Method for modifying biosynthetic gas vesicles by using polyethylene glycol and use thereof

Abstract

A method for modifying biosynthetic gas vesicles by using polyethylene glycol and use thereof. The preparation method comprises mixing a crosslinking agent, polyethylene glycol, and a buffer to obtain a mixed solution, performing a primary incubation, mixing the mixed solution after the primary incubation with gas vesicles, performing a secondary incubation, then performing centrifugation after the secondary incubation, and collecting upper-layer floating objects to obtain polyethylene glycol-modified gas vesicles, wherein the polyethylene glycol and the gas vesicles are fed in a ratio of 30-40 mg of polyethylene glycol:1 mL of gas vesicles with an OD500 of 3.0, and the gas vesicles are synthesized by microorganisms. According to the present application, it has been discovered that modification with different amounts of PEG can result in different metabolic durations of GVs in animals, and it has also been discovered that gas vesicles with good performance can be obtained without using excessive amounts of PEG for modification.

Description

A method for modifying biosynthetic gas vesicles using polyethylene glycol and its application Technical Field

[0001] The present application belongs to the field of biotechnology and relates to a method for modifying biosynthetic gas vesicles using polyethylene glycol and its application. Background Art

[0002] Due to their small particle size, nanobubbles can penetrate blood vessels and enter surrounding tissues, thereby improving ultrasound imaging effects and imaging time. There are also reports that nanobubbles can be used as an effective tool for drug and gene delivery. However, chemically synthesized nanobubbles are composed of a phospholipid or polymer shell and a gas core (such as an inert gas such as perfluoropropane or perfluorocarbon); therefore, the stability of chemically synthesized nanobubbles is easily affected by pH, ambient temperature, and ionic strength. When these factors change, it may cause bubble rupture and gas overflow, resulting in uneven particle size of the nanobubbles, which reduces the ultrasound imaging effect of the nanobubbles.

[0003] In recent years, Shapiro et al. have discovered genetically encoded nanoscale gas vesicles (GVs) in bacteria and archaea. GVs have a protein shell, mainly composed of GvpA and GvpC proteins. The hydrophobic GvpA forms a spindle-shaped skeleton, and the hydrophilic GvpC is arranged in the external structure of the protein shell. Studies have shown that the GVs from Halobacterium salinarum NRC-1 bacteria have a particle size width of 45-250nm and a length of 100-300nm, and exhibit excellent contrast imaging performance using clinical diagnostic ultrasound equipment under optimized parameters. However, because the GVs shell is composed of protein, it is easily phagocytosed by macrophages of the reticuloendothelial system (RES) and may bring some immunogenicity or side effects, which limits their use in future clinical practice.

[0004] Existing studies have shown that surface modification with PEG significantly reduces the absorption of nanoparticles by the RES and prolongs their circulation time in the body. PEG surface modification can also shield nanoparticle surface antigens, reducing the occurrence of immune responses. Excessive amounts of polyethylene glycol are typically used for modification to obtain PEG-modified GVs. However, due to the structural complexity of gas vesicles and their metabolic complexity in animals, the specific impact of PEG modification on their metabolism in animals remains unclear, resulting in a lack of efficient and cost-effective modification methods.

[0005] In summary, exploring the effect of PEG modification on the metabolism of gas vesicles in animals and developing efficient and economical modification methods are of great significance to the field of gas vesicles.

[0006] Summary of the Invention

[0007] The present application provides a method for modifying biosynthetic gas vesicles using polyethylene glycol and its application. The present application conducts an in-depth analysis of whether different amounts of PEG attached at the same molecular weight will affect the metabolic time of gas vesicles in animals, in order to develop an efficient and economical method for modifying biosynthetic gas vesicles using polyethylene glycol.

[0008] In a first aspect, the present application provides a method for modifying biosynthetic gas vesicles using polyethylene glycol, the method comprising:

[0009] A cross-linking agent, polyethylene glycol and a buffer solution are mixed to obtain a mixed solution, and the mixture is incubated once. The mixed solution is mixed with gas vesicles and incubated twice. After the second incubation, the mixture is centrifuged and the upper floating material is collected to obtain polyethylene glycol-modified gas vesicles; the feeding ratio of polyethylene glycol to gas vesicles is 30-40 mg:1 mL, wherein 30-40 mg of polyethylene glycol includes but is not limited to 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg or 39 mg, and the OD of the gas vesicles is 200 μg / mL. 500 is 3.0, and the gas vesicles are synthesized by microorganisms.

[0010] In this application, we conducted the first in-depth analysis of the effects of different PEG attachment amounts on the metabolism of gas vesicles in animals at the same molecular weight. We found that modification with different amounts of PEG can lead to different metabolic times of GVs in animals. This also breaks the conventional wisdom, showing that gas vesicles with good performance can be obtained without using a large amount of excess PEG for modification, providing new ideas for the development of efficient and economical modification methods.

[0011] Optionally, the cross-linking agent includes N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and / or N-hydroxysuccinimide (NHS).

[0012] Optionally, the addition ratio of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and polyethylene glycol is (1-5):(1-5):1, for example, it can be 4:1:1. The above numerical range is applicable to this application and is not listed one by one.

[0013] Optionally, the molecular weight of the polyethylene glycol is 2KD to 10KD, including but not limited to 3KD, 4KD, 5KD, 6KD, 7KD, 8KD, 9KD or 10KD.

[0014] Optionally, the buffer comprises PBS buffer and / or MES buffer.

[0015] Optionally, the temperature of the incubation is 20-30°C (for example, 21°C, 22°C, 23°C, 24°C, 25°C, 28°C or 29°C), and the time is 10-30 min (for example, 11 min, 12 min, 13 min, 15 min, 20 min, 25 min, 26 min, 28 min or 29 min).

[0016] Optionally, the temperature of the secondary incubation is 20-30°C (for example, 21°C, 22°C, 23°C, 24°C, 25°C, 28°C or 29°C), and the time is 8-12h (for example, 9h, 10h or 11h).

[0017] Optionally, the primary incubation and the secondary incubation are each independently performed under shaking conditions, and the shaking speed is 50-100 rpm, including but not limited to 60 rpm, 70 rpm, 80 rpm or 90 rpm.

[0018] Optionally, the centrifugation conditions are 2-8° C., 200-300×g, and 3-6 h.

[0019] Optionally, the step of washing is further included after collecting the upper floating matter.

[0020] Optionally, the washing method comprises:

[0021] The supernatant was mixed with a buffer solution, centrifuged, and the supernatant was collected. The process was repeated 3 to 4 times.

[0022] Optionally, the microorganisms include halophilic archaea and / or cyanobacteria.

[0023] Optionally, gas vesicles derived from halophilic archaea and / or cyanobacteria are applicable to the present application scheme, and the specific extraction method can be:

[0024] (1) Bacterial culture

[0025] a. Add 1 L of bacterial culture medium to a 2 L Erlenmeyer flask;

[0026] b. Inoculate 10 mL of the pink bacterial liquid starter into 1 L of fresh growth medium (1:100).

[0027] c. Place the bacterial solution in a constant temperature shaker. After inoculation, observe the bacterial solution daily until the inoculated culture is completely confluent and the medium changes from dark pink to bright pink.

[0028] (2) Extraction of gas vesicles

[0029] a. Slowly transfer the bacterial solution obtained from the previous portion to a separatory funnel and allow it to stand until the Halo bacteria containing gas vesicles float to the top layer of the culture medium, until a light pink ring visible to the naked eye forms on the top layer of the culture medium.

[0030] b. Discard excess culture medium, retaining only the top layer of light pink culture medium and the bacteria containing gas vesicles floating in it. Gently rinse off bacteria adhering to the flask wall with an equal volume of TMC lysis buffer.

[0031] c. Transfer the above solution to a 50 mL centrifuge tube and centrifuge at 300 g at 4°C for 4 h.

[0032] d. After centrifugation, the liquid in the centrifuge tube will separate into two layers: the upper layer is pinkish-white, representing the successfully extracted gas vesicles and some unlysed Halo bacteria, and the lower layer is pink, representing the lysed Halo bacteria. Use a syringe to slowly aspirate the pink solution from the lower layer, leaving only the pinkish-white solution in the centrifuge tube.

[0033] e. Take an appropriate amount of PBS to resuspend the white powder solution, centrifuge at 4°C, 300g for 4 hours; and

[0034] f. Repeat steps d and e until the lower layer of solution turns transparent. Terminate the centrifugation process, slowly aspirate the lower transparent solution with a syringe, and resuspend the upper white solution in an appropriate amount of PBS to obtain the purified gas vesicles. Aliquot the gas vesicle solution into 1.5 mL centrifuge tubes and store at 4°C.

[0035] As a preferred technical solution, the method of modifying biosynthetic gas vesicles using polyethylene glycol includes:

[0036] N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, polyethylene glycol and buffer were mixed to obtain a mixed solution, which was incubated once. The mixed solution after the first incubation was mixed with gas vesicles and incubated twice, wherein the feeding ratio of polyethylene glycol to gas vesicles was 30-40 mg polyethylene glycol: 1 mL OD 500 The gas vesicles are 3.0, and after the second incubation, centrifugation is performed to collect the upper floating matter; the upper floating matter is mixed with a buffer solution, centrifuged, and the upper floating matter is collected, and this is repeated 3 to 4 times to obtain polyethylene glycol-modified gas vesicles.

[0037] In a second aspect, the present application provides a polyethylene glycol-modified gas vesicle, which is prepared by the method of using polyethylene glycol to modify biologically synthesized gas vesicles as described in the first aspect.

[0038] In a third aspect, the present application provides the method for synthesizing biological gas vesicles modified with polyethylene glycol as described in the first aspect or the use of the polyethylene glycol-modified gas vesicles as described in the second aspect in the preparation of ultrasound contrast agents.

[0039] Compared with the prior art, this application has the following beneficial effects:

[0040] This application is the first to conduct an in-depth analysis of the effects of different PEG attachment amounts on the metabolism of gas vesicles in animals at the same molecular weight. It was found that modification with different amounts of PEG can lead to different metabolic times of GVs in animals. It also breaks with conventional cognition and finds that gas vesicles with good performance can be obtained without using excessive PEG for modification, providing new ideas for the development of efficient and economical modification methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 is a graph showing the amount of PEG attached to GVs.

[0042] Figure 2 shows the metabolic time of GVs modified with different amounts of PEG in mouse liver. DETAILED DESCRIPTION

[0043] To further illustrate the technical means and effects of this application, the following further describes this application in conjunction with examples and drawings. It should be understood that the specific implementation methods described herein are only used to explain this application, rather than to limit this application.

[0044] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0045] The gas vesicles used in the specific examples of this application are derived from the halophilic archaeon Halobacterium salinarum (ATCC 33170), and the extraction method includes:

[0046] (1) Bacterial culture

[0047] a. Add 1 L of bacterial culture medium to a 2 L Erlenmeyer flask;

[0048] b. Inoculate 10 mL of the pink bacterial liquid starter into 1 L of fresh growth medium (1:100).

[0049] c. Place the bacterial solution in a constant temperature shaker. After inoculation, observe the bacterial solution daily until the inoculated culture is completely confluent and the medium changes from dark pink to bright pink.

[0050] (2) Extraction of gas vesicles

[0051] a. Slowly transfer the bacterial solution obtained from the previous portion to a separatory funnel and allow it to stand until the Halo bacteria containing gas vesicles float to the top layer of the culture medium, until a light pink ring visible to the naked eye forms on the top layer of the culture medium.

[0052] b. Discard excess culture medium, retaining only the top layer of light pink culture medium and the bacteria containing gas vesicles floating in it. Gently rinse off bacteria adhering to the flask wall with an equal volume of TMC lysis buffer.

[0053] c. Transfer the above solution to a 50 mL centrifuge tube and centrifuge at 300 g at 4°C for 4 h.

[0054] d. After centrifugation, the liquid in the centrifuge tube will separate into two layers. The upper layer is pink and white, which contains the successfully extracted gas vesicles and some unlysed Halo bacteria. The lower layer is pink and contains the lysed Halo bacteria. Use a syringe to slowly aspirate the pink solution from the lower layer, leaving only the pink and white solution from the upper layer in the centrifuge tube.

[0055] e. Take an appropriate amount of PBS to resuspend the white powder solution and centrifuge at 4°C, 300g for 4 hours;

[0056] f. Repeat steps d and e until the lower solution turns transparent. Terminate the centrifugation process, slowly aspirate the lower transparent solution with a syringe, and resuspend the upper white solution in an appropriate amount of PBS to obtain the purified gas vesicles. Aliquot the gas vesicle solution into 1.5 mL centrifuge tubes and store at 4°C.

[0057] Example 1

[0058] This embodiment provides a polyethylene glycol-modified gas vesicle, and the specific preparation method is as follows:

[0059] (1) Weigh 5.25 mg of EDC (Aladdin, 25952-53-8) and 3.15 mg of NHS (Sigma-Aldrich, 6066-82-6) and dissolve them in 1 mL of PBS;

[0060] (2) Then, 35 mg of PEG (Melopeg, 020106) was weighed and added to the mixture in step (1), placed on a rolling shaker at 70 rpm, and incubated at 25°C for 20 min;

[0061] (3) Add 1 mL of OD 500 GVs of 3.0;

[0062] (4) The mixture from step (3) was placed on a rolling shaker at 70 rpm at 4°C overnight;

[0063] (5) Then centrifuge at low speed for 4 h (250 × g, 4°C). The solution will separate into three layers. Carefully aspirate the middle layer solution and the lower layer precipitate and discard them.

[0064] (6) The supernatant was resuspended in PBS buffer and centrifuged at low speed for 4 h (250 × g, 4°C), repeated three times; the supernatant was stored in PBS buffer and stored at 4°C until use.

[0065] Example 2

[0066] This embodiment provides a polyethylene glycol-modified gas vesicle, and the specific preparation method is as follows:

[0067] (1) Weigh 6 mg of EDC and 3.6 mg of NHS and dissolve them in 1 mL of PBS;

[0068] (2) Then, 40 mg of PEG was weighed and added to the mixture in step (1), placed on a rolling shaker at 50 rpm, and incubated at 25°C for 20 min;

[0069] (3) Add 1 mL of OD 500 GVs of 3.0;

[0070] (4) The mixture from step (3) was placed on a rolling shaker at 50 rpm at 4°C overnight;

[0071] (5) Then centrifuge at low speed for 4 h (250 × g, 4°C). The solution will separate into three layers. Carefully aspirate the middle layer solution and the lower layer precipitate and discard them.

[0072] (6) The supernatant was resuspended in PBS buffer and centrifuged at low speed for 5 h (300 × g, 5°C), repeated three times; the supernatant was stored in PBS buffer and stored at 4°C until use.

[0073] Example 3

[0074] This embodiment provides a polyethylene glycol-modified gas vesicle, and the specific preparation method is as follows:

[0075] (1) Weigh 4.5 mg of EDC and 2.7 mg of NHS and dissolve them in 1 mL of PBS;

[0076] (2) Then, 30 mg of PEG was weighed and added to the mixture in step (1), placed on a rolling shaker at 100 rpm, and incubated at 20°C for 30 min;

[0077] (3) Add 1 mL of OD 500 GVs of 3.0;

[0078] (4) The mixture from step (3) was placed on a rolling shaker at 100 rpm at 4°C overnight;

[0079] (5) Then centrifuge at low speed for 3 h (200 × g, 4°C). The solution will separate into three layers. Carefully aspirate the middle layer solution and the lower layer precipitate and discard them.

[0080] (6) The supernatant was resuspended in PBS buffer and centrifuged at low speed for 5 h (300 × g, 5°C), repeated three times; the supernatant was stored in PBS buffer and stored at 4°C until use.

[0081] Comparative Example 1

[0082] This comparative example provides a polyethylene glycol-modified gas vesicle, and the specific preparation method is as follows:

[0083] (1) Weigh 3 mg of EDC and 1.8 mg of NHS and dissolve them in 1 mL of PBS;

[0084] (2) Then, 20 mg of PEG was weighed and added to the mixture in step (1), placed on a rolling shaker at 70 rpm, and incubated at 25°C for 20 min;

[0085] (3) Add 1 mL of OD 500 GVs of 3.0;

[0086] (4) The mixture from step (3) was placed on a rolling shaker at 70 rpm at 4°C overnight;

[0087] (5) Then centrifuge at low speed for 4 h (250 × g, 4°C). The solution will separate into three layers. Carefully aspirate the middle layer solution and the lower layer precipitate and discard them.

[0088] (6) The supernatant was resuspended in PBS buffer and centrifuged at low speed for 4 h (250 × g, 4°C), repeated three times; the supernatant was stored in PBS buffer and stored at 4°C until use.

[0089] Comparative Example 2

[0090] This comparative example provides a polyethylene glycol-modified gas vesicle, and the specific preparation method is as follows:

[0091] (1) Weigh 1.5 mg of EDC and 0.9 mg of NHS, respectively, and dissolve them in 1 mL of PBS;

[0092] (2) Then, 10 mg of PEG was weighed and added to the mixture in step (1), placed on a rolling shaker at 70 rpm, and incubated at 25°C for 20 min;

[0093] (3) Add 1 mL of OD500 GVs of 3.0;

[0094] (4) The mixture from step (3) was placed on a rolling shaker at 70 rpm at 4°C overnight;

[0095] (5) Then centrifuge at low speed for 4 h (250 × g, 4°C). The solution will separate into three layers. Carefully aspirate the middle layer solution and the lower layer precipitate and discard them.

[0096] (6) The supernatant was resuspended in PBS buffer and centrifuged at low speed for 4 h (250 × g, 4°C), repeated 4 times; the supernatant was stored in PBS buffer and stored at 4°C for future use.

[0097] Comparative Example 3

[0098] This comparative example provides a polyethylene glycol-modified gas vesicle, and the specific preparation method is as follows:

[0099] (1) Weigh 0.75 mg of EDC and 0.45 mg of NHS, respectively, and dissolve them in 1 mL of PBS;

[0100] (2) Then, 5 mg of PEG was weighed and added to the mixture in step (1), placed on a rolling shaker at 70 rpm, and incubated at 25°C for 20 min;

[0101] (3) Add 1 mL of OD 500 GVs of 3.0;

[0102] (4) The mixture from step (3) was placed on a rolling shaker at 70 rpm at 4°C overnight;

[0103] (5) Then centrifuge at low speed for 4 h (250 × g, 4°C). The solution will separate into three layers. Carefully aspirate the middle layer solution and the lower layer precipitate and discard them.

[0104] (6) The supernatant was resuspended in PBS buffer and centrifuged at low speed for 4 h (250 × g, 4°C), repeated 4 times; the supernatant was stored in PBS buffer and stored at 4°C for future use.

[0105] Comparative Example 4

[0106] This comparative example provides a polyethylene glycol-modified gas vesicle, and the specific preparation method is as follows:

[0107] (1) Weigh 7.5 mg of EDC and 4.5 mg of NHS and dissolve them in 1 mL of PBS;

[0108] (2) Then, 50 mg of PEG was weighed and added to the mixture in step (1), placed on a rolling shaker at 70 rpm, and incubated at 25°C for 20 min;

[0109] (3) Add 1 mL of OD 500 GVs of 3.0;

[0110] (4) The mixture from step (3) was placed on a rolling shaker at 70 rpm at 4°C overnight;

[0111] (5) Then centrifuge at low speed for 4 h (250 × g, 4°C). The solution will separate into three layers. Carefully aspirate the middle layer solution and the lower layer precipitate and discard them.

[0112] (6) The supernatant was resuspended in PBS buffer and centrifuged at low speed for 4 h (250 × g, 4°C), repeated 4 times; the supernatant was stored in PBS buffer and stored at 4°C for future use.

[0113] Test Case

[0114] This test example analyzes the PEG connection amount and metabolism time in animals of the polyethylene glycol-modified gas vesicles prepared in Examples 1-3 and Comparative Examples 1-4.

[0115] (1) Analysis of PEG linkage amount

[0116] During quantification, fluorescently labeled PEG was used to connect to the gas vesicles. The prepared polyethylene glycol-modified gas vesicles were collected by centrifugation, and the upper floating material was diluted with PBS to an OD of 500 200 μL of the above solution was added to a 96-well plate, and the fluorescence was detected by an enzyme-labeled instrument to draw a curve of the connection amount of PEG and gas vesicles.

[0117] The results are shown in FIG1 . The fluorescence intensity of Examples 1-3 is significantly higher than that of the polyethylene glycol-modified gas vesicles prepared in Comparative Examples 1-3.

[0118] (2) Analysis of metabolic time in animals

[0119] Take the prepared polyethylene glycol modified gas vesicles, collect the upper floating matter by centrifugation and dilute it with PBS to OD 500 The concentration of the above solution was 3.0; 100 μL of the above solution was injected into mice (strain: C57BL / 6, age: 6-8 weeks, sex: female, purchased from Guangdong Medical Laboratory Animal Center) through the tail vein. Ultrasound molecular imaging of the mouse liver was performed using an ultrasound diagnostic system (Resona 7, Mindray, China). Ultrasound images were obtained and the imaging time was recorded.

[0120] The results are shown in Figure 2. The metabolic time of Examples 1-3 is significantly longer than that of the polyethylene glycol-modified gas vesicles prepared in Comparative Examples 1-3, indicating that the feeding ratio of polyethylene glycol to gas vesicles is controlled to be 30-40 mg polyethylene glycol: 1 mL gas vesicles (OD 500 3.0), which can significantly increase the metabolic time of gas vesicles in mouse liver. In addition, combined with the results of Comparative Example 4, the present application found that for the modification of gas vesicles, it is not necessary to use excessive amounts of polyethylene glycol as previously known. It is only necessary to control the feeding ratio of polyethylene glycol to gas vesicles to 30-40 mg polyethylene glycol: 1 mL OD 500 Gas vesicles with a density of 3.0 can obtain gas vesicles with good performance, which can significantly reduce costs and simplify the process.

[0121] In summary, this application is the first to conduct an in-depth analysis of the effects of different PEG attachment amounts on the metabolism of gas vesicles in animals at the same molecular weight. It was found that modification with different amounts of PEG can lead to different metabolic times of GVs in animals. Moreover, it breaks the conventional cognition and finds that gas vesicles with good performance can be obtained without using excessive PEG for modification, providing new ideas for the development of efficient and economical modification methods.

[0122] The applicant declares that while the above-mentioned embodiments are used to illustrate the detailed methods of the present application, the present application is not limited to the above-mentioned detailed methods, which does not mean that the present application must rely on the above-mentioned detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials of the present application's products, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. A method for modifying biosynthetic gas vesicles using polyethylene glycol, comprising: Mixing a cross-linking agent, polyethylene glycol and a buffer to obtain a mixed solution, performing a first incubation, mixing the mixed solution after the first incubation with gas vesicles, performing a second incubation, performing centrifugation after the second incubation, collecting the upper floating material, and obtaining polyethylene glycol-modified gas vesicles; The addition ratio of polyethylene glycol to gas vesicles is 30-40 mg:1 mL, and the OD of the gas vesicles is 500 is 3.0; Wherein, the gas vesicles are synthesized by microorganisms.

2. The method for modifying biosynthetic gas vesicles using polyethylene glycol according to claim 1, wherein: The cross-linking agent includes N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N-hydroxysuccinimide.

3. The method for modifying biosynthetic gas vesicles using polyethylene glycol according to claim 1, wherein: The addition ratio of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and polyethylene glycol is (1-5):(1-5):

1.

4. The method for modifying biosynthetic gas vesicles using polyethylene glycol according to any one of claims 1 to 3, wherein: The buffer comprises PBS buffer and / or MES buffer.

5. The method for modifying biosynthetic gas vesicles using polyethylene glycol according to any one of claims 1 to 4, wherein: The temperature of the first incubation is 20-30°C and the time is 10-30 minutes; Preferably, the secondary incubation temperature is 20-30°C and the time is 8-12h; Preferably, the primary incubation and the secondary incubation are each independently performed under shaking conditions, wherein the shaking speed is 50-100 rpm.

6. The method for modifying biosynthetic gas vesicles using polyethylene glycol according to any one of claims 1 to 5, wherein: The centrifugal conditions are 2-8°C, 200-300×g for 3-6 hours.

7. The method for modifying biosynthetic gas vesicles using polyethylene glycol according to any one of claims 1 to 6, wherein: The step of collecting the upper floating matter also includes a washing step; Preferably, the washing method comprises: The upper floating material is mixed with a buffer solution, centrifuged, and the upper floating material is collected. This process is repeated 3 to 4 times.

8. The method for modifying biosynthetic gas vesicles using polyethylene glycol according to any one of claims 1 to 7, wherein: The microorganisms include halophilic archaea and / or cyanobacteria.

9. The method for modifying biosynthetic gas vesicles using polyethylene glycol according to any one of claims 1 to 8, wherein: The method comprises: N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, polyethylene glycol and buffer are mixed to obtain a mixed solution, and the mixed solution after the first incubation is mixed with gas vesicles and incubated for a second time, wherein the feeding ratio of the polyethylene glycol to the gas vesicles is 30-40 mg polyethylene glycol: 1 mL OD 500 The gas vesicles are 3.0, and the secondary incubation is followed by centrifugation to collect the upper floating matter; the upper floating matter is mixed with a buffer solution, and the mixture is centrifuged to collect the upper floating matter, and the process is repeated 3 to 4 times to obtain the gas vesicles modified with polyethylene glycol.

10. A polyethylene glycol-modified gas vesicle, prepared by the method for synthesizing gas vesicles by using polyethylene glycol to modify organisms as described in any one of claims 1 to 9.

11. The method for synthesizing gas vesicles by using polyethylene glycol to modify organisms as described in any one of claims 1 to 9 or the use of the gas vesicles modified with polyethylene glycol as described in claim 10 in the preparation of ultrasound contrast agents.

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