Plasmid-carrying cationic lipid microbubbles and method for preparing the same

Abstract

The application relates to the technical field of lipid microbubble, and is a plasmid-carrying cationic lipid microbubble and a preparation method thereof. The plasmid-carrying cationic lipid microbubble is composed of a lipid shell and a gas core, and is obtained by the following method: after di-stearoyl phosphatidylcholine, di-stearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, dipalmitoyl phosphatidylcholine and DC cholesterol are mixed according to a required proportion and dissolved in chloroform, the formed lipid membrane is subjected to vacuum extraction, hydration, vacuum extraction again, gas replacement and mechanical oscillation to obtain the plasmid-carrying cationic lipid microbubble. The prepared cationic lipid microbubble has high electric potential and small particle size, has high plasmid carrying capacity and carrying rate, has the advantages of low price, simple operation, storage convenience and the like, and provides a reference for the preparation of other gene-carrying microbubbles.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lipid microbubble, and is a cationic lipid microbubble carrying plasmid and a preparation method thereof. BACKGROUND

[0002] Microbubbles emit harmonic waves under high acoustic energy ultrasound, and compared with normal tissues, microbubbles are more taken up by diseased tissues, and the blood flow signal and signal-to-noise ratio are significantly increased, and the gray scale is enhanced. Therefore, ultrasound can realize real-time monitoring of the circulation of microbubbles in the body. In addition to being used as a contrast agent, microbubbles are considered as a therapeutic drug in the field of drug delivery. Ultrasound-induced cavitation makes the gas core of the microbubble expand and compress, at the same time, changes the blood flow mode in the blood vessel and increases the permeability of the endothelial cells in the blood vessel intima, thereby allowing the drug to extravasate and improving the uptake of the drug into the cells by enhancing the membrane permeability of the nearby cells. In addition to drug delivery, microbubbles can also deliver genes or plasmids carrying target genes to realize gene-level treatment.

[0003] Microbubbles are generally composed of a shell membrane made of protein, lipid or polymer and a gas core wrapped in the shell membrane, and are used as a carrier to carry drugs or genes, and have been widely used in the fields of anti-tumor treatment, thrombolysis, drug and gene therapy, etc. As a carrier, the ability of microbubbles to carry genes is the most basic prerequisite for improving gene transfection efficiency, and the charge amount of lipid microbubbles is one of the main factors affecting the saturation and carrying capacity of the carrier. At present, the charge amount of lipid microbubbles and the carrying capacity of genes through electrostatic adsorption are both low, which limits their application. SUMMARY

[0004] The present application provides a cationic lipid microbubble carrying plasmid and a preparation method thereof, which overcomes the shortcomings of the prior art and effectively solves the problem of low plasmid carrying rate of existing microbubbles.

[0005] One of the technical solutions of the present application is achieved by the following measures: a cationic lipid microbubble carrying plasmid, the basic structure of the cationic lipid microbubble includes a lipid shell and a gas core, the composition of the lipid shell includes distearoylphosphatidylcholine (DSPC), distearoylphosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoylphosphatidylcholine (DPPC) and DC cholesterol (DC-CHOL), the gas core is a perfluoropropane (C3F8) core, and the outer surface of the lipid shell is connected with plasmid by electrostatic adsorption method.

[0006] The following is a further optimization or / and improvement of the above-mentioned one of the technical solutions:

[0007] The mass ratio of the above-mentioned distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoyl phosphatidyl choline (DPPC) and DC cholesterol (DC-CHOL) is (2.5 to 3.5):(0.5 to 1.5):(2.5 to 3.5):(5.5 to 6.5).

[0008] The cationic lipid microbubble with plasmid is obtained by the following method:

[0009] S1, distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoyl phosphatidyl choline (DPPC) and DC cholesterol (DC-CHOL) are mixed in the required ratio, and then dissolved in chloroform to form a mother liquor;

[0010] S2, the mother liquor is preheated in a test tube, and then the preheated mother liquor is oscillated while being filled with nitrogen, and a white lipid film is formed on the wall of the test tube;

[0011] S3, after the lipid film is vacuumed, the prepared mixed solution is added, and after ultrasonic oscillation, the lipid film is completely dissolved to obtain a hydration solution;

[0012] S4, after the hydration solution is vacuumed, perfluoropropane gas is filled, and then mechanical oscillation is carried out to obtain a microbubble suspension, and then the microbubble suspension is centrifuged, the upper layer is discarded, and the cationic lipid microbubble is obtained;

[0013] S5, the cationic lipid microbubble and the plasmid are mixed in the required ratio, and then incubated at room temperature to obtain the cationic lipid microbubble with plasmid.

[0014] In the above step S2, the preheating temperature of the mother liquor is 60-70℃, and the preheating time is 2-3s.

[0015] In the above step S3, the vacuum time of the lipid film is 3.0-3.5h, and the mixed solution is prepared by mixing Tris buffer, glycerol and 1,2-propanediol in a volume ratio of (7-9):1:1.

[0016] In the above step S4, the vacuum time of the hydration solution is 1.0-1.5h, the perfluoropropane gas is filled for 3-4s, and this step is repeated 3-4 times.

[0017] In the above step S5, the volume ratio of the cationic lipid microbubble to the plasmid is 5:2, and the incubation time is 30-35min.

[0018] The above-mentioned plasmid is pEGFP-N1 plasmid.

[0019] The second technical solution of the present application is realized by the following measures: a preparation method of cationic lipid microbubbles carrying plasmids, which is performed according to the following method:

[0020] S1, distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoylphosphatidylcholine (DPPC) and DC cholesterol (DC-CHOL) are mixed in the required proportion, and after being dissolved in chloroform, a mother liquor is formed;

[0021] S2, the mother liquor is placed in a test tube for preheating, and then the preheated mother liquor is oscillated while being filled with nitrogen, and a white lipid film is formed on the wall of the test tube;

[0022] S3, after the lipid film is vacuumed, the prepared mixed solution is added, and after ultrasonic oscillation, the lipid film is completely dissolved, and a hydration solution is obtained;

[0023] S4, after the hydration solution is vacuumed, perfluoropropane gas is filled, and then mechanical oscillation is performed, and a microbubble suspension is obtained, and then the microbubble suspension is centrifuged, the upper layer is discarded, and cationic lipid microbubbles are obtained;

[0024] S5, the cationic lipid microbubbles and the plasmid are mixed in the required proportion, and after incubation at room temperature, cationic lipid microbubbles carrying plasmids are obtained.

[0025] The cationic lipid microbubbles prepared by the present application have high electric potential and small particle size, have high plasmid carrying capacity and carrying rate, have the advantages of low price, simple operation, and easy storage, and provide a reference for the preparation of other gene-loaded microbubbles. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 FIG. 1 is a structural schematic diagram of cationic lipid microbubbles carrying plasmids obtained by the present application;

[0027] Figure 2 FIG. 3 is a morphology and structure diagram of the cationic lipid microbubbles obtained by the present application;

[0028] Figure 3 FIG. 5 is a combination of the cationic lipid microbubbles obtained by the present application and the pEGFP-N1 plasmid;

[0029] Figure 4 FIG. 7 is an agarose gel electrophoresis spectrum of the cationic lipid microbubbles obtained by the present application and the pEGFP-N1 plasmid carrying capacity;

[0030] Figure 5 FIG. 9 is a flow cytometry spectrum of the cationic lipid microbubbles obtained by the present application and different amounts of pEGFP-N1 plasmid combination. DETAILED DESCRIPTION

[0031] The present application is not limited by the following examples, and the specific embodiments can be determined according to the technical solutions of the present application and the actual situation. The various chemical reagents and chemical products mentioned in the present application are common chemical reagents and chemical products known in the prior art unless otherwise specified; the percentages in the present application are mass percentages unless otherwise specified; the solutions in the present application are aqueous solutions with water as the solvent unless otherwise specified, for example, a hydrochloric acid solution is an aqueous hydrochloric acid solution; the normal temperature and room temperature in the present application generally refer to a temperature of 15-25°C, and are generally defined as 25°C.

[0032] The present application will be further described below in conjunction with examples:

[0033] Example 1: The plasmid-carrying cationic lipid microbubble, the basic structure of the cationic lipid microbubble includes a lipid shell and a gas core, the composition of the lipid shell includes distearoylphosphatidylcholine (DSPC), distearoylphosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoylphosphatidylcholine (DPPC) and DC cholesterol (DC-CHOL), the gas core is a perfluoropropane (C3F8) core, and the outer surface of the lipid shell is connected with the plasmid by electrostatic adsorption method.

[0034] Example 2: As an optimization of the above-mentioned example, the mass ratio of distearoylphosphatidylcholine (DSPC), distearoylphosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoylphosphatidylcholine (DPPC) and DC cholesterol (DC-CHOL) is (2.5-3.5):(0.5-1.5):(2.5-3.5):(5.5-6.5).

[0035] Example 3: As an optimization of the above-mentioned example, the plasmid-carrying cationic lipid microbubble is obtained by the following method:

[0036] S1, distearoylphosphatidylcholine (DSPC), distearoylphosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoylphosphatidylcholine (DPPC) and DC cholesterol (DC-CHOL) are mixed in the required proportion, and chloroform is added to dissolve to form a mother liquor;

[0037] S2, the mother liquor is preheated in a test tube, and the preheated mother liquor is oscillated while being filled with nitrogen, and a white lipid film is formed on the wall of the test tube;

[0038] S3, after the lipid film is vacuumed, the prepared mixed solution is added, and after ultrasonic oscillation, the lipid film is completely dissolved to obtain a hydration solution;

[0039] S4, after the hydration solution is vacuumed, the perfluoropropane gas is filled, and then the mechanical oscillation is carried out, so as to obtain the microbubble suspension, and then the microbubble suspension is centrifuged, the upper layer is discarded, and the cationic lipid microbubble is obtained;

[0040] S5, the cationic lipid microbubble and the plasmid are mixed according to the required proportion, and then incubated at room temperature, so as to obtain the plasmid-carrying cationic lipid microbubble.

[0041] In the step S2 of the embodiment 4, the preheating temperature of the mother solution is 60-70 DEG C, and the preheating time is 2-3 s.

[0042] In the step S3 of the embodiment 5, the vacuum time of the lipid membrane is 3.0-3.5 h, and the mixed solution is mixed by Tris buffer, glycerol and 1,2-propanediol with the volume ratio of (7-9):1:1.

[0043] In the step S4 of the embodiment 6, the vacuum time of the hydration solution is 1.0-1.5 h, and the perfluoropropane gas is filled for 3-4 s, and the step is repeated for 3-4 times.

[0044] In the step S5 of the embodiment 7, the volume ratio of the cationic lipid microbubble and the plasmid is 5:2, and the incubation time is 30-35 min.

[0045] In the embodiment 8, the plasmid is pEGFP-N1 plasmid.

[0046] Compared with the prior art, the embodiment has the beneficial effects that:

[0047] Firstly, the cationic lipid microbubble can be coupled with the negative DNA through electrostatic coupling, so as to protect the DNA from being degraded by the nuclease in advance, and increase the genetic load near the target cell.

[0048] Secondly, the plasmid-carrying cationic lipid microbubble prepared by the DSPC, DSPE-PEG2000, DPPC and DC-Chol has high electric potential, small particle size, high plasmid carrying capacity and carrying rate, and good repeatability of the microbubble preparation technology.

[0049] Embodiment 9:

[0050] The plasmid-carrying cationic lipid microbubble is obtained by the following method:

[0051] S1, mixing distearoylphosphatidylcholine (DSPC), distearoylphosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoylphosphatidylcholine (DPPC) and DC cholesterol (DC-CHOL) in a mass ratio of 2.5:0.5:2.5:5.5, dissolving in chloroform to form a mother liquor;

[0052] S2, preheating the mother liquor in a test tube at 60℃ for 2s, oscillating the preheated mother liquor while filling with nitrogen, and forming a white lipid film on the test tube wall;

[0053] S3, after vacuumizing the lipid film for 3.0h, adding the prepared mixed solution, ultrasonic oscillation to completely dissolve the lipid film, and obtaining a hydration solution; wherein the mixed solution is prepared by mixing Tris buffer, glycerol and 1,2-propanediol in a volume ratio of 7:1:1;

[0054] S4, after vacuumizing the hydration solution for 1.0h, filling with perfluoropropane gas for 3s, repeating the step for 3 times, then, oscillating the hydration solution filled with perfluoropropane gas to obtain a microbubble suspension, centrifuging the microbubble suspension, and discarding the upper layer to obtain cationic lipid microbubbles;

[0055] S5, mixing the cationic lipid microbubbles and pEGFP-N1 plasmid in a volume ratio of 5:2, incubating at room temperature for 30min to obtain cationic lipid microbubbles carrying plasmid.

[0056] Example 10:

[0057] The cationic lipid microbubbles carrying plasmid are obtained by the following method:

[0058] S1, mixing distearoylphosphatidylcholine (DSPC), distearoylphosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoylphosphatidylcholine (DPPC) and DC cholesterol (DC-CHOL) in a mass ratio of 3.5:1.5:3.5:6.5, dissolving in chloroform to form a mother liquor;

[0059] S2, preheating the mother liquor in a test tube at 70℃ for 3s, oscillating the preheated mother liquor while filling with nitrogen, and forming a white lipid film on the test tube wall;

[0060] S3, after vacuumizing the lipid film for 3.5h, adding the prepared mixed solution, ultrasonic oscillation to completely dissolve the lipid film, and obtaining a hydration solution; wherein the mixed solution is prepared by mixing Tris buffer, glycerol and 1,2-propanediol in a volume ratio of 9:1:1;

[0061] S4, after the hydration solution is vacuumed for 1.5h, the perfluoropropane gas is filled for 4s, the step is repeated for 4 times, then, the hydration solution filled with the perfluoropropane gas is mechanically oscillated to obtain a microbubble suspension, and the microbubble suspension is centrifuged to discard the upper layer to obtain the cationic lipid microbubble;

[0062] S5, after the cationic lipid microbubble and the pEGFP-N1 plasmid are mixed in a volume ratio of 5:2, the plasmid-carrying cationic lipid microbubble is obtained after being incubated at room temperature for 35min.

[0063] Example 11:

[0064] The plasmid-carrying cationic lipid microbubble is obtained by the following method:

[0065] S1, the distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dipalmitoylphosphatidylcholine (DPPC) and DC cholesterol (DC-CHOL) are mixed in a mass ratio of 3:1:3:6, and chloroform is added to form a mother liquor;

[0066] S2, the mother liquor is preheated in a test tube at 65℃ for 2s, and the preheated mother liquor is oscillated while being filled with nitrogen to form a white lipid film on the test tube wall;

[0067] S3, after the lipid film is vacuumed for 3.0h, the prepared mixed solution is added, and the lipid film is completely dissolved after ultrasonic oscillation to obtain a hydration solution; wherein the mixed solution is mixed by Tris buffer, glycerol and 1,2-propanediol in a volume ratio of 8:1:1;

[0068] S4, after the hydration solution is vacuumed for 1.0h, the perfluoropropane gas is filled for 3s, the step is repeated for 3 times, then, the hydration solution filled with the perfluoropropane gas is mechanically oscillated to obtain a microbubble suspension, and the microbubble suspension is centrifuged to discard the upper layer to obtain the cationic lipid microbubble;

[0069] S5, after the cationic lipid microbubble and the pEGFP-N1 plasmid are mixed in a volume ratio of 5:2, the plasmid-carrying cationic lipid microbubble is obtained after being incubated at room temperature for 30min.

[0070] The plasmid-carrying cationic lipid microbubble obtained by the example 10 of the application is shown in the schematic structural diagram of Figure 1 .

[0071] Comparative example 1:

[0072] The cationic lipid microbubble differs from Example 10 of the present application in that step S1 is modified as "mixing distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and DC cholesterol (DC-CHOL) in a mass ratio of 3:1:6, dissolving in chloroform to form a mother liquor", and the remaining steps are the same.

[0073] Comparative Example 2:

[0074] The cationic lipid microbubble differs from Example 10 of the present application in that step S1 is modified as "mixing distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and DC cholesterol (DC-CHOL) in a mass ratio of 3:1:6, dissolving in chloroform to form a mother liquor", and the remaining steps are the same.

[0075] The following is an investigation of the morphology, physicochemical properties, combination of the cationic lipid microbubble obtained by the present application with pEGFP-N1 plasmid, optimal loading capacity, optimal loading capacity and loading rate.

[0076] Test 1: Morphology of cationic lipid microbubble

[0077] The morphology of the cationic lipid microbubble (hereinafter referred to as CMBs) obtained by Example 10 of the present application was investigated by optical microscope and scanning electron microscope. The test results are shown in Figure 2 , wherein A is the photo of the hydrated liquid before mechanical oscillation, B is the photo of CMBs after mechanical oscillation, C is the microscope image of the CMBs of the present application, and D is the scanning electron microscope image of the CMBs of the present application, as known from Figure 2 , the CMBs of the present application are spherical and regular in shape.

[0078] Test 2: Combination of cationic lipid microbubble with pEGFP-N1 plasmid

[0079] The combination of the CMBs obtained by Example 10 of the present application with pEGFP-N1 plasmid was investigated by fluorescence microscope. The test results of the combination are shown in Figure 3 , wherein A is the fluorescence microscope image of the plasmid-loaded cationic lipid microbubble (hereinafter referred to as ECMBs) under bright field conditions, B is the fluorescence microscope image of the Dil-labeled CMBs under excitation spectrum 549 nm, C is the fluorescence microscope image of the DAPI-labeled pEGFP-N1 plasmid under excitation spectrum 364 nm, and D, E are the scanning electron microscope images of the ECMBs, as known from Figure 3It can be seen that, at the same location under bright-field microscope conditions, the CMBs of this invention not only express red fluorescence under a 549 nm excitation spectrum, but also blue fluorescence under a 488 nm excitation spectrum, indicating that the pEGFP-N1 plasmid has been successfully bound to the surface of CMBs.

[0080] Experiment 3: Physicochemical properties of cationic lipid microvesicles

[0081] The physicochemical properties of the CMBs obtained in Example 10 of this invention were tested, including average particle size, potential, and concentration. The experimental results are shown in Table 1. Table 1 shows that the surface potential of the CMBs was 44.5 ± 1.8 mV, the particle size was 459.6 ± 27.1 nm, and the concentration was 3.30 ± 0.52 × 10⁻⁶. 7 / mL; the surface potential of ECMBs saturated with pEGFP-N1 plasmid was -9.0±1.7mV, the particle size was 808.6±76.2nm, and the concentration was 3.54±0.24×10⁻⁶ / mL. 7 The changes in potential and particle size of CMBs per mL indirectly reflect that CMBs have been successfully loaded with the pEGFP-N1 plasmid.

[0082] Experiment 4: Saturation loading capacity of cationic lipid microvesicles and pEGFP-N1 plasmid

[0083] 24.4 µL of the CMBs obtained in Example 10 of this invention was pipetted and diluted with 13.1 µL of distilled water. The CMBs were then serially diluted at twice the concentration obtained at this time. Next, 3.5 µL of 45 ng / µL pEGFP-N1 plasmid was incubated with 7.5 µL of CMBs at different concentrations at room temperature for 30 min. After gel electrophoresis, the plasmid loading of the CMBs was observed using a gel imaging system. The experimental results are as follows: Figure 4 As shown, by Figure 4 It can be seen that when the number of CMBs is 0.067 × 10 4 At that time, 3.5 µL of pEGFP-N1 plasmid (45 ng / µL) could be saturated with the plasmid, and the saturated loading amount of CMBs was 31.30 µg / 10 6 CMBs.

[0084] Experiment 5: Investigating the physicochemical properties of different cationic lipid microvesicles and their saturation loading capacity with pEGFP-N1 plasmid.

[0085] The physicochemical properties of the CMBs obtained in Example 10 of this invention were tested, and the CMBs obtained in Comparative Example 1 and Comparative Example 2 were compared. The experimental results are shown in Table 2. As can be seen from Table 2, the CMBs obtained using Example 10 of this invention have high potential and small particle size, and have a high plasmid loading capacity.

[0086] Experiment 6: Saturation loading rate of cationic lipid microvesicles and pEGFP-N1 plasmid

[0087] Take 0 µL, 2.5 µL, 5 µL, 10 µL, 20 µL, and 40 µL of pEGFP-N1 plasmid (1.5 µg / µL) obtained in Example 10 of this invention, respectively. Add 150 µL of DAPI (10 µg / mL) to each pEGFP-N1 plasmid and incubate on ice for 10 min. Then add 50 µL of the CMBs obtained in Example 10 of this invention and unequal volumes of distilled water to prepare a mixture with a total volume of 500 µL. Incubate at room temperature for 30 min. Before flow cytometry, mix the mixture in the flow cytometer tube thoroughly. The results of the flow cytometry analysis are as follows: Figure 5 As shown, by Figure 5 It can be seen that when 50µL of the CMBs obtained in Example 10 of this invention are combined with 0µL, 2.5µL, 5µL, 10µL, 20µL, and 40µL of pEGFP-N1 plasmid (1.5µg / µL), the binding rates of the CMBs in each group are 4.5%, 51.33%, 65.37%, 97.57%, 99.9%, and 99.9%, respectively. When the amount of pEGFP-N1 plasmid is 20µL, the binding rate reaches saturation, and the highest plasmid loading rate of CMBs is 99.9%.

[0088] In summary, the cationic lipid microbubbles prepared by this invention have high potential and small particle size, high plasmid loading capacity and loading rate, and advantages such as low price, simple operation, and easy storage, providing a reference for the preparation of other gene-carrying microbubbles.

[0089] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A plasmid-carrying cationic lipid microbubble, characterized by The basic structure of the cationic lipid microbubble comprises a lipid shell and a gas core, the lipid shell is composed of distearoylphosphatidylcholine, distearoylphosphatidyl ethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylcholine and DC cholesterol with a mass ratio of 2.5-3.5:0.5-1.5:2.5-3.5:5.5-6.5, and the gas core is perfluoropropane, and the outer surface of the lipid shell is connected with a plasmid by electrostatic adsorption.

2. The plasmid-carrying cationic liposome microbubble according to claim 1, characterized by The following method is used: S1, distearoylphosphatidylcholine, distearoylphosphatidyl ethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylcholine and DC cholesterol are mixed in the required proportion, and then dissolved in chloroform to form a mother liquor; S2, the mother liquor is placed in a test tube for preheating, and then the preheated mother liquor is oscillated while being filled with nitrogen, and a white lipid film is formed on the wall of the test tube; S3, after the lipid film is vacuumed, the prepared mixed solution is added, and after ultrasonic oscillation, the lipid film is completely dissolved to obtain a hydration solution; S4, after the hydration solution is vacuumed, perfluoropropane gas is filled, and then mechanical oscillation is carried out to obtain a microbubble suspension, and then the microbubble suspension is centrifuged, the upper layer is discarded, and a cationic lipid microbubble is obtained; S5, the cationic lipid microbubble and the plasmid are mixed in the required proportion, and then incubated at room temperature to obtain a plasmid-carrying cationic lipid microbubble.

3. The plasmid-carrying cationic liposome microbubble according to claim 2, characterized by In step S2, the preheating temperature of the mother liquor is 60-70℃, and the preheating time is 2-3s.

4. The plasmid-carrying cationic liposome microbubble according to claim 2 or 3, characterized by In step S3, the lipid film is vacuumed for 3.0-3.5h, and the mixed solution is prepared by mixing Tris buffer, glycerol and 1,2-propanediol in a volume ratio of 7-9:1:

1.

5. The plasmid-carrying cationic liposome microbubble according to claim 2 or 3, characterized by In step S4, the hydration solution is vacuumed for 1.0-1.5h, and perfluoropropane gas is filled for 3-4s, and this step is repeated 3-4 times.

6. The plasmid-carrying cationic liposome microbubble according to claim 4, characterized by In step S4, the hydration solution is vacuumed for 1.0-1.5h, and perfluoropropane gas is filled for 3-4s, and this step is repeated 3-4 times.

7. The plasmid-carrying cationic liposome microbubble according to claim 2 or 3 or 6, characterized by In step S5, the volume ratio of cationic lipid microbubble to plasmid is 5:2, and the incubation time is 30-35min.

8. The plasmid-carrying cationic liposome microbubble according to claim 4, characterized by In step S5, the volume ratio of cationic lipid microbubble to plasmid is 5:2, and the incubation time is 30-35min.

9. The plasmid-carrying cationic liposome microbubble according to claim 5, characterized by In step S5, the volume ratio of cationic lipid microbubble to plasmid is 5:2, and the incubation time is 30-35min.

10. The plasmid-carrying cationic liposome microbubble according to claim 1 or 2 or 3 or 6 or 8 or 9, characterized by The plasmid is pEGFP-N1 plasmid.

11. A method for producing the cationic liposome microbubble carrying the plasmid according to any one of claims 1, 3 to 10, characterized by The following method is used: S1, distearoylphosphatidylcholine, distearoylphosphatidyl ethanolamine-polyethylene glycol 2000, dipalmitoylphosphatidylcholine and DC cholesterol are mixed in the required proportion, and then dissolved in chloroform to form a mother liquor; S2, the mother liquor is placed in a test tube for preheating, and then the preheated mother liquor is oscillated while being filled with nitrogen, and a white lipid film is formed on the wall of the test tube; S3, after the lipid film is vacuumed, the prepared mixed solution is added, and after ultrasonic oscillation, the lipid film is completely dissolved to obtain a hydration solution; S4, after the hydration solution is vacuumed, full fluorine propane gas is filled, mechanical oscillation is carried out, microbubble suspension is obtained, the microbubble suspension is centrifuged, the upper layer is discarded, and cationic lipid microbubbles are obtained; S5, the cationic lipid microbubbles and the plasmid are mixed according to a required proportion, incubated at room temperature, and cationic lipid microbubbles carrying plasmids are obtained.

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