Polyethylene glycol modified gas vesicles, methods of making and using the same

Abstract

The application discloses a polyethylene glycol (PEG) modified gas vesicle and a preparation method and application thereof. The PEG modified gas vesicle comprises a gas vesicle synthesized by halophilic archaea Halobacterium NRC-1 and PEG connected to the surface of the gas vesicle. The PEG is covalently connected to the gas vesicle synthesized by halophilic archaea Halobacterium NRC-1, and an ultrasonic contrast agent for blood vessel wall imaging is successfully prepared, the ultrasonic contrast agent has good ultrasonic imaging effect, high biological safety and small particle size, and the novel nanoscale ultrasonic contrast agent can enter the inside of an atherosclerotic plaque through neovessels in the plaque, which provides a basis for connecting a targeted molecular probe to accurately judge the dangerous degree of the plaque and to perform targeted drug delivery treatment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a polyethylene glycol-modified gas vesicle, its preparation method and application, and particularly to the application of polyethylene glycol-modified gas vesicles in the preparation of ultrasound contrast agents for vascular wall imaging. Background Technology

[0002] Ultrasound molecular imaging technology has been widely used in the diagnosis of tumors, organ damage, and cardiovascular diseases, with contrast agents playing a crucial role in diagnostic accuracy. Vascular ultrasound contrast imaging can increase the accuracy of detecting vascular pathological changes, including atherosclerotic plaques, intima-media thickness, and the detection of nourishing vessels in the vessel wall. Taking atherosclerotic plaques as an example, atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall. In the early stages of AS, influenced by inflammation and lipid deposition, lipoproteins infiltrate the intima, leading to endothelial cell dysfunction and intima-media thickening. Macrophages take up lipids to form foam cells, resulting in atherosclerotic plaques. As the disease progresses, in late-stage AS, foam cells are prone to death, releasing their contents into the extracellular space, thus worsening the inflammatory state and plaque structural stability. Unstable plaques rupture, leading to secondary thrombosis, further narrowing of the blood vessel, and triggering acute cardiovascular and cerebrovascular diseases. Utilizing ultrasound contrast agents for vascular wall imaging holds promise for improving the diagnostic efficiency of atherosclerotic plaques in their early stages.

[0003] Currently, the micron-sized ultrasound contrast agents that have been commercially available in clinical settings are mainly chemically synthesized agents such as SonoVue and Snonazoid. When these micron-sized ultrasound contrast agents are used in angiography, due to the characteristics of their blood pool imaging, the contrast signal is prone to overflow, masking early vascular lesions and missing the opportunity for early prevention and treatment.

[0004] In conclusion, developing contrast agents capable of imaging blood vessel walls while avoiding signal spillover during micron-level contrast agent blood pool imaging is of great significance for the diagnosis and treatment of vascular diseases. Summary of the Invention

[0005] In view of the shortcomings of existing technologies and practical needs, this invention provides a polyethylene glycol-modified gas vesicle, its preparation method and application, with the aim of developing an ultrasound contrast agent capable of imaging blood vessel walls.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a polyethylene glycol-modified gas vesicle, the polyethylene glycol-modified gas vesicle comprising a gas vesicle synthesized by the halophilic archaea Halobacterium NRC-1 and polyethylene glycol attached to its surface.

[0008] This invention discovers that gas vesicles (GVs) with a particle size of approximately 200 nm isolated from the halophilic archaea Halobacterium NRC-1 can be used for angiography imaging, and the angiographic signal is concentrated on the vessel wall rather than in the blood pool. This phenomenon effectively compensates for the inaccuracy in the diagnosis of vascular wall lesions caused by signal extravasation during blood pool imaging with micron-sized contrast agents. It provides the possibility for the accurate diagnosis of vascular wall lesions such as atherosclerotic plaques and arterial dissections. In addition, modifying the gas vesicles with polyethylene glycol can reduce in vivo clearance and significantly prolong the imaging efficiency and imaging time in blood vessels.

[0009] Preferably, the ratio of gas vesicles synthesized by the halophilic archaea Halobacterium NRC-1 to polyethylene glycol in the polyethylene glycol-modified gas vesicle 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, and more preferably 4900-5100.

[0011] In this invention, modification with polyethylene glycol of a specific average molecular weight can further improve imaging performance.

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

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

[0014] The cross-linking agent and gaseous vesicles synthesized from the halophilic archaea Halobacterium NRC-1 were mixed to obtain a mixture, which was then incubated once. Polyethylene glycol was mixed with a buffer solution to obtain a polyethylene glycol solution. The mixture was then mixed with the polyethylene glycol solution and incubated a second time. After the second incubation, the mixture was centrifuged and the upper white suspension was collected to obtain the polyethylene glycol-modified gaseous vesicles.

[0015] Preferably, the feeding ratio of polyethylene glycol to gas vesicles synthesized from the halophilic archaea Halobacterium NRC-1 is 500-600 mg polyethylene glycol: 1 mL OD. 500 The gas vesicles are 3.5.

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

[0017] Preferably, the feeding 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 The gas vesicles are 3.5.

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

[0019] Preferably, 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 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℃ (e.g., 2.2℃, 2.5℃, 2.8℃, 3℃, 3.2℃, 3.5℃, 4℃, 4.5℃, 4.8℃, 5℃, 5.5℃, or 5.8℃), and the time is 8-12 hours, including but not limited to 8.2 hours, 8.5 hours, 8.8 hours, 9 hours, 9.2 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, or 11.8 hours.

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

[0022] Halobacteria NRC-1 was cultured, and the Halobacteria NRC-1 was collected and mixed with cell lysate. The mixture was centrifuged to collect gas vesicles. The gas vesicles were washed with buffer and collected by centrifugation.

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

[0024] (1) Inoculate Halobacteria NRC-1 into the culture medium and culture it. Place the culture medium in a separatory funnel and let it stand until Halobacteria NRC-1 floats on the surface of the liquid. Remove the lower layer of culture medium and isolate Halobacteria NRC-1.

[0025] (2) Mix the isolated Halobacteria NRC-1 bacteria with cell lysis buffer, centrifuge, repeat centrifugation 2 to 4 times to separate gas vesicles, then wash the gas vesicles with PBS, centrifuge, repeat centrifugation 2 to 4 times to obtain the final gas vesicles.

[0026] Thirdly, the present invention provides the application of the polyethylene glycol-modified gas vesicles described in the first aspect in the preparation of products for vascular wall imaging.

[0027] In this invention, it was discovered that gas vesicles (GVs) with a particle size of approximately 200 nm isolated from the halophilic archaea Halobacterium NRC-1 can be used for angiography imaging. Furthermore, the angiographic signal is concentrated on the vessel wall, rather than in the blood pool. This allows for the development of ultrasound contrast agents for vessel wall imaging, potentially improving the diagnostic efficiency of atherosclerotic plaques in their early stages and enabling timely control of disease progression. Targeted modification and nano-drug delivery technologies also offer possibilities for the diagnosis and even treatment of atherosclerotic plaques.

[0028] Compared to contrast agents currently used in clinical diagnostics, gas vesicles (GVs) offer advantages such as high adaptability to vascular wall imaging and efficient adaptation to vascular lesions. Polyethylene glycol-modified gas vesicles (PEG-GVs) effectively reduce the immunogenicity of GVs and prolong their in vivo circulation time, enhancing their ability to evade immune clearance and improving their stability for in vivo vascular imaging. This invention prepares a novel ultrasound contrast agent with the aim of its future application in the diagnosis of vascular lesions.

[0029] Fourthly, the present invention provides an ultrasound contrast agent for imaging blood vessel walls, the ultrasound contrast agent comprising the polyethylene glycol-modified gas vesicles described in the first aspect.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention discovers that gas vesicles (GVs) with a particle size of approximately 200 nm isolated from the halophilic archaea Halobacterium NRC-1 can be used for angiography imaging, and the angiography signal is concentrated on the vessel wall rather than in the blood pool. This phenomenon effectively compensates for the shortcomings of existing micron-sized contrast agents in blood pool imaging, which cause signal spillover and inaccurate diagnosis of vascular wall lesions. It can be used for the early diagnosis of vascular wall lesions (such as atherosclerotic plaques).

[0032] (2) This invention is the first to combine gas vesicles synthesized by the halophilic archaea Halobacterium NRC-1 with PEG and apply them to vascular wall imaging, thereby prolonging the circulation and metabolism time and compensating for the lack of immunogenicity of contrast agents.

[0033] (3) The novel ultrasound contrast agent for imaging blood vessel walls provided by this invention has advantages such as small particle size, strong stability, low preparation difficulty and low cost compared with the currently commercialized micron-sized contrast agents. Attached Figure Description

[0034] Figure 1 Particle size distribution of contrast agent GVs and PEG-GVs linked to PEG;

[0035] Figure 2 Potential diagrams of contrast agent GVs and PEG-GVs linked to PEG;

[0036] Figure 3 In vitro ultrasound images of contrast agents GVs and PEG-GVs linked to PEG;

[0037] Figure 4 In vivo ultrasound images of the left carotid artery of a normal rat with clinical contrast agent SonoVue, contrast agent GVs, and PEG-GVs linked to PEG.

[0038] Figure 5 In vivo ultrasound imaging of the left carotid artery of rats with plaques containing clinical contrast agent SonoVue, contrast agent GVs, and PEG-GVs linked to PEG. Detailed Implementation

[0039] 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.

[0040] 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 obtainable through legitimate channels.

[0041] Example 1

[0042] This embodiment prepares polyethylene glycol-modified gas vesicles.

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

[0044] Dissolve EDC (4 mg) and NHS (6 mg) in 1 mL OD 500 3.5% GVs were activated at 25°C for 2 hours. Separately, 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 hours. Then, the mixture was centrifuged 4 times at 250 g to remove free PEG, EDC, and NHS, resulting in polyethylene glycol-modified gas vesicles (PEG-GVs).

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

[0046] Example 2

[0047] This embodiment prepares polyethylene glycol-modified gas vesicles.

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

[0049] Dissolve EDC (3 mg) and NHS (7 mg) in 1 mL OD 5003.5% GVs were activated at 20°C for 2 hours. Separately, 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 hours. Then, the mixture was centrifuged 4 times at 250 g to remove free PEG, EDC, and NHS, yielding polyethylene glycol-modified gas vesicles (PEG-GVs).

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

[0051] Example 3

[0052] This embodiment prepares polyethylene glycol-modified gas vesicles.

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

[0054] Dissolve 5 mg of EDC and 5 mg of NHS in 1 mL of OD 500 3.5% GVs were activated at 30°C for 2 hours. Separately, 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 hours. Then, the mixture was centrifuged 4 times at 250 g to remove free PEG, EDC, and NHS, yielding polyethylene glycol-modified gas vesicles (PEG-GVs).

[0055] Add indocyanine green (ICG-NHS) (30 μg / mL) to the GVs solution or PEG-GVs, incubate at 25°C for 2 h, and then centrifuge 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 embodiment.

[0058] Transmission electron microscopy (TEM) revealed that GVsHE PEG-GVs were monodisperse, exhibiting rugby ball-shaped structures with relatively regular morphology and uniform size. 1 mL of the contrast agent solution was placed in a special dish for a laser particle size analyzer to detect its particle size, distribution, and zeta potential. Taking the product prepared in the example as an example, the results are as follows... Figure 1 and Figure 2 As shown, Zetasizer measurements indicate that the particle size distribution is relatively uniform, approximately 217.45 ± 4.65 nm, and the potential is approximately -26.70 ± 1.80 mV. This demonstrates that the contrast agent of the present invention has a small particle size and good stability; the PEG-GVs particle size is approximately 272.65 ± 8.95 nm, and the potential is approximately -1.67 ± 0.13 mV.

[0059] Evaluation of in vitro ultrasound imaging: Using PBS as a control, different concentrations (OD) were added... 500 GVs or PEG-GVs (prepared in Example 1) at concentrations of 1.0-2.0% were placed in wells of a 1% agarose model. A linear array probe (3-11 MHz) of a Mindray Reson 7 clinical ultrasound diagnostic device was placed on one side of the agarose model, and imaging was performed in ultrasound contrast imaging mode, producing a strong and stable contrast ultrasound signal. The ultrasound signal became stronger with increasing concentration (see...). Figure 3 ).

[0060] Evaluation of in vivo ultrasound imaging: Healthy male SD rats were injected via the tail vein with 300 μL of SonoVue (manufacturer: BRACCO) prepared from 5 mL of 0.9% w / v sodium chloride solution and 300 μL of LOD. 500 For GVs and PEG-GVs (prepared in Example 1) with a concentration of 3.5, video imaging was performed simultaneously at least half an hour after the contrast signal completely disappeared, until the ultrasound contrast signal disappeared. Subsequently, ImageJ software was used to perform quantitative analysis of the vessel wall, such as peak time, peak intensity, and metabolic time. It was found that SonoVue imaging mode is blood pool imaging, while the novel ultrasound contrast agent GVs imaging mode is vessel wall imaging. Compared with PEG-GVs, the novel ultrasound contrast agent GVs has a longer circulation and metabolic time in the vessel wall (see...). Figure 4 Healthy male SD rats underwent left carotid artery endothelial injury via balloon catheter after a week of high-fat feeding. Following this, they were fed a high-fat diet until an atherosclerotic plaque model was established, and in vivo imaging was performed to observe contrast agent accumulation at the plaque site. It was found that due to the overflow phenomenon of SonoVue, the plaque location could not be accurately identified, while GVs, due to their vessel wall imaging characteristics, could accurately identify the plaque. Furthermore, PEG-modified GVs exhibited a longer circulation time within the plaque (see...). Figure 5 ).

[0061] In summary, this invention successfully prepared PEG-GVs by covalently linking PEG to gas vesicles synthesized from the halophilic archaea Halobacterium NRC-1. These PEG-GVs exhibit good ultrasound imaging performance, high biocompatibility, and small particle size. This novel nanoscale ultrasound contrast agent can penetrate the atherosclerotic plaque through neovascularization, providing a foundation for accurately assessing plaque risk levels and delivering targeted drugs via targeted molecular probes.

[0062] 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. The application of a polyethylene glycol-modified gas vesicle in the preparation of products for vascular wall imaging, characterized in that, The polyethylene glycol-modified gas vesicles include gas vesicles synthesized by the halophilic archaea Halobacterium NRC-1 and polyethylene glycol attached to their surface; The feeding ratio of the gas vesicles synthesized from polyethylene glycol and the halophilic archaea Halobacterium NRC-1 is 500-600 mg polyethylene glycol : 1 mL OD 500 The gas vesicles are 3.5 cm in size; The method for preparing the polyethylene glycol-modified gas vesicles includes: The cross-linking agent and gas vesicles synthesized by the halophilic archaea Halobacterium NRC-1 were mixed to obtain a mixture, which was then incubated once. Polyethylene glycol was mixed with a buffer solution to obtain a polyethylene glycol solution. The mixture was then mixed with the polyethylene glycol solution and incubated a second time. After the second incubation, the mixture was centrifuged and the upper white suspension was collected to obtain polyethylene glycol-modified gas vesicles. The crosslinking agent includes N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; The feeding ratio of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and polyethylene glycol is (3~5) mg:(5~7) mg:(500~600) mg; The average molecular weight of the polyethylene glycol is 4000~6000.

2. The application according to claim 1, characterized in that, The buffers include PBS buffer and / or MES buffer.

3. The application according to claim 1, characterized in that, The incubation temperature is 20~30℃ and the time is 1~3 h.

4. The application according to claim 1, characterized in that, The secondary incubation is carried out at a temperature of 2-6°C for 8-12 hours.

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