEG, and 0.6 mg of cell membrane protein, the signal intensity decreased significantly at 15 minutes of imaging. This indicates that when the phospholipid content is fixed, the more cell membrane is added, the worse the stability of the microbubbles. In order to balance the stability and targeting of the microbubbles, it was finally determined that 0.3 mg of cell membrane protein was added to the synthetic phospholipids (10 mg DPPC, 2000 4 mg DSPE-PEG).

[0079] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing ultrasound contrast microbubbles targeting CD93 molecules of tumor vascular endothelium, characterized in that, The method comprises the following steps: Constructing a recombinant expression vector capable of expressing MMRN2 protein, and transfecting the recombinant expression vector into HEK293T cells to obtain HEK293T cells expressing MMRN2 protein; Extracting cell membranes of the HEK293T cells expressing MMRN2 protein by using a differential centrifugation method to obtain HEK293T cell membranes expressing MMRN2 protein; Mixing dipalmitoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and chloroform, adding DiI, and preparing a liposome film by using a thin film hydration method; Mixing the HEK293T cell membranes expressing MMRN2 protein and the liposome film, performing ice bath ultrasonication to obtain a mixed solution, mixing the mixed solution with perfluoropropane in a sealed device, and performing mechanical oscillation to obtain tumor vascular endothelial CD93 molecule ultrasound contrast microbubbles; and the nucleotide sequence of the recombinant gene capable of expressing MMRN2 protein is shown in SEQ ID NO.

1.

2. The production method according to claim 1, characterized by, The method for constructing the recombinant expression vector capable of expressing MMRN2 protein comprises the following steps: Constructing a recombinant gene capable of expressing MMRN2 protein into a basic plasmid to obtain a recombinant expression vector expressing MMRN2 protein; the recombinant gene comprises, from 3' end to 5' end, a restriction site Hind III, a Kozak fragment, a signal peptide, an MMRN2 fragment, a PTGFRN fragment, a FLAG tag and a restriction site Kpn I; and the basic plasmid comprises pcDNA3.1(+).

3. The preparation method according to claim 1, characterized in that, The method for extracting cell membranes comprises the following steps: trypsinizing the HEK293T cells expressing MMRN2 protein 24 hours after transfection, centrifuging, resuspending the precipitate with a hypotonic lysis buffer, lysing, homogenizing and centrifuging, removing cell nuclei and unbroken cells, and then centrifuging the supernatant, separating mitochondria, and performing ultracentrifugation on the obtained supernatant to obtain HEK293T cell membranes expressing MMRN2 protein.

4. The method of claim 1, wherein, The mass of the dipalmitoyl phosphatidylcholine, the mass of the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, the volume of the chloroform and the volume of the DiI are (5-15) mg:(3-5) mg:(1-3) mL:(8-12) µL; and the concentration of the DiI is 930 µg / mL.

5. The preparation method according to claim 1, characterized in that, The method for preparing the liposome film by using a thin film hydration method comprises the following steps: Mixing dipalmitoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and chloroform, adding DiI under light shielding conditions, performing rotary evaporation after dissolving, adding a hydration solution into the dry lipid film, and oscillating to obtain a liposome film.

6. The method of claim 1, wherein, The added amount of the HEK293T cell membrane expressing the MMRN2 protein is based on the added amount of dipalmitoyl phosphatidylcholine and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, and the mass ratio of dipalmitoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and the HEK293T cell membrane expressing the MMRN2 protein calculated according to the mass of protein quantification is (5-15):(3-5):(0.25-0.35).

7. The preparation method according to claim 1, characterized in that, The power of the ultrasound is 100 W, the frequency of the ultrasound is 40-45 kHz, and the time of the ultrasound is 2-4 min.

8. The ultrasound contrast microbubble targeting the CD93 molecule of tumor vascular endothelium prepared by the preparation method of any one of claims 1-7.

9. The use of the ultrasound contrast microbubble targeting the CD93 molecule of tumor vascular endothelium prepared by the preparation method of any one of claims 1-7 or the ultrasound contrast microbubble of claim 8 in the preparation of a product for evaluating the immune microenvironment of a tumor; the evaluation of the immune microenvironment of the tumor comprises monitoring the immune-related hemodynamic changes or reflecting the immune infiltration of the tumor through a targeted signal.

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