Modification method of lipid vesicles, modified lipid vesicles and application of modified lipid vesicles

Abstract

The invention belongs to the technical field of biology, and particularly relates to a modification method of lipid vesicles, modified lipid vesicles and application of the modified lipid vesicles. The modification method of the lipid vesicles comprises the following steps: S1, mixing a lipid vesicle solution and a liquid organic modifier at 0-60 DEG C to obtain a mixture; s2, fully and uniformly mixing the mixture to enable the modifier and the lipid vesicles to fully act, and standing to obtain a water phase, namely the modified lipid vesicles. According to the method, the delivery efficiency of the lipid vesicles can be improved while the structure and the function of the original lipid vesicles are not influenced. The modified lipid vesicle prepared by the method disclosed by the invention can be used as an ultrasonic synergistic material and has ultrasonic synergistic (radiography and treatment) effects. The modified lipid vesicles prepared by the method disclosed by the invention have stronger permeability, active ingredients carried by the lipid vesicles are favorably delivered to the deep part of a tumor, and the treatment effect is enhanced.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for modifying lipid vesicles, modified lipid vesicles and applications thereof. Background Art

[0002] Ultrasound imaging is based on the principle of sound wave propagation in physics. When sound waves propagate inside the human body, they encounter tissues of different densities, which reflect and refract the sound waves in different ways. By receiving these reflected and refracted sound waves, images of the human body can be generated. These images can provide information about the structure, function and pathological conditions of the human body. Ultrasound is anisotropic and can penetrate opaque substances. It has been widely used in the diagnosis and treatment of various diseases such as cancer and cardiovascular disease.

[0003] However, the resolution of ordinary ultrasound is low, which leads to certain limitations in its clinical application. In order to obtain better imaging effects of ultrasound imaging, ultrasound microbubble contrast agents are usually used, such as SonoVue. However, the size of the microbubbles of ultrasound microbubble contrast agents is micron-sized, with a diameter usually between 1-10μm, which makes it difficult to enter deep into the tumor blood vessels and is not targeted.

[0004] To solve the above problems, there have been many studies on nano-sized contrast agents in recent years, such as liquid halothane nanoparticles. Due to the high permeability and long retention effect, nanobubbles may penetrate the gaps in the vascular walls of tumors and achieve imaging of extravascular tissues through the liquid-gas phase change of liquid halothane. However, its targeting still needs to be solved.

[0005] Lipid vesicles (especially extracellular vesicles including exosomes) have become a good choice of nanomaterials for imaging enhancement due to their good tissue compatibility, easy access to natural sources, and natural targeting. However, the material properties and nanoscale size of extracellular vesicles make their ultrasound effect weak, and the efficiency of loading substances such as gas or liquid fluorocarbon is not high, and it will affect the original lipid vesicle structure and content composition, limiting its application in the field of ultrasound enhancement. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for modifying lipid vesicles, modified lipid vesicles and applications thereof, so as to improve the efficiency of loading / carrying the modifier without changing the original lipid vesicle structure and content components.

[0007] To achieve the above objectives, the scheme of this application is as follows:

[0008] In a first aspect, the present invention provides a method for modifying a lipid vesicle, comprising the following steps:

[0009] S1. mixing the lipid vesicle solution and the liquid organic modifier at a temperature in the range of 0-60° C. to obtain a mixture;

[0010] S2. mixing the mixture thoroughly to allow the modifier to fully interact with the lipid vesicles;

[0011] S3. Let stand (maybe no separation occurs, if separation occurs, discard the organic phase), the resulting aqueous phase contains the modified lipid vesicles.

[0012] Optionally, in step S1, the liquid organic modifier is selected from alcohol substances, ester substances or combinations thereof having carbon atoms greater than or equal to 4 and less than or equal to 8.

[0013] It should be noted that, in the present application, alcohol substances include, for example, butanol (including isomers), pentanol (including isomers), hexanol (including isomers), heptanol (including isomers), octanol (including isomers), cyclobutanol, cyclopentanol, cyclohexanol, cycloheptanol, cyclooctanol, etc. Esters include, for example, ethyl acetate, methyl propionate, etc.

[0014] Optionally, in the mixture of step S1, the volume proportion of the liquid organic modifier is ≥1%.

[0015] In a second aspect, the present invention also provides a modified lipid vesicle prepared according to the method described above.

[0016] In a third aspect, the present invention also provides the use of the modified lipid vesicles prepared according to the method as described above in ultrasound angiography or in the preparation of drugs for treating tumors.

[0017] In a fourth aspect, the present invention further provides an ultrasound contrast method, comprising the following steps:

[0018] The modified lipid vesicles prepared according to the method described above were heated and subjected to ultrasonic irradiation.

[0019] Optionally, the modified lipid vesicles prepared according to the method described above are heated up before imaging, and the temperature increase is greater than or equal to 5°C.

[0020] Optionally, the frequency of the ultrasonic irradiation is ≥10 MHz.

[0021] In a fifth aspect, the present invention also provides a drug carrier for treating tumors, wherein the drug carrier comprises a modified lipid vesicle prepared according to the method described above and content components loaded by the modified lipid vesicle.

[0022] Optionally, the modified lipid vesicles are used as carriers to deliver the content components into tumor tissues and cells.

[0023] Beneficial effects of the present invention:

[0024] The present invention only modifies the lipid vesicles, does not destroy the membrane and structure of the lipid vesicles, has no effect on the substances carried by the lipid vesicles and the biological molecules on the membrane, and retains the original biological characteristics of the lipid vesicles.

[0025] The modified lipid vesicles prepared by the invention can be used as ultrasound enhancement materials and have ultrasound enhancement (contrast imaging and treatment) effects.

[0026] The modified lipid vesicles prepared by the present invention have stronger tissue / cell penetration, which is beneficial for delivering the content components carried by the lipid vesicles to the deep part of the tumor (including the deep part of the tissue block and the inside of the cell), thereby enhancing the therapeutic effect.

[0027] The method of the present invention has a wide application range and can process lipid vesicles without being limited by the preparation method / process of lipid vesicle nanomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 1A is a schematic diagram of the principle of the present invention, 1B is a schematic diagram of the structure of the lipid vesicles after modification and the separation of the organic phase and the aqueous phase of the solution, 1C is a schematic diagram of the structure of the modified lipid vesicles after the aqueous phase solution is heated, and 1D is a schematic diagram of the structure of the modified lipid vesicles after irradiation;

[0029] Figure 2 2A is an electron microscopic image of the OMV before and after modification, and 2B is an electron microscopic image of the OMV after modification with n-butanol.

[0030] Figure 3 Figure 3 is the ultrasound contrast imaging result of OMV before and after modification. The upper part is the B-ultrasound signal diagram, and the lower part is the ultrasound contrast imaging signal diagram. 3A is the conventional B-ultrasound and ultrasound contrast imaging result diagram of OMV before modification. 3B is the ultrasound contrast imaging result diagram of OMV after n-butanol modification without heating treatment. 3C is the ultrasound contrast imaging result diagram of OMV after n-butanol modification with heating.

[0031] Figure 4 The conventional B-ultrasound and ultrasound contrast imaging results of OMV before and after modification in the tumor-bearing part of mice, the upper row is the B-ultrasound signal diagram, the lower row is the ultrasound contrast imaging signal diagram, 4A is the ultrasound contrast imaging result diagram of OMV before modification injected into the tumor-bearing mouse for 6 hours and then subjected to infrared heating treatment, 4B is the ultrasound contrast imaging result diagram of OMV after n-butanol modification and infrared heating treatment;

[0032] Figure 5Figures 5A and 5B are electrophoresis results of OMV protein extraction before and after modification, 5A is a marker, 5B is OMV protein extraction before modification, 5C is OMV protein extraction after 4:1 n-butanol modification (i.e., Example 1), and 5B is OMV protein extraction after 16:1 n-butanol modification (i.e., Example 2);

[0033] Figure 6 The laser confocal scanning results of OMV penetration into the cell cluster before and after modification. The dimensions marked from left to right are the scanning depths from the surface to the inside of the cell cluster. 6A is the penetration distribution of OMV (red) at different depths of the cell cluster before modification; 6B is the penetration distribution of OMV (red) at different depths of the cell cluster after n-butanol modification;

[0034] Figure 7 The laser confocal scanning results of OMV penetration / uptake into cells before and after modification, from left to right are cell nucleus staining (blue), OMV staining (green) and fusion images, 7A is the laser confocal scanning result of OMV (green) in cells before modification; 7B is the laser confocal scanning result of OMV (green) in cells after n-butanol modification;

[0035] Figure 8 Figure 8 is the in vivo imaging result of the distribution of OMV in tumor-bearing mice before and after modification. Figure 8A is the in vivo imaging result of OMV injected into tumor-bearing mice at different times before modification; Figure 8B is the in vivo imaging result of OMV injected into tumor-bearing mice at different times after n-butanol modification;

[0036] Fig. 9 Figure 9 is the measurement result of the effect of OMV before and after modification on the tumor size of mice. 9A shows the change in tumor size of the control group injected with PBS; 9B shows the change in tumor size of the group injected with PBS modified by n-butanol; 9C shows the change in tumor size of the group injected with OMV before modification; 8D shows the change in tumor size of the group injected with OMV modified by n-butanol;

[0037] Fig.10 The ultrasound contrast imaging results of OMVs modified with different organic reagents are shown in the upper row, the B-ultrasound signal diagram is shown in the lower row, 10A is the ultrasound contrast imaging result of OMVs modified with n-propanol; 10B is the ultrasound contrast imaging result of OMVs modified with n-butanol; 10C is the ultrasound contrast imaging result of OMVs modified with n-pentanol; 10D is the ultrasound contrast imaging result of OMVs modified with n-hexanol;

[0038] Fig.11Figure 11 is the ultrasound contrast imaging result of n-butanol-modified OMV after heating at different temperatures. The upper part is the B-ultrasound signal diagram, and the lower part is the ultrasound contrast imaging signal diagram. Figure 11A is the ultrasound contrast imaging result after treatment at room temperature (preparation and storage temperature); Figure 10B is the ultrasound contrast imaging result after heating to 37°C; Figure 11C is the ultrasound contrast imaging result after heating to 47°C; Figure 11D is the ultrasound contrast imaging result after heating to 57°C; and Figure 11E is the ultrasound contrast imaging result after heating to 67°C. DETAILED DESCRIPTION

[0039] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0040] It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0041] like Figure 1 As shown, one embodiment of the present application provides a method for modifying lipid vesicles, comprising the following steps:

[0042] S1. Mix lipid vesicles (such as Figure 1 A) solution and a liquid organic modifier to obtain a mixture in which the volume proportion of the liquid organic modifier is ≥ 1%, wherein the liquid organic modifier is selected from alcohol substances, ester substances or a combination thereof having a carbon number greater than or equal to 4 and less than or equal to 8;

[0043] S2. The mixture is thoroughly mixed to allow the modifier to fully react with the lipid vesicles, and the mixture is allowed to stand. The resulting aqueous phase contains the modified lipid vesicles.

[0044] After the modification, the lipid vesicles have organic modifier molecules inside and on their membranes, but the lipid vesicles and their bimolecular membrane structures remain intact (e.g. Figure 1 B).

[0045] Another embodiment of the present application also provides the use of the modified lipid vesicles prepared according to the method as described above in ultrasound angiography or in the preparation of drugs for treating tumors.

[0046] Another embodiment of the present application further provides an ultrasound contrast method, comprising the following steps:

[0047] After the modified lipid vesicles prepared according to the above method are heated (the temperature increase is greater than or equal to 5°C), the organic modifier molecules carried by the modified lipid vesicles change (such as Figure 1 C);

[0048] Ultrasound irradiation was performed at a frequency of ≥10 MHz, and the modified lipid vesicles reflected or scattered high-frequency ultrasound contrast imaging (such as Figure 1 D).

[0049] Another embodiment of the present application also provides a drug for treating tumors, which includes modified lipid vesicles prepared according to the method as described above and content components loaded by the modified lipid vesicles. The modified lipid vesicles are used as carriers to deliver the content components to tumor tissues and cells.

[0050] In the following description, a large amount of details are discussed to provide a more thorough explanation of the embodiments of the present invention, however, it is obvious to those skilled in the art that embodiments of the present invention can be implemented without these specific details. The following only enumerates the specific circumstances of n-butyl alcohol as the liquid organic modifier, and those skilled in the art may also select the liquid organic modifier except n-butyl alcohol according to actual conditions, for example isobutyl alcohol, neobutyl alcohol, n-pentanol, isopentanol, neopentyl alcohol, n-hexanol, isohexanol, neohexanol, enanol (containing isomers), octanol (containing isomers), cyclobutanol, cyclopentanol, cyclohexanol, cycloheptanol, cyclooctanol etc. or isobutyl alcohol, neobutyl alcohol, n-pentanol, isopentanol, neopentyl alcohol, n-hexanol, isohexanol, neohexanol, enanol (containing isomers), octanol (containing isomers), cyclobutanol, cyclopentanol, cyclohexanol, cycloheptanol, cyclooctanol etc. at least one composition formed with n-butyl alcohol is as the liquid organic modifier.

[0051] Example 1

[0052] S1. At room temperature, the E. coli OMV solution was mixed in a volume ratio of 4:1 (the specific extraction steps of the E. coli OMV solution are as follows: take the E. coli colony and inoculate it into 10mL of LB liquid culture medium, shake and culture it at 37℃ and 220rpm for 6h, take five portions of 2mL of bacterial solution and add them into 5 triangular flasks containing 400mL of LB broth, shake and culture it at 37℃ and 220rpm for 24h; collect the bacterial solution, centrifuge it at 4℃ and 5000g for 15min, and collect the supernatant; filter the collected supernatant through a 0.45μm filter and a 0.22μm filter in turn, and add the obtained filtrate to 50mL 100 kDa ultrafiltration tube, centrifuged at 3500 g for 5 min to concentrate, recovered the concentrate, centrifuged at 4 ° C and 150000 g for 3 h, the obtained precipitate was resuspended with PBS buffer, and centrifuged again at 4 ° C and 150000 g for 3 h, and the obtained precipitate was resuspended with PBS buffer to obtain OMVs solution) and n-butanol to obtain a mixture with a volume proportion of n-butanol of 20%;

[0053] S2. The mixture is thoroughly mixed by vortexing to allow the modifier to fully react with the lipid vesicles, and the mixture is allowed to stand, and the aqueous phase is collected. The aqueous phase obtained contains n-butanol-modified OMVs;

[0054] The morphology of OMV before and after modification was observed using an electron microscope. Figure 2 As shown, 2A is an electron microscope scanning image of OMV before modification, and 2B is an electron microscope scanning image of modified OMV.

[0055] Depend on Figure 2 It can be seen that there is no significant difference in the morphology of OMV before and after modification. This result shows that the modification method of the present application does not destroy the membrane and structure of the lipid vesicles.

[0056] S3. The unmodified OMV and the n-butanol modified OMV were subjected to high-frequency ultrasound irradiation at a frequency of 20 MHz. The results are as follows: Figure 3 As shown in A and 3B, the upper part is the ultrasound signal diagram, and the lower part is the contrast signal diagram. 3A is the high-frequency ultrasound irradiation contrast image of OMV before modification, and 3B is the high-frequency ultrasound irradiation contrast image of modified OMV without heating treatment;

[0057] The aqueous phase containing n-butanol-modified OMV was heated to 52°C and subjected to high-frequency ultrasound irradiation at a frequency of 20 MHz. Figure 3 As shown in C.

[0058] Depend on Figure 3 It can be seen that direct high-frequency ultrasound irradiation of OMV has no echo and no contrast effect (such as Figure 3 A), high-frequency ultrasound irradiation of n-butanol-modified OMVs showed no echo or contrast effects (e.g. Figure 3B), after heating the n-butanol-modified OMV and then irradiating it with high-frequency ultrasound, obvious echoes were produced, which had a good contrast effect (such as Figure 3 C). The results show that after the modification, the modified OMV is heated and then irradiated with high-frequency ultrasound, which can produce a good ultrasound enhancement (such as contrast imaging) effect.

[0059] S4. The unmodified OMVs and n-butanol-modified OMVs were injected into tumor-bearing mice, respectively. After 6 hours, the tumor tissues were enriched and high-frequency ultrasound irradiation was performed at a frequency of 20 MHz. The results are shown in Figure 4 As shown in A and 4B, the upper row is the B-ultrasound signal image, and the lower row is the ultrasound contrast imaging signal image. 4A is the high-frequency ultrasound irradiation imaging result image after the unmodified OMV was injected into the tumor-bearing mouse for 6 hours and then subjected to infrared heating treatment. 4B is the high-frequency ultrasound irradiation imaging image after the n-butanol-modified OMV was injected into the tumor-bearing mouse for 6 hours and then subjected to infrared heating treatment.

[0060] Depend on Figure 4 It can be seen that the unmodified OMVs were heated in the tumor site in vivo and then irradiated with high-frequency ultrasound, without echo and contrast effect (e.g. Figure 4 A); n-butanol-modified OMVs were heated in vivo and then irradiated with high-frequency ultrasound, producing obvious echoes and having good contrast effects (such as Figure 4 B). The results show that after the OMVs are modified and heated at the tumor site in vivo and then irradiated with high-frequency ultrasound, they can also produce good ultrasound enhancement (such as contrast imaging) effects in vivo.

[0061] Example 2

[0062] The difference between this embodiment and embodiment 1 is that the volume ratio of Escherichia coli OMV and n-butanol is 16:1.

[0063] The OMV proteins in the OMV before modification and the OMV proteins in the aqueous phase obtained in Example 1 and Example 2 were extracted respectively. The specific steps were as follows: take the resuspended OMVs, add lysis buffer (2% SDS, 50mM Tris-HCl, pH 6.8, 10% glycerol) at a volume ratio of 1:1, gently vortex mix, put the sample on ice after ice bathing for 15 minutes, use an ultrasonic crusher for lysis treatment, the ultrasonic instrument parameters are 50% power, ultrasonic 5 times, each time for 10 seconds, interval 10 seconds, avoid overheating. The sample was centrifuged at 4°C and 14000g for 10 minutes, and the supernatant was collected as the total protein extracted from the OMVs, and electrophoresis was performed. The electrophoresis results are as follows Figure 5 As shown, 5A is a marker, 5B is an OMV extracted protein extracted from the OMV before modification, 5C is an OMV extracted protein extracted from the aqueous phase obtained in Example 1, and 5B is an OMV extracted protein extracted from the aqueous phase obtained in Example 2.

[0064] Depend on Figure 5 It can be seen that the bands of OMV extracted proteins extracted from the aqueous phase obtained in Example 1 and Example 2 are similar to those of OMV extracted proteins in OMV before modification. This result shows that the modification treatment has no effect on the content components such as proteins carried by OMV.

[0065] The OMVs before modification and the aqueous phase obtained in Example 2 were co-incubated with the suspended 4T1 cell clusters at 37°C and 5% carbon dioxide, and the distribution of the OMVs in the cell clusters was scanned by laser confocal scanning. Figure 6 As shown, the dimensions marked from left to right are the scanning depths of the cell cluster from the surface to the inside, 6A is the penetration distribution of OMV (red) at different depths of the cell cluster before modification; 6B is the penetration distribution of the water phase (red) obtained in Example 2 at different depths of the cell cluster.

[0066] Depend on Figure 6 It can be seen that the penetration depth of OMV in the cell mass increased after n-butanol modification compared with that before modification. This result shows that OMV after n-butanol modification can penetrate deeper into the cell mass.

[0067] The OMVs before modification and the aqueous phase obtained in Example 2 were co-incubated with adherent cultured 4T1 cells at 37°C and 5% carbon dioxide, and the distribution of OMVs in the cells was scanned by laser confocal microscopy. Figure 7 Shown, from left to right are cell nucleus staining (blue), OMV staining (green) and fusion image.

[0068] Depend on Figure 7 It can be seen that there are few OMVs (green) in the cells before modification (7A), and more OMVs (green) in the cells after modification with n-butanol (7B). This result shows that the OMVs modified with n-butanol have a stronger ability to be taken up by cells or penetrate into cells.

[0069] The OMVs before modification and the aqueous phase obtained in Example 2 were injected into tumor-bearing mice, respectively, and a blank group and a n-butanol + PBS treatment group were set up. The blank group used an equal volume of PBS buffer to replace the OMVs, and the n-butanol + PBS treatment group used an equal volume of PBS buffer to replace the OMVs in the aqueous phase preparation process obtained in Example 2 (i.e., except that the PBS buffer was used to replace the OMVs, the treatment was performed in the same manner as in Example 2). The distribution of OMVs in tumor-bearing mice was detected by in vivo fluorescence imaging. The results are shown in FIG. Figure 8 shown.

[0070] Depend on Figure 8It can be seen that before modification, OMVs were significantly enriched in the tumor site (red circle) after 6 hours in tumor-bearing mice. After modification with n-butanol, OMVs were significantly enriched in the tumor site (red circle) after 1 hour in tumor-bearing mice, and maintained a high fluorescence intensity from 3 to 24 hours. This result shows that the modified OMVs can be enriched in tumor tissues faster and can remain in tumor tissues for a longer time.

[0071] The tumor sizes of the blank control group (PBS), the n-butanol + PBS treatment group, the OMV before modification, and the tumor-bearing mice injected with the aqueous phase obtained in Example 2 were measured. The results are as follows: Fig. 9 As shown, 9A is the tumor size change of the blank control group; 9B is the tumor size change of the aqueous phase obtained in Example 2; 9C is the tumor size change of the OMV group injected before modification; 9D is the tumor size change of the n-butanol + PBS treatment group.

[0072] Depend on Fig. 9 It can be seen that E. coli OMV has a significant inhibitory effect on tumor growth, and the tumor size of the tumor-bearing mice injected with the aqueous phase obtained in Example 2 is significantly reduced compared with that before modification. This result shows that OMV modified with n-butanol has a stronger ability to inhibit tumor growth.

[0073] Example 3

[0074] Using n-propanol, n-butanol, n-pentanol and n-hexanol as modifiers respectively, the aqueous phase was obtained in the same manner as in Example 1 (ignore if there is no stratification).

[0075] The four aqueous phases were heated to 52°C and subjected to high-frequency ultrasound irradiation at 20 MHz. Fig.10 As shown, the upper row is B-ultrasound signal diagram, the lower row is ultrasound contrast imaging signal diagram, 10A is the ultrasound irradiation contrast imaging result diagram of OMV modified with n-propanol; 10B is the ultrasound irradiation contrast imaging result diagram of OMV modified with n-butanol; 10C is the ultrasound irradiation contrast imaging result diagram of OMV modified with n-pentanol; 10D is the ultrasound irradiation contrast imaging result diagram of OMV modified with n-hexanol.

[0076] Depend on Fig.10 It can be seen that the OMV modified with n-butanol has no echo and no contrast effect (10A), the OMV modified with n-butanol has echo and contrast effect (10B), the OMV modified with n-pentanol has echo and contrast effect (10C), and the OMV modified with n-hexanol has echo and contrast effect (10D). The results show that n-butanol and alcohols with longer carbon chains can effectively modify OMV to give it ultrasound contrast function.

[0077] Example 4

[0078] The aqueous phase was obtained according to the method of Example 1, and the aqueous phase was divided into five parts. Four of the parts were heated to 37°C, 57°C, 57°C, and 67°C, respectively, and then subjected to high-frequency ultrasound irradiation imaging at a frequency of 20 MHz. The remaining part was directly subjected to high-frequency ultrasound irradiation imaging at a frequency of 20 MHz at room temperature. The results are shown in FIG. Fig.11 As shown, the upper part is the B-ultrasound signal image, and the lower part is the ultrasound contrast imaging signal image.

[0079] Depend on Fig.11 It can be seen that after being treated at room temperature, high-frequency ultrasound irradiation imaging showed no echo and no contrast effect (11A), and after being heated to 37°C, high-frequency ultrasound irradiation imaging showed echo and contrast effect (11B); after being heated to 47°C, high-frequency ultrasound irradiation imaging showed echo and contrast effect (11C); after being heated to 57°C, high-frequency ultrasound irradiation imaging showed echo and contrast effect (11D); after being heated to 67°C, high-frequency ultrasound irradiation imaging showed echo and contrast effect (11E). The results show that n-butanol-modified OMV has ultrasound contrast function after being heated above 5°C.

[0080] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for modifying lipid vesicles, characterized in that: The following steps are involved: S1. mixing the lipid vesicle solution and the liquid organic modifier at a temperature in the range of 0-60° C. to obtain a mixture; S2. The mixture is thoroughly mixed to allow the modifier to fully react with the lipid vesicles, and the mixture is allowed to stand. The resulting aqueous phase contains the modified lipid vesicles.

2. The method for modifying lipid vesicles according to claim 1, wherein In step S1, the liquid organic modifier is selected from alcohol substances, ester substances or combinations thereof having carbon atoms greater than or equal to 4 and less than or equal to 8.

3. The method for modifying lipid vesicles according to claim 1, wherein In the mixture of step S1, the volume proportion of the liquid organic modifier is ≥ 1%.

4. The modified lipid vesicle prepared according to the method according to any one of claims 1 to 3.

5. Use of the modified lipid vesicles prepared according to the method according to any one of claims 1 to 3 in ultrasound contrast imaging or in the preparation of drugs for treating tumors.

6. An ultrasound angiography method, characterized in that: The following steps are involved: The modified lipid vesicles prepared according to any one of claims 1 to 3 are heated and subjected to ultrasonic irradiation.

7. The ultrasound contrast method according to claim 6, characterized in that: The modified lipid vesicles prepared according to the method according to any one of claims 1 to 3 are heated up for imaging, wherein the temperature increase is greater than or equal to 5°C.

8. The ultrasound contrast method according to claim 6, characterized in that: The frequency of the ultrasonic irradiation is ≥10 MHz.

9. A drug for treating tumors, characterized in that: The drug comprises the modified lipid vesicles prepared according to the method according to any one of claims 1 to 3 and the content components loaded by the modified lipid vesicles.

10. The drug for treating tumors according to claim 9, characterized in that: The modified lipid vesicle is used as a carrier to deliver the content components into tumor tissues and cells.

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