Polyethylene glycol-modified gas vesicle, preparation method therefor, and use thereof

Abstract

Provided are a polyethylene glycol (PEG)-modified gas vesicle, a preparation method therefor, and use thereof. The PEG-modified gas vesicle comprises a gas vesicle synthesized by a halophilic archaeon Halobacterium NRC-1 and PEG linked to the surface thereof. The PEG is covalently linked to the gas vesicle synthesized by the halophilic archaeon Halobacterium NRC-1, thereby successfully preparing an ultrasound contrast agent for vascular wall imaging, which has a good ultrasound imaging effect, high biosafety, and a small particle size. The nano-scale novel ultrasound contrast agent can enter the interior of atherosclerotic plaques by means of neovessels within the plaques, which provides a basis for connecting a targeted molecular probe to accurately judge the risk degree of the plaques and for targeted drug delivery therapy.

Description

A polyethylene glycol-modified gas vesicle and its preparation method and application Technical Field

[0001] The present application belongs to the field of biotechnology and relates to a polyethylene glycol-modified gas vesicle and a preparation method and application thereof, and in particular to the application of the polyethylene glycol-modified gas vesicle in the preparation of an ultrasound contrast agent for vascular wall imaging. Background Art

[0002] Ultrasound molecular imaging technology has been widely used in the diagnosis of tumors, organ damage, and cardiovascular diseases, with contrast agents playing a key role in diagnostic accuracy. Vascular ultrasound imaging can improve the accuracy of detecting vascular pathological changes, including atherosclerotic plaques, intima-media thickness, and the detection of vasa vasorum. Taking atherosclerotic plaques as an example, atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall. In the early stages of AS, inflammatory and lipid deposition lead to lipoprotein infiltration into the intima, endothelial cell dysfunction, and thickening of the intima-media. These lipoproteins are then taken up by macrophages to form lipid-laden foam cells, forming atherosclerotic plaques. As the disease progresses, in advanced AS, foam cells tend to die and release their contents into the extracellular space, exacerbating the inflammatory state and plaque structural stability. This can lead to rupture of unstable plaques, subsequent thrombosis, further vascular stenosis, and the development of acute cardiovascular and cerebrovascular diseases. Using ultrasound contrast agents for vascular wall imaging has the potential to improve the diagnostic efficiency of atherosclerotic plaques at an early stage.

[0003] At present, the micron-level ultrasound contrast agents that have been used in clinical commercialization are mainly chemically synthesized SonoVue, Snonazoid, etc. When these micron-level ultrasound contrast agents are used for angiography examinations, due to their blood pool imaging characteristics, the contrast signal is prone to overflow and mask early vascular lesions, missing the opportunity for early prevention and treatment.

[0004] In summary, the development of contrast agents that can image the blood vessel wall while avoiding signal spillover during imaging of the micron-scale contrast agent blood pool is of great significance for the diagnosis and treatment of vascular diseases.

[0005] Summary of the Invention

[0006] The present application provides a polyethylene glycol-modified gas vesicle, a preparation method and application thereof, in order to develop an ultrasound contrast agent capable of imaging the blood vessel wall.

[0007] In a first aspect, the present application provides a polyethylene glycol-modified gas vesicle, wherein the polyethylene glycol-modified gas vesicle comprises a gas vesicle synthesized by the halophilic archaeon Halobacterium NRC-1 and polyethylene glycol connected to the surface thereof.

[0008] The present application discovered that gas vesicles (GVs) with a particle size of approximately 200 nm isolated from the halophilic archaeon Halobacterium NRC-1 can be used for angiographic imaging, and the angiographic signal is concentrated on the vascular wall rather than in the blood pool. This phenomenon effectively compensates for the deficiency of inaccurate diagnosis of vascular wall lesions caused by signal spillover during existing micron-level contrast agent blood pool imaging, and provides the possibility for accurate diagnosis of vascular wall lesions such as atherosclerotic plaques and arterial dissections. In addition, modification of the gas vesicles with polyethylene glycol can reduce clearance in the body, significantly prolong the imaging efficiency, and extend the imaging time in the blood vessels.

[0009] Preferably, the ratio of the gas vesicles synthesized by the halophilic archaeon Halobacterium NRC-1 and polyethylene glycol in the polyethylene glycol-modified gas vesicles is 500-600 mg polyethylene glycol: 1 mL OD 500 =3.5 gas vesicles.

[0010] Preferably, the average molecular weight of the polyethylene glycol is 4000-6000, including but not limited to 4100, 4200, 4500, 4800, 5000, 5200, 5500, 5600, 5800 or 5900, preferably 4800-5200, more preferably 4900-5100.

[0011] In the present application, modification with polyethylene glycol of a specific average molecular weight can further improve the imaging effect.

[0012] Preferably, the linking method includes covalent linking.

[0013] In a second aspect, the present application provides a method for preparing the polyethylene glycol-modified gas vesicles described in the first aspect, the preparation method comprising:

[0014] A cross-linking agent and gas vesicles synthesized by the halophilic archaeon Halobacterium NRC-1 are mixed to obtain a mixed solution, which is then incubated once; polyethylene glycol is mixed with a buffer solution to obtain a polyethylene glycol solution, and the mixed solution is mixed with the polyethylene glycol solution and incubated twice; after the second incubation, the mixture is centrifuged and the upper white suspension is collected to obtain polyethylene glycol-modified gas vesicles.

[0015] Preferably, the feeding ratio of polyethylene glycol to the gas vesicles synthesized by halophilic archaeon Halobacterium NRC-1 is 500-600 mg polyethylene glycol: 1 mL OD 500 It is a gas vesicle of 3.5.

[0016] Preferably, the cross-linking agent includes N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0017] Preferably, the addition ratio of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and polyethylene glycol is (3-5) mg: (5-7) mg: 1 mL OD 500 It is a gas vesicle of 3.5.

[0018] Preferably, the buffer comprises PBS buffer and / or MES buffer.

[0019] Preferably, the temperature of the primary incubation is 20-30°C, including but not limited to 21°C, 22°C, 23°C, 24°C, 25°C, 28°C or 29°C; the time is 1-3 hours, including but not limited to 1.2 hours, 1.5 hours, 1.8 hours, 2 hours, 2.2 hours, 2.5 hours, 2.8 hours or 2.9 hours;

[0020] Preferably, the temperature of the secondary incubation is 2-6°C, including but not limited to 2.2°C, 2.5°C, 2.8°C, 3°C, 3.2°C, 3.5°C, 4°C, 4.5°C, 4.8°C, 5°C, 5.5°C or 5.8°C; the time is 8-12h, including but not limited to 8.2h, 8.5h, 8.8h, 9h, 9.2h, 9.5h, 10h, 10.5h, 11h, 11.5h or 11.8h.

[0021] Preferably, the method for preparing the gas vesicles synthesized by the halophilic archaeon Halobacterium NRC-1 comprises:

[0022] Halobacterium NRC-1 is cultured, Halobacterium NRC-1 is collected, mixed with a cell lysate, and centrifuged to collect gas vesicles. The gas vesicles are washed with a buffer and collected by centrifugation.

[0023] Preferably, the method for preparing the gas vesicles synthesized by the halophilic archaeon Halobacterium NRC-1 specifically comprises:

[0024] (1) inoculating Halobacterium NRC-1 into a culture medium and culturing the culture solution. The culture solution is placed in a separatory funnel and allowed to stand until Halobacterium NRC-1 floats to the surface of the liquid. The lower layer of culture solution is removed to isolate Halobacterium NRC-1.

[0025] (2) The separated Halobacterium NRC-1 bacteria were mixed with the cell lysate, and the mixture was centrifuged and repeated 2 to 4 times to separate the gas vesicles. The gas vesicles were then washed with PBS and centrifuged and repeated 2 to 4 times to obtain the final gas vesicles.

[0026] In a third aspect, the present application provides the use of the polyethylene glycol-modified gas vesicles described in the first aspect in preparing a product for vascular wall imaging.

[0027] In this application, we discovered that gas vesicles (GVs) with a particle size of approximately 200 nm, isolated from the halophilic archaeon Halobacterium NRC-1, can be used for angiographic imaging. The imaging signal is concentrated on the vessel wall, rather than in the blood pool. This allows for the development of ultrasound contrast agents for vascular wall imaging. For example, this could improve the diagnosis of atherosclerotic plaques at an early stage and help control disease progression. Through targeted modification and nano-drug delivery technologies, this could also open up possibilities for the diagnosis and even treatment of atherosclerotic plaques.

[0028] Compared to contrast agents currently used in clinical diagnostics, GVs offer advantages for vascular wall imaging and are highly compatible with vascular lesions. Polyethylene glycol-modified gas vesicles (PEG-GVs) can effectively reduce the immunogenicity of GVs and prolong their circulation time in vivo, improving their ability to evade immune clearance and stability for in vivo vascular imaging. This application describes the preparation of a novel ultrasound contrast agent with the goal of future application in the diagnosis of vascular lesions.

[0029] In a fourth aspect, the present application provides an ultrasound contrast agent for vascular wall imaging, wherein the ultrasound contrast agent comprises the polyethylene glycol-modified gas vesicles described in the first aspect.

[0030] Compared with the prior art, this application has the following beneficial effects:

[0031] The present application discovered that gas vesicles (GVs) with a particle size of approximately 200 nm, isolated from the halophilic archaeon Halobacterium NRC-1, can be used for angiographic imaging, with the angiographic signal concentrated on the vessel wall rather than in the blood pool. This phenomenon effectively compensates for the inaccurate diagnosis of vascular wall lesions caused by signal spillover during existing micron-sized contrast agent blood pool imaging, and can be used for the early diagnosis of vascular wall lesions (such as atherosclerotic plaques).

[0032] (2) This application is the first to combine the gas vesicles synthesized by the halophilic archaeon Halobacterium NRC-1 with PEG for vascular wall imaging, thereby extending the circulation and metabolism time and compensating for the lack of immunogenicity of the contrast agent;

[0033] (3) Compared with the currently commercialized micron-sized contrast agents, the novel ultrasound contrast agent for vascular wall imaging provided in this application has the advantages of small particle size, strong stability, low preparation difficulty and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG1 is a graph showing the particle size of contrast agent GVs and PEG-linked contrast agent PEG-GVs.

[0035] FIG2 is a potential diagram of contrast agent GVs and PEG-GVs connected with PEG.

[0036] FIG3 is an in vitro ultrasound imaging diagram of contrast agent GVs and PEG-GVs connected with PEG.

[0037] FIG4 is an in vivo ultrasound image of the left carotid artery of a normal rat using the clinical contrast agent SonoVue, contrast agent GVs, and PEG-linked contrast agent PEG-GVs.

[0038] FIG5 is an in vivo ultrasound imaging of the left carotid artery of a rat with plaques of the clinical contrast agent SonoVue, contrast agent GVs, and PEG-GVs linked to PEG. DETAILED DESCRIPTION

[0039] 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.

[0040] 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 available through regular channels.

[0041] Example 1

[0042] In this example, polyethylene glycol-modified gas vesicles were prepared.

[0043] Halobacterium NRC-1 (Halo) was added to ATCC culture medium and cultured in a shaker at 37°C at 220 rpm for 8 days. The culture medium was then placed in a separatory funnel and allowed to stand until Halo floated to the surface of the liquid. The lower culture medium was removed to separate the Halo bacteria. The separated Halo bacteria were added to an equal volume of TMC lysis buffer and centrifuged at 300g for 4 hours, repeated 3 times, to separate gas bubbles (GVs) from the Halo bacteria. The GVs were then washed with PBS and centrifuged at 250g for 4 hours, repeated 3 times to obtain white GVs, which were stored in a 4°C refrigerator for future use.

[0044] EDC (4 mg) and NHS (6 mg) were dissolved in 1 mL OD 500 =3.5 in GVs activated at 25°C for 2 h. In addition, 523 mg of PEG-5000 was dissolved in PBS to obtain a PEG solution. The activated GVs were slowly added to the PEG solution and incubated at 4°C for 10 h. The mixture was then centrifuged four times at 250 g to remove free PEG, EDC and NHS to obtain polyethylene glycol-modified gas vesicles (PEG-GVs).

[0045] Indocyanine green ICG-NHS (30 μg / mL) was added to the GVs solution or PEG-GVs, incubated at 25° C. for 2 h, and then centrifuged three times at 250 g to obtain the ICG-labeled GVs solution or PEG-GVs solution.

[0046] Example 2

[0047] In this example, polyethylene glycol-modified gas vesicles were prepared.

[0048] Halobacterium NRC-1 (Halo) was added to ATCC culture medium and cultured in a shaker at 37°C at 220 rpm for 7 days. The culture medium was then placed in a separatory funnel and allowed to stand until Halo floated to the surface of the liquid. The lower culture medium was removed to separate the Halo bacteria. The separated Halo bacteria were added to an equal volume of TMC lysis buffer and centrifuged at 300g for 4 hours, repeated 4 times, to separate gas bubbles (GVs) from the Halo bacteria. The GVs were then washed with PBS and centrifuged at 250g for 4 hours, repeated 4 times to obtain white GVs, which were stored in a 4°C refrigerator for future use.

[0049] EDC (3 mg) and NHS (7 mg) were dissolved in 1 mL OD 500 =3.5 in GVs activated at 20°C for 2 h. In addition, 600 mg of PEG-4000 was dissolved in PBS to obtain a PEG solution. The activated GVs were slowly added to the PEG solution and incubated at 6°C for 8 h. Subsequently, the mixture was centrifuged four times at 250 g to remove free PEG, EDC and NHS to obtain polyethylene glycol-modified gas vesicles (PEG-GVs).

[0050] Indocyanine green ICG-NHS (30 μg / mL) was added to the GVs solution or PEG-GVs, incubated at 25° C. for 2 h, and then centrifuged three times at 250 g to obtain the ICG-labeled GVs solution or PEG-GVs solution.

[0051] Example 3

[0052] In this example, polyethylene glycol-modified gas vesicles were prepared.

[0053] Halobacterium NRC-1 (Halo) was added to ATCC culture medium and cultured in a shaker at 37°C at 220 rpm for 8 days. The culture medium was then placed in a separatory funnel and allowed to stand until Halo floated to the surface of the liquid. The lower culture medium was removed to separate the Halo bacteria. The separated Halo bacteria were added to an equal volume of TMC lysis buffer and centrifuged at 300g for 4 hours, repeated 3 times, to separate gas bubbles (GVs) from the Halo bacteria. The GVs were then washed with PBS and centrifuged at 250g for 4 hours, repeated 3 times to obtain white GVs, which were stored in a 4°C refrigerator for future use.

[0054] EDC (5 mg) and NHS (5 mg) were dissolved in 1 mL OD 500 3.5 GVs were activated at 30°C for 2 h. In addition, 500 mg of PEG-6000 was dissolved in PBS to obtain a PEG solution. The activated GVs were slowly added to the PEG solution and incubated at 2°C for 12 h. Subsequently, the mixture was centrifuged four times at 250g to remove free PEG, EDC and NHS to obtain polyethylene glycol-modified gas vesicles (PEG-GVs).

[0055] Indocyanine green ICG-NHS (30 μg / mL) was added to the GVs solution or PEG-GVs, incubated at 25° C. for 2 h, and then centrifuged three times at 250 g to obtain the ICG-labeled GVs solution or PEG-GVs solution.

[0056] Test Case

[0057] This test example tests the polyethylene glycol-modified gas vesicles prepared in each example.

[0058] Transmission electron microscope (TEM) observation of GVsHE PEG-GVs showed that they were monodisperse, had a rugby-shaped structure, and were more regular in morphology and uniform in size. 1 mL of contrast agent solution was placed in a special dish of a laser particle size analyzer to detect its particle size, distribution, and zeta potential. Taking the product prepared in Example as an example, the results are shown in Figures 1 and 2. Zetasizer measured that its particle size distribution was relatively uniform, about 217.45 ± 4.65 nm, and the potential was about -26.70 ± 1.80 mV. It shows that the contrast agent particle size of the present application is small and has good stability. The PEG-GVs particle size is about 272.65 ± 8.95 nm, and the potential is about -1.67 ± 0.13 mV.

[0059] Evaluation of in vitro ultrasound imaging: Using PBS as a control, different concentrations (OD 500GVs (1.0-2.0) or PEG-GVs (prepared in Example 1) were placed in a 1% agarose model well. A Mindray Reson 7 linear array probe (3-11 MHz) was used on one side of the agarose model for imaging using ultrasound contrast imaging. This generated a strong and stable contrast ultrasound signal. The ultrasound signal became stronger as the concentration increased (see Figure 3).

[0060] Evaluation of in vivo ultrasound imaging: 300 μL of SonoVue (manufacturer: BRACCO) prepared with 5 mL of 0.9% w / v sodium chloride solution and 300 μL of OD 500 For GVs and PEG-GVs (prepared in Example 1) with a GV density of 3.5, video of the imaging was captured for more than half an hour after the complete disappearance of the contrast contrast signal until the ultrasound contrast signal disappeared. Subsequently, Image J software was used to quantitatively analyze the vascular wall, such as peak time, peak intensity, and metabolic time. It was found that the SonoVue imaging method was blood pool imaging, while the new ultrasound contrast agent GVs imaging method was vascular wall imaging. Compared with PEG-GVs, the new ultrasound contrast agent GVs had a longer circulation and metabolic time in the vascular wall (see Figure 4). Healthy male SD rats were fed a high-fat diet for one week and then underwent left carotid artery endothelial injury via a balloon catheter. After the surgery, the rats were fed a high-fat diet until an atherosclerotic plaque model was formed. In vivo imaging was performed to observe the accumulation of contrast agent in the plaque. It was found that due to the overflow of SonoVue, the plaque location could not be accurately identified. However, GVs were able to accurately identify the plaque due to their imaging characteristics of the vessel wall. In addition, GVs modified with PEG had a longer circulation time in the plaque (see Figure 5).

[0061] In summary, this application successfully prepared PEG-GVs by covalently linking PEG to gas vesicles synthesized by the halophilic archaeon Halobacterium NRC-1. PEG-GVs have good ultrasound imaging effects, high biosafety, and small particle size. This new nanoscale ultrasound contrast agent can enter the interior of atherosclerotic plaques through the new blood vessels in the plaques, which provides a basis for connecting targeted molecular probes to accurately judge the risk level of plaques and for targeted drug delivery therapy.

[0062] 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 polyethylene glycol-modified gas vesicle, comprising a gas vesicle synthesized by the halophilic archaeon Halobacterium NRC-1 and polyethylene glycol attached to the surface of the gas vesicle.

2. The polyethylene glycol-modified gas vesicle according to claim 1, wherein The average molecular weight of the polyethylene glycol is 4000-6000.

3. A method for preparing polyethylene glycol-modified gas vesicles according to claim 1 or 2, wherein: The preparation method comprises: A cross-linking agent and gas vesicles synthesized by the halophilic archaeon Halobacterium NRC-1 are mixed to obtain a mixed solution, which is then incubated once; polyethylene glycol is mixed with a buffer solution to obtain a polyethylene glycol solution, and the mixed solution is mixed with the polyethylene glycol solution and incubated twice; after the second incubation, the mixture is centrifuged and the upper white suspension is collected to obtain polyethylene glycol-modified gas vesicles.

4. The method for preparing polyethylene glycol-modified gas vesicles according to claim 3, wherein: The feeding ratio of polyethylene glycol to the gas vesicles synthesized by halophilic archaeon Halobacterium NRC-1 is 500-600 mg polyethylene glycol: 1 mL OD 500 It is a gas vesicle of 3.

5.

5. The method for preparing polyethylene glycol-modified gas vesicles according to claim 3 or 4, wherein: The cross-linking agents include N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

6. The method for preparing polyethylene glycol-modified gas vesicles according to claim 5, wherein: The addition ratio of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and polyethylene glycol is (3-5) mg: (5-7) mg: 1 mL OD 500 It is a gas vesicle of 3.

5.

7. The method for preparing polyethylene glycol-modified gas vesicles according to any one of claims 3 to 6, wherein: The buffer comprises PBS buffer and / or MES buffer.

8. The method for preparing polyethylene glycol-modified gas vesicles according to any one of claims 3 to 7, wherein: The temperature of the first incubation is 20-30°C and the time is 1-3 hours; Preferably, the secondary incubation temperature is 2-6° C. and the time is 8-12 h.

9. Use of the polyethylene glycol-modified gas vesicles according to claim 1 or 2 in preparing a product for vascular wall imaging.

10. An ultrasound contrast agent for vascular wall imaging, comprising the polyethylene glycol-modified gas vesicles according to claim 1 or 2.

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