Method for modifying biosynthetic gas vesicles with polyethylene glycol and uses thereof

Abstract

The application discloses a method for modifying biosynthetic gas vesicles by polyethylene glycol and application thereof. The method comprises the following steps: mixing a crosslinking agent, polyethylene glycol and a buffer to obtain a mixed solution, carrying out primary incubation, mixing the mixed solution with gas vesicles, carrying out secondary incubation, carrying out centrifugation after the secondary incubation, and collecting upper floating matter, so as to obtain polyethylene glycol modified gas vesicles, wherein the feeding ratio of the polyethylene glycol to the gas vesicles is 30-40 mg polyethylene glycol: 1 mL OD 500 3.0, and the gas vesicles are synthesized by microorganisms. It is found that different amounts of PEG can result in different metabolic times of GVs in animals, and good gas vesicles can be obtained without using excessive PEG for modification, thereby providing a new idea for developing an efficient and economical modification method.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for modifying biosynthetic gas vesicles with polyethylene glycol and its application. Background Technology

[0002] Due to their small particle size, nanobubbles can penetrate blood vessels and enter surrounding tissues, thereby improving ultrasound imaging effectiveness and time. There are also reports of nanobubbles serving as an effective tool for drug and gene delivery. However, chemically synthesized nanobubbles consist of a phospholipid or polymer shell and a gaseous core (such as inert gases like perfluoropropane or perfluorocarbon). Therefore, the stability of chemically synthesized nanobubbles is easily affected by pH, ambient temperature, and ionic strength. Changes in these factors can lead to bubble rupture and gas leakage, resulting in non-uniform nanobubble size and reduced ultrasound imaging effectiveness.

[0003] In recent years, Shapiro et al. have discovered genetically encoded nanoscale gas vesicles (GVs) in bacteria and archaea. GVs possess a protein shell, primarily composed of GvpA and GvpC proteins. Hydrophobic GvpA forms a spindle-shaped backbone, while hydrophilic GvpCs are arranged in the outer structure of the protein shell. Studies have shown that GVs from *Halobacter* NRC-1 bacteria have a particle size of 45-250 nm and a length of 100-300 nm, and exhibit excellent contrast imaging performance using clinical diagnostic ultrasound equipment under optimized parameters. However, because the GV shell is composed of proteins, it is easily phagocytosed by macrophages of the reticuloendothelial system (RES), potentially leading to immunogenicity or side effects, thus limiting their future clinical application.

[0004] Existing research indicates that PEG surface modification significantly reduces the absorption of nanoparticles by RES and prolongs the circulation time of nanoparticles in vivo. Simultaneously, PEG surface modification can also shield the surface antigens of nanoparticles, reducing the occurrence of immune responses. Current techniques typically employ excessive polyethylene glycol for modification to obtain PEG-modified gas vesicles (GVs). However, due to the complexity of the gas vesicle structure and its metabolism in animals, the specific effects of PEG modification on gas vesicle metabolism remain unclear, thus lacking an efficient and economical modification method.

[0005] In conclusion, exploring the effects 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. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a method for modifying biosynthetic gas vesicles with polyethylene glycol and its application. This invention analyzes in depth whether different amounts of PEG linkage at the same molecular weight affect the metabolic time of gas vesicles in animals, aiming to develop an efficient and economical method for modifying biosynthetic gas vesicles with polyethylene glycol.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0009] A crosslinking agent, polyethylene glycol, and buffer solution are mixed to obtain a mixture, which is then incubated once. This mixture is then mixed with gas vesicles and incubated a second time. After the second incubation, the mixture is centrifuged, and the supernatant is collected to obtain polyethylene glycol-modified gas vesicles. The polyethylene glycol to gas vesicle feeding ratio is 30–40 mg polyethylene glycol (including but not limited to 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, or 39 mg): 1 mL OD 500 The gas vesicles are 3.0 in size and are synthesized by microorganisms.

[0010] This invention provides the first in-depth analysis of the effect of different PEG linkage amounts on the metabolism of gas vesicles in animals at the same molecular weight. It finds that modifying different amounts of PEG can lead to different metabolic times of GVs in animals, and breaks with conventional knowledge that high-performance gas vesicles can be obtained without using large amounts of excessive PEG for modification, providing a new idea for developing efficient and economical modification methods.

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

[0012] Optionally, the feeding 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 the present invention and will not be 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 may include PBS buffer and / or MES buffer.

[0015] Optionally, the temperature of the incubation is 20-30°C (e.g., 21°C, 22°C, 23°C, 24°C, 25°C, 28°C, or 29°C), and the time is 10-30 min (e.g., 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 (e.g., 21°C, 22°C, 23°C, 24°C, 25°C, 28°C, or 29°C), and the time is 8-12 hours (e.g., 9 hours, 10 hours, or 11 hours).

[0017] Optionally, the primary incubation and the secondary incubation are each carried out independently under oscillation conditions, wherein the oscillation speed is 50 to 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 for 3-6 hours.

[0019] Optionally, the process of collecting the upper floating matter further includes a washing step.

[0020] Optionally, the washing method includes:

[0021] Mix the upper floating matter with buffer solution, centrifuge, collect the upper floating matter, and repeat 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 suitable for the present invention, and the specific extraction method may be as follows:

[0024] (1) Bacterial culture

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

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

[0027] c. Place the bacterial suspension in a constant temperature shaker. After inoculation, observe the state of the bacterial suspension every day until the inoculated culture is completely fused and the culture medium changes from dark pink to bright pink.

[0028] (2) Extraction of gas vesicles

[0029] a. Slowly transfer the bacterial solution obtained in the previous part to the separatory funnel and let it stand until the Halo bacteria containing gas vesicles float to the upper layer of the culture medium until a light pink ring visible to the naked eye is formed on the top layer of the culture medium.

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

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

[0032] d. After centrifugation, the liquid in the centrifuge tube will be divided into two layers; the upper layer is pinkish-white, which is the successfully extracted gas vesicles and some Halo bacteria that have not yet been lysed, and the lower layer is pink, which is the lysed Halo bacteria. Use a syringe to slowly aspirate the lower pink solution, leaving only the upper pinkish-white solution in the centrifuge tube.

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

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

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

[0036] N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, polyethylene glycol, and buffer solution were mixed to obtain a mixture, which was then incubated once at a polyethylene glycol to gas vesicle feeding ratio of 30-40 mg polyethylene glycol: 1 mL OD 500 The gas vesicles with a density of 3.0 were mixed with the mixture and incubated a second time. After the second incubation, the mixture was centrifuged and the upper floating matter was collected. The upper floating matter was mixed with the buffer solution, centrifuged, and the upper floating matter was collected. This process was repeated 3 to 4 times to obtain polyethylene glycol modified gas vesicles.

[0037] In a second aspect, the present invention provides a polyethylene glycol-modified gas vesicle, which is prepared by the method of modifying biosynthetic gas vesicles with polyethylene glycol as described in the first aspect.

[0038] Thirdly, the present invention provides the method for modifying biosynthetic gas vesicles with polyethylene glycol as described in the first aspect or the application of polyethylene glycol-modified gas vesicles as described in the second aspect in the preparation of ultrasound contrast agents.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This invention provides the first in-depth analysis of the effect of different PEG linkage amounts on the metabolism of gas vesicles in animals at the same molecular weight. It finds that different amounts of PEG modification can lead to different metabolic times of GVs in animals, and breaks with conventional knowledge by showing that high-performance gas vesicles can be obtained without using excessive PEG modification, providing a new approach for developing efficient and economical modification methods. Attached Figure Description

[0041] Figure 1 A graph showing the connection amount between PEG and GVs;

[0042] Figure 2 The figure shows the metabolic time of GVs modified with different amounts of PEG in mouse liver. Detailed Implementation

[0043] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] In a specific embodiment of the present invention, the gas vesicles used are derived from the halophilic archaea *Halobacterium salinarum* (ATCC 33170), and the extraction method includes:

[0046] (1) Bacterial culture

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

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

[0049] c. Place the bacterial suspension in a constant temperature shaker. After inoculation, observe the state of the bacterial suspension every day until the inoculated culture is completely fused and the culture medium changes from dark pink to bright pink.

[0050] (2) Extraction of gas vesicles

[0051] a. Slowly transfer the bacterial solution obtained in the previous part to the separatory funnel and let it stand until the Halo bacteria containing gas vesicles float to the upper layer of the culture medium until a light pink ring visible to the naked eye is formed on the top layer of the culture medium.

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

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

[0054] d. After centrifugation, the liquid in the centrifuge tube will be divided into two layers; the upper layer is pinkish-white, which is the successfully extracted gas vesicles and some Halo bacteria that have not yet been lysed, and the lower layer is pink, which is the lysed Halo bacteria. Use a syringe to slowly aspirate the lower pink solution, leaving only the upper pinkish-white solution in the centrifuge tube.

[0055] e. Resuspend the powdery white solution in an appropriate amount of PBS, and centrifuge at 4℃ and 300g for 4 hours;

[0056] f. Repeat steps d and e until the lower layer solution is transparent. Then stop the centrifugation process, slowly aspirate the lower transparent solution with a syringe, and resuspend the upper white solution with an appropriate amount of PBS to obtain the purified gas vesicles. Aliquot the gas vesicle solution into 1.5mL 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 dissolve 3.15 mg of NHS (Sigma-Aldrich, 6066-82-6) in 1 mL of PBS;

[0060] (2) Then weigh 35 mg PEG (Melopeg, 020106) and add it to the mixture in step (1). Place it on a 70 rpm rolling shaker and incubate at 25°C for 20 min.

[0061] (3) Add 1 mL of OD to the mixture from step (2). 500 The gas vesicles are 3.0 in size;

[0062] (4) Place the mixture from step (3) on a 70 rpm rotating shaker and incubate overnight at 4°C.

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

[0064] (6) The upper floating matter was resuspended in PBS buffer and centrifuged at low speed for 4 hours (250×g, 4℃). This was repeated 3 times. The precipitate was stored in PBS buffer at 4℃ for later 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 dissolve 3.6 mg of NHS in 1 mL of PBS;

[0068] (2) Then weigh 40 mg of PEG and add it to the mixture in step (1), place it on a 50 rpm rolling shaker, and incubate at 25°C for 20 min;

[0069] (3) Add 1 mL of OD to the mixture from step (2). 500 GVs of 3.0;

[0070] (4) Place the mixture from step (3) on a 50 rpm rotating shaker and incubate overnight at 4°C.

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

[0072] (6) The upper floating matter was resuspended in PBS buffer and centrifuged at low speed for 5 h (300×g, 5℃). This was repeated 3 times. The precipitate was stored in PBS buffer at 4℃ for later 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 dissolve 2.7 mg of NHS in 1 mL of PBS;

[0076] (2) Then weigh 30 mg of PEG and add it to the mixture in step (1), place it on a 100 rpm rolling shaker, and incubate at 20°C for 30 min;

[0077] (3) Add 1 mL of OD to the mixture from step (2). 500 GVs of 3.0;

[0078] (4) Place the mixture from step (3) on a 100 rpm rotating shaker and incubate overnight at 4°C.

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

[0080] (6) The upper floating matter was resuspended in PBS buffer and centrifuged at low speed for 5 h (300×g, 5℃). This was repeated 3 times. The precipitate was stored in PBS buffer at 4℃ for later 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 dissolve 1.8 mg of NHS in 1 mL of PBS;

[0084] (2) Then weigh 20 mg of PEG and add it to the mixture in step (1), place it on a 70 rpm rolling shaker, and incubate at 25°C for 20 min;

[0085] (3) Add 1 mL of OD to the mixture from step (2). 500 GVs of 3.0;

[0086] (4) Place the mixture from step (3) on a 70 rpm rotating shaker and incubate overnight at 4°C.

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

[0088] (6) The upper floating matter was resuspended in PBS buffer and centrifuged at low speed for 4 hours (250×g, 4℃). This was repeated 3 times. The precipitate was stored in PBS buffer at 4℃ for later 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 dissolve 0.9 mg of NHS in 1 mL of PBS;

[0092] (2) Then weigh 10 mg of PEG and add it to the mixture in step (1), place it on a 70 rpm rolling shaker, and incubate at 25°C for 20 min;

[0093] (3) Add 1 mL of OD to the mixture from step (2). 500GVs of 3.0;

[0094] (4) Place the mixture from step (3) on a 70 rpm rotating shaker and incubate overnight at 4°C.

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

[0096] (6) The upper floating matter was resuspended in PBS buffer and centrifuged at low speed for 4 hours (250×g, 4℃). This was repeated 4 times. The precipitate was stored in PBS buffer at 4℃ for later 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 dissolve 0.45 mg of NHS in 1 mL of PBS;

[0100] (2) Then weigh 5 mg of PEG and add it to the mixture in step (1), place it on a 70 rpm rolling shaker, and incubate at 25°C for 20 min;

[0101] (3) Add 1 mL of OD to the mixture from step (2). 500 GVs of 3.0;

[0102] (4) Place the mixture from step (3) on a 70 rpm rotating shaker and incubate overnight at 4°C.

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

[0104] (6) The upper floating matter was resuspended in PBS buffer and centrifuged at low speed for 4 hours (250×g, 4℃). This was repeated 4 times. The precipitate was stored in PBS buffer at 4℃ for later 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 dissolve 4.5 mg of NHS in 1 mL of PBS;

[0108] (2) Then weigh 50 mg of PEG and add it to the mixture in step (1), place it on a 70 rpm rolling shaker, and incubate at 25°C for 20 min;

[0109] (3) Add 1 mL of OD to the mixture from step (2). 500 GVs of 3.0;

[0110] (4) Place the mixture from step (3) on a 70 rpm rotating shaker and incubate overnight at 4°C.

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

[0112] (6) The upper floating matter was resuspended in PBS buffer and centrifuged at low speed for 4 hours (250×g, 4℃). This was repeated 4 times. The precipitate was stored in PBS buffer at 4℃ for later use.

[0113] Test case

[0114] This test case analyzes the PEG linkage amount and metabolic duration in animals of the polyethylene glycol-modified gas vesicles prepared in Examples 1-3 and Comparative Examples 1-4.

[0115] PEG linkage analysis

[0116] For quantification, fluorescently labeled PEG was conjugated to gas vesicles. The prepared polyethylene glycol-modified gas vesicles were collected by centrifugation, and the supernatant was diluted with PBS to OD. 500 The value was 3.0; 200 μL of the above solution was added to a 96-well plate, and the fluorescence was detected by an ELISA reader to plot the connection amount between PEG and gas vesicles.

[0117] The results are as follows Figure 1 As shown, 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] Analysis of metabolic duration in animals

[0119] The prepared polyethylene glycol-modified gas vesicles were collected by centrifugation, and the supernatant was diluted with PBS to OD200. 500 The value 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 Provincial Center for Medical Laboratory Animals) via the tail vein. Ultrasound molecular imaging of the mouse liver was performed using an ultrasound diagnostic system (Resona 7, Mindray, China). The ultrasound images were acquired and the imaging time was recorded.

[0120] The results are as follows Figure 2 As shown, the metabolic time of Examples 1-3 was significantly longer than that of the polyethylene glycol-modified gas vesicles prepared in Comparative Examples 1-3, indicating that controlling the feeding ratio of polyethylene glycol to gas vesicles to be 30-40 mg polyethylene glycol: 1 mL OD500 The 3.0 g / mL gas vesicle significantly increased the metabolic time of the gas vesicles in mouse liver. Furthermore, combined with the results of Comparative Example 4, this invention found that the modification of gas vesicles does not require the use of excessive amounts of polyethylene glycol as previously understood; simply controlling the polyethylene glycol to gas vesicle feeding ratio to low-speed centrifugation for 4 hours (250×g, 4℃) and 30–40 mg polyethylene glycol: 1 mL OD 500 A gas vesicle with a size of 3.0 can be used to obtain a high-performance gas vesicle, which can significantly reduce costs and simplify the process.

[0121] In summary, this invention provides the first in-depth analysis of the effect of different PEG linkage amounts on the metabolism of gas vesicles in animals at the same molecular weight. It finds that modifying different amounts of PEG can lead to different metabolic times of GVs in animals, and breaks with conventional understanding by showing that high-performance gas vesicles can be obtained without using excessive amounts of PEG for modification. This provides a new approach for developing efficient and economical modification methods.

[0122] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for modifying biosynthetic gas vesicles with polyethylene glycol, characterized in that, The method includes: N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, polyethylene glycol, and buffer solution were mixed to obtain a mixture, which was then incubated once at a polyethylene glycol to gas vesicle feeding ratio of 30-40 mg polyethylene glycol: 1 mL OD 500 The mixture of the gas vesicles with a density of 3.0 was mixed with the gas vesicles and incubated twice. After the second incubation, the mixture was centrifuged and the supernatant was collected. The supernatant was mixed with the buffer solution, centrifuged, and the supernatant was collected. This process was repeated 3 to 4 times to obtain polyethylene glycol modified gas vesicles. The gas vesicles are synthesized by microorganisms; The molecular weight of the polyethylene glycol is 2KD~5KD; The centrifugation conditions are 2~8℃, 200~300×g for 3~6 h; The temperature for each incubation is 20~30℃, and the time is 10~30 min; The secondary incubation was carried out at a temperature of 4°C for 8–12 hours. The first and second incubations are each carried out independently under oscillation conditions, with the oscillation speed being 50~100 rpm; The microorganisms include halophilic archaea.

2. The method for modifying biosynthetic gas vesicles with polyethylene glycol according to claim 1, characterized in that, The buffers include PBS buffer and / or MES buffer.

3. The method for modifying biosynthetic gas vesicles with polyethylene glycol according to claim 1, characterized in that, The process of collecting the floating debris from the upper layer also includes a washing step.

4. The method for modifying biosynthetic gas vesicles with polyethylene glycol according to claim 3, wherein the washing method comprises: Mix the upper floating matter with buffer solution, centrifuge, collect the upper floating matter, and repeat 3-4 times.

5. A polyethylene glycol-modified gas vesicle, characterized in that, The gas vesicles are prepared by the method of modifying biosynthetic gas vesicles with polyethylene glycol as described in any one of claims 1-4.

6. The method of modifying biosynthetic gas vesicles with polyethylene glycol according to any one of claims 1-4, or the application of the polyethylene glycol-modified gas vesicles according to claim 5 in the preparation of ultrasound contrast agents.

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