A gasbag protein complex and its use in tumor imaging

Abstract

The present application relates to a kind of gasbag protein complex, comprising: gasbag protein;And dye molecule;The dye molecule is coupled to gasbag protein by cationic polymer.This application gasbag protein complex as imaging preparation can be good repeatability, stable performance, can be used as multiple tumor dual-mode imaging;And preparation method is simple, can be mass-produced in short time, with the characteristics of low cost, high efficiency.

Description

Technical Field

[0001] This invention relates to the medical field, and in particular to an air sac protein complex and its application in tumor imaging. Background Technology

[0002] Gliomas are characterized by their difficulty in detection, high morbidity, high recurrence rate, and high disability rate, making them a serious threat to human life and quality of life worldwide. Data from clinical surveys in my country shows that gliomas account for 45% of all brain tumor cases. Because gliomas lack a clear boundary with normal brain tissue, complete resection is difficult or even impossible, resulting in a relatively limited range of treatment options and an annual death toll of 30,000. Therefore, early detection of gliomas is crucial and can effectively improve patient survival rates. Currently, various diagnostic agents are available for the early detection of gliomas; however, due to the presence of the blood-brain barrier or intercellular spaces, traditional agents still suffer from poor stability and low resolution in clinical diagnosis and evaluation. Therefore, designing and developing novel, multifunctional glioma imaging contrast agents is essential for the efficient diagnosis of gliomas.

[0003] As a low-cost, convenient, and rapid imaging method, ultrasound imaging is often used in conjunction with other imaging modalities as part of bimodal imaging, providing a non-invasive and relatively inexpensive way to generate images of internal organs and tissues. Importantly, ultrasound imaging, as a commonly used complementary imaging modality in bimodal imaging, can provide real-time and highly detailed images of soft tissues and organs, and has been widely used in clinical practice. For example, in obstetrics and gynecology, ultrasound imaging is an important tool for displaying the uterus, ovaries, and other reproductive organs, and when combined with other imaging modalities such as CT and MRI scans, it can provide more complete images of the reproductive system and its function. In cardiology, ultrasound imaging is widely used in the diagnosis and treatment of cardiovascular diseases. When combined with other imaging modalities, it can provide more complete images of the heart and blood vessels, including structure, function, and blood flow. In oncology, ultrasound imaging is also often combined with other imaging modalities, such as NIRF and fluorescence imaging, to improve the accuracy of cancer diagnosis and treatment planning. For example, ultrasound-guided observation of tumors in the body can provide real-time images, helping doctors accurately locate specific areas, while fluorescence imaging modality can provide more detailed images of the tissues and organs surrounding the tumor. In summary, ultrasound imaging plays a vital role in bimodal imaging by providing real-time images of soft tissues and organs, enabling a more complete and accurate understanding of the patient's condition, which is crucial for accurate diagnosis and the development of detailed treatment plans.

[0004] Gas vesicles (GVs) are intracellular structures found in many different cyanobacteria and archaea. They are hollow protein clusters that help some algal cells and archaea regulate buoyancy and position in aquatic environments. In recent years, there has been increasing interest in the potential applications of GVs across various fields, with one research area focusing on their use as contrast agents in medical imaging. Due to their morphological characteristics, GVs can scatter and reflect ultrasound waves, thus possessing the potential to be used as ultrasound contrast agents. However, because GVs undergo cavitation under high ultrasound or pressure, causing their structure to collapse, researchers have been working to optimize the production and modification of gas vesicles to improve their imaging performance and make them more effective for clinical use. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention proposes a method for preparing an air sac protein complex or complex system, comprising: obtaining extracts of *Microcystis aeruginosa*, aquatic bacteria, or archaea; adding a long-chain cationic polymer to an air sac protein suspension and shaking for 1.5-3 hours; adding cyanine dye molecules and shaking for 14-20 hours; wherein the addition ratio of the *Microcystis aeruginosa*, aquatic bacteria, or archaea extract to the cyanine dye molecules is (0.5-3.0 OD500): (3-30 μg / mL); the addition ratio of the *Microcystis aeruginosa*, aquatic bacteria, or archaea extract to the cationic polymer and cyanine dye molecules is (0.5-3.0 OD500). 500 ): (1.5-30 μg / mL): (3-30 μg / mL); and to obtain air sac protein complexes or complex systems.

[0006] The preparation method described above, wherein the method for obtaining the extract of Microcystis aeruginosa is an osmotic shock method, further comprising: obtaining Microcystis aeruginosa; adding 30% hydrogen peroxide, wherein the volume ratio of hydrogen peroxide to Microcystis aeruginosa is 1:10; shaking culture under light at 20-28°C for 2-4 hours; obtaining the supernatant after low-speed centrifugation, mixing it with PBS; dialyzing using a 2.5-4 kDa dialysis bag for 3-6 hours; and obtaining the extract of Microcystis aeruginosa.

[0007] The preparation method described above includes air sac proteins in the extracts of Microcystis aeruginosa, aquatic bacteria, or archaea.

[0008] In the preparation method described above, the ratio of the extract of Microcystis aeruginosa, aquatic bacteria, or archaea to the cationic polymer and cyanine dye molecules is 2OD. 500 7.5 μg / mL: 15 μg / mL.

[0009] In the preparation method described above, the long-chain cationic polymer is a cationic polymer; preferably, the cationic polymer is selected from: polyethyleneimine and polylysine.

[0010] As described in preparation method 1 above, the cyanine dye molecules are selected from CY3, CY5, and CY7.

[0011] A composite system comprising: extracts of Microcystis aeruginosa, aquatic bacteria or archaea; long-chain cationic polymers; and cyanine dye molecules.

[0012] An air sac protein complex includes: an air sac protein; and a dye molecule; said dye molecule being coupled to the air sac protein via a long-chain cationic polymer.

[0013] In the preparation method described above, the air sac protein is derived from Microcystis aeruginosa, aquatic bacteria, or archaea.

[0014] The air sac protein complex prepared by any of the above preparation methods, or the air sac protein complex system as described above, or the dual-modal imaging formulation of the air sac protein complex as described above; wherein the dual modality is an ultrasound modality and a fluorescence modality.

[0015] Application of air sac protein complexes prepared by any of the preparation methods described above, or air sac protein complex systems as described above, or air sac protein complexes as described above, or dual-modal imaging agents as described above, in tumor imaging.

[0016] In the application described above, the tumor is selected from: brain tumors, subcutaneous tumors; preferably, the tumor is a glioma.

[0017] The air sac protein complex of this application has good reproducibility and stable performance as an imaging formulation, and can be used for dual-modal imaging of various tumors; moreover, the preparation method is simple and can be mass-produced in a short time, with the characteristics of low cost and high efficiency. Attached Figure Description

[0018] The preferred embodiments of the present invention will now be described in further detail with reference to the accompanying drawings, wherein:

[0019] Figure 1 A is a GV characterization of a PBS buffer dispersed according to an embodiment of the present invention.

[0020] Figure 1 B is a characterization of GV extracted from Microcystis aeruginosa under a transmission electron microscope (TEM) according to an embodiment of the present invention;

[0021] Figure 2 This is a characterization of the dual-modal imaging probe GV@pCY5 under a transmission electron microscope (TEM) according to an embodiment of the present invention;

[0022] Figure 3 Fluorescence imaging of an in vitro gel model grafted with different concentrations of GV and CY5 according to an embodiment of the present invention;

[0023] Figure 4 This is a comparison of ultrasound output power of GV@pCY5 at different output powers according to an embodiment of the present invention;

[0024] Figure 5 Fluorescence imaging of mouse orthotopic gliomas at different time points according to an embodiment of this application; and

[0025] Figure 6 This is a comparison of ultrasound imaging of mice with gliomas after injection of different nanomaterials, according to one embodiment of this application. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In the following detailed description, reference can be made to the accompanying drawings, which form part of this application and illustrate specific embodiments of the present application. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the present application are described in sufficient detail below to enable those skilled in the art to implement the technical solutions of the present application. It should be understood that other embodiments or modifications to the embodiments of the present application may also be utilized.

[0028] Given that GV will undergo cavitation when exposed to high ultrasound or pressure, causing its own structure to collapse, the inventors of this application have explored the use of GV as a scaffold for constructing nanoparticles and other materials by taking advantage of GV's unique structural properties and ease of modification. They have also studied the potential applications of GV in nanobiotechnology and materials life sciences, and achieved unexpected technical results.

[0029] The definitions of the technical terms used in this article are as follows:

[0030] The term "tumor" as used herein refers to a tumor that can be observed using ultrasound or dual-modal imaging. In some embodiments, the tumor referred to herein may be a brain tumor, a subcutaneous tumor, etc. Further, in a specific embodiment, the tumor is a glioma.

[0031] The “dual-modal imaging” mentioned in this article refers to an imaging modality that simultaneously combines the high-resolution structural imaging of ultrasound with the high-contrast capabilities of fluorescence.

[0032] The term "gas vesicles" used in this article refers to gas vesicles, abbreviated as GV. Gas vesicles (GV) are intracellular structures found in many different cyanobacteria, aquatic bacteria, and archaea. They are hollow protein clusters that help some algal cells and archaea regulate buoyancy and position in aquatic environments. In recent years, there has been increasing interest in the potential applications of GV across various fields, with one research area focusing on its use as a contrast agent in medical imaging. Due to its morphological characteristics, GV can scatter and reflect ultrasound waves, thus possessing the potential to be used as an ultrasound contrast agent.

[0033] This application does not limit the source of the air sac protein. In some embodiments, the air sac protein is derived from archaea, aquatic bacteria, or Microcystis aeruginosa.

[0034] The term "cationic polymer" as used herein refers to a polymer that functions as a complexing agent and dispersant, promoting the linkage between GV and CY5. In some embodiments, the cationic polymer is a long-chain cationic polymer. In some embodiments, the cationic polymer is selected from: polyethyleneimine (hereinafter referred to as PEI), polylysine, etc. Preferably, the cationic polymer is polyethyleneimine.

[0035] The term "dye molecule" as used herein refers to reactive dyes that can be used to label the amino groups of peptides, proteins, and oligonucleotides. In some embodiments, the dye molecule may be a cyanine dye. Cyanine dyes are derivatives of polymethyl cyanide dyes, which are chromophores with intercalation of amidine ions between N and N atoms. They belong to the category of organic dyes. When the two nitrogen atoms and part of the polymethyl cyanide chain are part of a heterocyclic core, a typical cyanine dye is formed. According to the molecular structure type, they are classified into linear chain, bridged chain, cyclobutenedione, and ketone types, etc. Further, in some embodiments, the dye molecule is selected from CY3, CY5, and CY7. Preferably, the dye molecule is CY5.

[0036] Furthermore, the “pCY5” mentioned in this article refers to CY5 coupled with PEI.

[0037] The "GV@pCY5" mentioned in this article refers to a complex formed by grafting CY5 onto the surface of GV via PEI. GV can penetrate the blood-brain barrier to achieve targeted targeting of brain tumors, and PEI enhances its effect.

[0038] This application relates to an air sac protein complex, comprising: an air sac protein; and dye molecules; wherein the dye molecules are coupled to the air sac protein via a cationic polymer. The air sac protein is derived from *Microcystis aeruginosa*; the cationic polymer is a long-chain cationic polymer; preferably, the cationic polymer is selected from the following polymers: polyethyleneimine, polylysine, etc. The dye is selected from cyanine dyes; preferably, the dye molecules are selected from the following molecules: CY3, CY5, CY7.

[0039] This application relates to a composite system comprising: an extract of Microcystis aeruginosa, aquatic bacteria or archaea; a long-chain cationic polymer; and an anthocyanin dye molecule.

[0040] To prepare the air bladder protein complex or complex system, first obtain extracts of air bladder protein or *Microcystis aeruginosa*, aquatic bacteria, or archaea; then add polyethyleneimine to the air bladder protein suspension and shake for approximately 2 hours; finally, add CY5 and shake for approximately 16 hours to obtain the air bladder protein complex or complex system. Further, in the complex or complex system, the ratio of the extract of *Microcystis aeruginosa*, aquatic bacteria, or archaea to cyanine dye molecules is (0.5-3.0 OD). 500 (3-30 μg / mL); or the ratio of extracts of Microcystis aeruginosa, aquatic bacteria, or archaea to the cationic polymer and cyanine dye molecules is (0.5-3.0 OD). 500 ): (1.5-30 μg / mL): (3-30 μg / mL), preferably 2.0 OD: 7.5 μg / mL: 5 μg / mL.

[0041] The method for obtaining the extract of Microcystis aeruginosa is an osmotic shock method, which further includes: obtaining Microcystis aeruginosa; adding 30% hydrogen peroxide, wherein the ratio of hydrogen peroxide to Microcystis aeruginosa is 1:10; shaking culture under light at 20-28℃ for 2-4 hours; obtaining the supernatant after low-speed centrifugation, mixing it with PBS; dialyzing using a 2.5-4 kDa dialysis bag for 3-6 hours; and obtaining the extract of Microcystis aeruginosa.

[0042] In some embodiments, this application also relates to a dual-modal imaging formulation comprising an air sac protein complex prepared as described in this application, or an air sac whitening complex or complex system as described in this application. The dual-modality comprises an ultrasonic modality and a fluorescence modality.

[0043] The air sac protein complex of this application can be used in tumor imaging, particularly tumors that can be imaged using ultrasound / fluorescence dual-modality imaging. Furthermore, the air sac protein complex of this application can be applied to the imaging of brain tumors, etc. Furthermore, in some embodiments, it can be used for the imaging of gliomas.

[0044] This application will illustrate the technical solution through the following embodiments:

[0045] Example 1: Isolation and purification of GV from Microcystis aeruginosa

[0046] According to one embodiment of this application, *Microcystis aeruginosa* cultured for 15 days was used to separate and purify its volatile organic compounds (GV). GV was obtained using an osmotic shock method. *Microcystis aeruginosa* FACHB-2329 grown in the logarithmic phase was added with 30% hydrogen peroxide at a volume ratio of 10:1. The reactants were placed in a 25°C light incubator and shaken at 70 rpm for 3 hours to allow for complete reaction. After the reaction, a refrigerated centrifuge was set to 400 g / min and 4°C for 4 hours. White floating matter appeared on the top layer of the centrifuge tube. This white floating matter was mixed with PBS at a 1:1 ratio and centrifuged again at low speed for 4 hours. This process was repeated multiple times for purification. The purified GV was then dialyzed at 4°C for 4 hours using a 3 kDa dialysis bag. Finally, the extracted GV was stored at 4°C.

[0047] like Figure 1 As shown in Figure A, the GVs purified by repeated low-speed centrifugation were dispersed in PBS buffer, forming a white emulsion that floated on top after prolonged standing. The concentration of GVs was adjusted to OD0.05. 500 =0.5, added dropwise to a 300-mesh copper grid, dried, and then negatively stained with a 2% phosphotungstic acid solution before observation under a transmission electron microscope. For example... Figure 1 The TEM image of B shows that the extracted GVs are rod-shaped, uniform in size, with a diameter of about 200 nm and a length of about 600 nm.

[0048] Example 2: Construction method of dual-modal imaging probe GV@pCY5

[0049] According to one embodiment of this application, 0.0025 g of PEI was weighed and dissolved in 10 mL of deionized water to a concentration of 250 μg / mL for later use (PEI must be freshly prepared). The CY5 solution was set to 2.5 mg / mL, and the GV concentration per milliliter was set to approximately OD. 500 =2. When preparing a PEI concentration of 7.5 μg / mL, add 30 μL of PEI solution to 1 mL of GV solution, vortex for 2 hours, then add 15 μg of CY5 solution, and continue vortexing for 16 hours to obtain GV@pCY5, which is then stored at 4℃. Figure 1 A comparison of transmission electron microscopy (TEM) images of GV in section B clearly shows that the surface of GV grafted with PEI and CY5 has changed from smooth and transparent to rougher, with granular material appearing on the surface. This difference from the TEM images of GV directly demonstrates that CY5 has been successfully grafted onto the surface of GV via PEI.

[0050] Example 3: Validation of the in vitro imaging function of the dual-modal imaging probe GV@pCY5

[0051] According to one embodiment of this application, a 1cm deep 1% agarose gel is cast into a square double-sided gel casting frame as the imaging base. After solidification, a 0.8cm diameter circular pipette is vertically inserted into the gel to remove excess cylindrical gel pieces. Multiple regular small holes are then formed in sequence based on the number of samples. Different concentrations of GV samples are mixed 1:1 with 1% agarose at approximately 55°C in a molten state, and 300μL of the mixture is quickly poured into the simulated cylindrical holes. GV@pCY5 sample gel models are prepared using the same method. After all samples are added and solidified, 1% agarose is slowly poured in to seal the model holes, ensuring a smooth and bubble-free top. PBS, different concentrations of GV, GV@pCY5 samples, and 1% agarose are mixed in a 1:1 ratio and slowly injected into the gel holes. Using a Mindray Resona 9 ultrasound imaging system, different output powers and different pseudo-color patterns (gray pseudo-color and wheat yellow pseudo-color) are adjusted to perform ultrasound imaging of the gel model. In addition, PBS, different concentrations of GV and different concentrations of GV@pCY5 samples were mixed with 1% agarose in a 1:1 ratio and slowly injected into the gel wells to perform fluorescence imaging on the gel model.

[0052] like Figure 3 As shown, when the GV concentration is 3OD 500 At that time, the fluorescence intensity was 20.38 × 10⁻⁶. 9 When the GV concentration is 2OD, the fluorescence intensity is 18.26 × 10⁻⁶. 9 When the GV concentration was 1.5 OD, the fluorescence intensity was 18.28 × 10⁻⁶. 9 When the GV concentration is 1 OD, the fluorescence intensity is 14.92 × 10⁻⁶. 9 When the GV concentration is 0.5 OD, the fluorescence intensity is 17.10 × 10⁻⁶. 9 There were no significant differences between the groups; therefore, the concentration of GV had little effect on the fluorescence intensity of CY5.

[0053] like Figure 4 As shown, when the ultrasound power is 46%, the overall intensity of the image reaches approximately 4.62 × 10⁻⁶. 5 When the ultrasound power is 55%, the overall intensity of the image reaches approximately 6.14 × 10⁻⁶. 5 When the ultrasound power is 67%, the overall intensity of the image reaches approximately 2.53 × 10⁻⁶. 5 When the ultrasound power is 89%, the overall intensity of the image reaches approximately 3.22 × 10⁻⁶. 5Therefore, when the ultrasound power is 55%, the resulting image has the highest overall intensity and can relatively maintain the stability of GV@pCY5 without producing GV cavitation.

[0054] Example 4: In vivo imaging function verification of the dual-modal imaging probe GV@pCY5

[0055] According to one embodiment of this application, mice were anesthetized by intraperitoneal injection of 70-100 μL of 1.5% sodium pentobarbital, and 100 μL of 1×PBS solution and 100 μL of 2OD solution were prepared. 500 Three samples were injected via tail vein into mice with constructed brain tumors: GV solution, 100 μL of freshly prepared GV@pCY5 solution, and GV solution. Using a Mindray Resona 9 ultrasound imaging system with parameters adjusted to 7.5 MHz, 55% output power, and mechanical index MI = 1-2, and with a suitable ultrasound probe, ultrasound imaging of the brain tumor implantation sites was performed.

[0056] like Figure 5 As shown, fluorescence appeared in the brain tumor area at the 4-hour time point, and the average fluorescence intensity in the brain of mice treated with GV@pCY5 reached 14.78 × 10⁻⁶. 8 The fluorescence intensity was 2.08 times that of the CY5-treated group at the same time point; at the 6-hour time point, the average fluorescence intensity in the brains of mice in the GV@pCY5-treated group reached 22.90 × 10⁻⁶. 8 It is 1.13 times that of the CY5 treatment group at the same time point.

[0057] like Figure 6 As shown, no obvious signal appeared in the brain of mice injected with PBS; the brain of mice injected with GV showed a blurry signal without precise edges and sparse light spots, the overall tumor imaging was not smooth and the resolution of the tumor tissue structure was low; while the ultrasound images of the brain of mice injected with GV@pCY5 showed a high intensity signal with precise edges and clear and dense light spots, the overall tumor imaging was smooth and the resolution of the tumor tissue structure was high.

[0058] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention.

Claims

1. A method for preparing an air sac protein complex or complex system, comprising: Obtain extracts of Microcystis aeruginosa, aquatic bacteria, or archaea; wherein the extracts of Microcystis aeruginosa, aquatic bacteria, or archaea include air sac proteins; Add long-chain cationic polymers to the air bladder protein suspension and shake for 1.5-3 hours; Add cyanine dye molecules and shake for 14-20 hours; wherein the cyanine dye molecules are selected from: CY3, CY5, CY7; The ratio of the extract of Microcystis aeruginosa, aquatic bacteria, or archaea to the cyanine dye molecules is (0.5-3.0 OD). 500 ): (3-30 μg / mL); the ratio of the extract of Microcystis aeruginosa, aquatic bacteria or archaea to the cationic polymer and anthocyanin dye molecules is (0.5-3.0 OD). 500 ): (1.5-30 μg / mL): (3-30 μg / mL); and Obtain air sac protein complexes or complex systems.

2. The preparation method according to claim 1, wherein the method for obtaining the extract of Microcystis aeruginosa is an osmotic shock method, comprising: Microcystis aeruginosa was obtained; Add 30% hydrogen peroxide, wherein the volume ratio of hydrogen peroxide to Microcystis aeruginosa is 1:10; Incubate with shaking under light at 20-28℃ for 2-4 hours; The supernatant obtained after low-speed centrifugation was mixed with PBS; Dialysis for 3-6 hours using a 2.5-4 kDa dialysis bag; as well as An extract of Microcystis aeruginosa was obtained.

3. The preparation method according to claim 1, wherein, The ratio of the extract of Microcystis aeruginosa, aquatic bacteria, or archaea to the cationic polymer and anthocyanin dye molecules is 2 OD. 500 7.5 μg / mL: 15 μg / mL.

4. The preparation method according to claim 1, wherein the cationic polymer is selected from: polyethyleneimine and polylysine.

5. A composite system comprising: Extracts of Microcystis aeruginosa, aquatic bacteria, or archaea; The extracts of Microcystis aeruginosa, aquatic bacteria, or archaea mentioned above include air sac proteins. Long-chain cationic polymers; and Cyanide dye molecules; wherein the cyanide dye molecules are selected from: CY3, CY5, CY7; The ratio of the extract of *Microcystis aeruginosa*, aquatic bacteria, or archaea to the cyanine dye molecules is (0.5-3.0 OD). 500 ): (3-30 μg / mL); the ratio of the extract of Microcystis aeruginosa, aquatic bacteria or archaea to the cationic polymer and cyanine dye molecules is (0.5-3.0 OD). 500 ): (1.5-30 μg / mL): (3-30 μg / mL); The cyanine dye molecules are coupled to the air sac protein via a long-chain cationic polymer.

6. An air sac protein complex prepared by the preparation method according to any one of claims 1-4, comprising: Air sac protein; as well as Dye molecules; The dye molecules mentioned therein are cyanine dye molecules; the cyanine dye molecules are selected from: CY3, CY5, CY7; The dye molecules are coupled to the air sac protein via a long-chain cationic polymer.

7. The air sac protein complex according to claim 6, wherein the air sac protein is derived from Microcystis aeruginosa, aquatic bacteria, or archaea.

8. A dual-modal imaging formulation comprising an air sac protein complex or air sac protein complex system prepared by any of the preparation methods described in claims 1-4, or a complex system as described in claim 5, or an air sac protein complex as described in claim 6 or 7; wherein the dual modality is an ultrasound modality and a fluorescence modality.

9. The use of the dual-modal imaging formulation according to claim 8 in the preparation of tumor imaging agents.

10. The application according to claim 9, wherein the tumor is selected from: brain tumors, subcutaneous tumors.

11. The application according to claim 9, wherein the tumor is a glioma.

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