A nano-ultrasound contrast agent targeting CD147 and a preparation method and application thereof

Abstract

This invention provides a CD147-targeting nano-ultrasound contrast agent, its preparation method, and its application. The contrast agent comprises: microbial-derived gas vesicles and CD147 nanoantibodies linked to the surface of the gas vesicles via a coupling agent. The hollow nanostructure of the gas vesicles in this invention endows the contrast agent with highly efficient acoustic reflection capabilities, significantly improving the ultrasound signal intensity. The CD147 nanoantibodies possess high affinity and high specificity, enabling precise recognition of the CD147 antigen on the surface of tumor cells. This invention uses biosynthesized gas vesicles instead of traditional chemically synthesized gas microbubbles, combined with a simple and efficient coupling process, significantly reducing preparation difficulty and equipment dependence. The contrast agent of this invention overcomes the shortcomings of existing technologies in imaging performance, targeting, preparation process, safety, and multifunctional diagnostic and therapeutic capabilities, providing a novel technical pathway for precision diagnosis and treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular imaging, specifically relating to a CD147-targeting nano-ultrasound contrast agent, its preparation method, and its application. Background Technology

[0002] In existing molecular imaging techniques, traditional ultrasound contrast agents are mainly made of gas microbubbles encapsulated in materials such as phospholipids and proteins, and are widely used in vascular imaging and disease diagnosis. However, their micron-sized particle size limits their penetration into the intervascular spaces of tumors, making it impossible to achieve efficient targeted imaging of tumor tissue. Meanwhile, while targeted contrast agents (such as antibody-based molecular probes) can enhance imaging specificity by targeting specific antigens, they suffer from problems such as large relative molecular mass, poor tissue permeability, and complex preparation processes.

[0003] Studies targeting CD147 have shown that it is a transmembrane glycoprotein widely expressed on the surface of various tumor cells, possessing biological functions such as inducing matrix metalloproteinase (MMP) production and promoting tumor invasion and metastasis, thus becoming a hot topic in tumor diagnosis and treatment. However, traditional monoclonal antibody-based targeting strategies are limited by its large molecular structure, resulting in difficulties in penetrating the blood-brain barrier and low targeting efficiency.

[0004] The shortcomings of existing technologies include: (1) Particle size limitation: The particle size of existing microbubble contrast agents is mainly in the micrometer range, which makes it difficult to penetrate the gap between tumor blood vessels and results in low imaging efficiency. (2) Insufficient targeting: Ordinary contrast agents cannot specifically bind to tumor antigens, resulting in poor imaging sensitivity. (3) Antibody limitations: Monoclonal antibodies have a large molecular weight, long circulation time, and low permeability, making it difficult to achieve targeted imaging of deep tumor tissues. (4) Complex preparation process: The preparation of traditional contrast agents requires high-precision equipment, and the steps are complex and costly. (5) Safety issues: Some contrast agents may have immunogenicity risks or non-specific binding to normal tissues, affecting clinical application.

[0005] Therefore, providing a targeted ultrasound contrast agent with good imaging performance, strong targeting, safety and high efficiency has important application value in ultrasound molecular imaging and diagnosis of tumor tissues. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a CD147-targeted nano-ultrasound contrast agent, its preparation method, and its applications. This invention combines microbial-derived gas vesicles with CD147 nanobodies to develop an ultrasound molecular imaging and diagnostic tool targeting tumor tissue.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a CD147-targeting nano-ultrasound contrast agent, the contrast agent comprising: gas vesicles derived from microorganisms and CD147 nanoantibodies connected to the surface of the gas vesicles by a coupling agent.

[0009] This invention introduces biosynthesized nanoscale gas bubbles (derived from halophilic archaea, Serratia, or their genetically engineered forms) and CD147 nanobodies with high specificity and high affinity, combined with targeted molecular modification, which can effectively overcome the bottlenecks of existing technologies and achieve precise ultrasound molecular imaging and diagnosis of tumor tissues.

[0010] Preferably, the coupling agent is selected from any one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide (EDC / NHS), maleimide polyethylene glycol active ester or azido polyethylene glycol active ester (N3-PEG-NHS).

[0011] Preferably, the coupling agent is maleimide polyethylene glycol active ester, wherein the N-hydroxysuccinimide ester in the maleimide polyethylene glycol active ester is connected to the gas vesicles; and the maleimide in the maleimide polyethylene glycol active ester is connected to the CD147 nanobody.

[0012] In this invention, the N-hydroxysuccinimide ester in the maleimide polyethylene glycol active ester is connected to gas vesicles. The NHS active ester group can react with amino groups (-NH2) to form stable amide bonds.

[0013] In this invention, the maleimide in the maleimide polyethylene glycol active ester is linked to the CD147 nanobody. The maleimide group can undergo a Michael addition reaction with a thiol group to form a stable thioether bond.

[0014] In this invention, the polyethylene glycol (PEG) chain provides flexibility and water solubility, enhances the water solubility and biocompatibility of the modified molecule, and reduces non-specific adsorption and aggregation.

[0015] In this invention, gaseous vesicles (GVs) serve as carriers, with their surfaces chemically modified to incorporate Mal-PEG-NHS. The CD147 nanobody undergoes a coupling reaction between its thiol groups and the maleimide groups on the Mal-PEG-NHS, forming a stable bond. The coupled CD147-targeting nano-ultrasound contrast agent exhibits uniform particle size, high binding efficiency, and demonstrates good stability and targeting in both in vitro and in vivo experiments.

[0016] In this invention, the working principle of the CD147-targeting nano-ultrasound contrast agent includes: Targeting: The CD147 nanobody can specifically bind to the highly expressed CD147 protein on the surface of tumor cells, enabling the contrast agent to efficiently accumulate at the tumor site. Good imaging performance: The gas vesicles, through their internal gas properties, generate strong echo signals under ultrasound excitation, thereby significantly improving imaging contrast. Therapeutic integration: The hollow structure and surface properties of the gas vesicles can be further loaded with anticancer drugs, achieving integrated diagnosis and treatment.

[0017] Preferably, the molecular weight of polyethylene glycol in the maleimide polyethylene glycol active ester is 1-20 kDa.

[0018] In this invention, the preferred polyethylene glycol has a molecular weight of 2000-5000 Da. The moderate chain length of PEG 2000-5000 results in relatively small steric hindrance, preventing excessive obstruction of the approach between reactive groups and thus ensuring the efficiency of the linkage reaction. In maleimide polyethylene glycol active ester (Mal-PEG-NHS), the chain length of PEG 2000 does not excessively encapsulate or block the maleimide groups or NHS active ester groups, allowing them to react smoothly with thiol or amino groups. Simultaneously, PEG 2000-5000 effectively reduces the non-specific binding of contrast agents to plasma proteins, lowering the likelihood of recognition and clearance by the immune system, thereby prolonging the residence time of the contrast agent in blood circulation and enhancing imaging effects.

[0019] Preferably, the particle size of the gas vesicles is in the range of 100-300 nm.

[0020] Existing ultrasound contrast agents, due to particle size limitations or insufficient imaging signal intensity, struggle to achieve high-contrast imaging of deep tissues. This invention introduces gas vesicles (GVs) as carriers, leveraging their unique nanostructure and acoustic properties to significantly improve the intensity and stability of ultrasound signals while reducing signal attenuation, thus meeting the needs of deep tumor tissue imaging.

[0021] In this invention, the gas vesicles have a particle size of approximately 100-300 nm. This nanoscale size range gives the gas vesicles strong penetrating power, enabling them to pass through the intercellular spaces of vascular endothelial cells and enter the interstitial space, achieving converging imaging within and outside the blood vessels, thus obtaining a finer image contrast. This characteristic allows the gas vesicles to reach the lesion site more effectively, improving imaging resolution and sensitivity. They can also generate strong scattered signals over a wider range of ultrasound frequencies, making them suitable for high-resolution ultrasound imaging. This allows the gas vesicles to exhibit good imaging results under different ultrasound imaging equipment and parameters, enhancing their adaptability in clinical applications.

[0022] Preferably, the CD147 nanobody has a molecular weight of 15-19 kDa.

[0023] Existing contrast agents have limited targeting ability to tumor tissues, and traditional monoclonal antibodies suffer from drawbacks such as large molecular weight and poor permeability. This invention utilizes high-affinity CD147 nanobody to bind to gas vesicles, achieving highly specific recognition of tumor cells that highly express CD147, thereby improving targeting and enabling the contrast agent to more precisely accumulate at the tumor site.

[0024] Preferably, the amino acid sequence of the CD147 nanobody is shown in SEQ ID NO:1.

[0025] The CD147 nanobody screened and prepared by the present invention through phage display technology can bind to the CD147 antigen highly expressed on the surface of tumor cells with high affinity, thereby significantly improving the targeted recognition efficiency.

[0026] Some traditional contrast agents may bind nonspecifically to normal tissues, posing a certain risk of immunogenicity. The CD147 nanobody used in this invention, due to its small molecular weight and low immunogenicity, and through the loading and protective effect of gas vesicles, can significantly reduce interference with normal tissues, further improving safety and meeting clinical requirements.

[0027] In a second aspect, the present invention provides the application of the CD147-targeting nano-ultrasound contrast agent described in the first aspect in the preparation of a drug, wherein the drug is obtained by loading an antitumor drug onto the surface of the CD147-targeting nano-ultrasound contrast agent described in the first aspect.

[0028] In this invention, the surface loading method includes: utilizing the physical properties of the contrast agent surface, such as electrostatic or hydrophobic effects, to adsorb antitumor drugs onto the contrast agent surface.

[0029] Preferably, the antitumor drug is selected from chemotherapy drugs and / or nucleic acid fragments.

[0030] The CD147-targeting nano-ultrasound contrast agent developed in this invention not only improves ultrasound imaging performance but also possesses the potential for integrated diagnosis and treatment. By further modifying the surface of the gas vesicles with anti-tumor drugs (such as chemotherapy drugs or gene fragments), drug delivery can be achieved, providing an integrated solution for the precise diagnosis and treatment of tumors.

[0031] Thirdly, the present invention provides a method for preparing the CD147-targeting nano-ultrasound contrast agent as described in the first aspect, the method comprising:

[0032] (1) The coupling agent is mixed with the gas vesicle solution and reacted, and the free coupling agent is removed by dialysis to obtain the coupling agent modified gas vesicles.

[0033] (2) The CD147 nanobody was reduced; the reduced CD147 nanobody was incubated with a gas vesicle solution modified with a coupling agent to prepare a CD147-targeted nano-ultrasound contrast agent.

[0034] Existing ultrasound contrast agents are complex to prepare, relying on various high-precision equipment, resulting in high processing costs and difficulties in large-scale production. This invention combines biosynthesized gas vesicles with a CD147 nanobody conjugation process, designing a more efficient and economical preparation method. This reduces reliance on high-end equipment, lowers production costs, and improves product reproducibility and stability.

[0035] Preferably, in step (1), the OD of the gas vesicle solution is... 500 It is 2.5-3.0, for example, it can be 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0, etc.

[0036] Preferably, in step (1), the final concentration of the coupling agent in the gas vesicle solution is 5-20 mg / mL, for example, it can be 5 mg / mL, 10 mg / mL, 15 mg / mL or 20 mg / mL, etc.

[0037] Preferably, in step (1), the reaction conditions are 35-37℃ for 3-5 hours, wherein 35-37℃ can be, for example, 35℃, 36℃ or 37℃, and 3-5 hours can be, for example, 3 hours, 4 hours or 5 hours.

[0038] Preferably, in step (2), TCEP is added to the CD147 nanobody solution for reduction.

[0039] Preferably, the final concentration of the CD147 nanobody solution is 3-5 mg / mL, for example, it can be 3 mg / mL, 4 mg / mL or 5 mg / mL, etc.

[0040] Preferably, the final concentration of TCEP in the solution is 1-20 mM, for example, it can be 1 mM, 5 mM, 15 mM or 20 mM.

[0041] Preferably, the reduction conditions are reduction at 20-25℃ for 2-3 hours, wherein 20-25℃ can be, for example, 20℃, 21℃, 22℃, 23℃, 24℃ or 25℃, etc.

[0042] Preferably, in step (2), the OD of the coupling agent-modified gas vesicle solution is... 500 It is 2.5-3.0, for example, it can be 2.5, 2.6, 2.7, 2.8, 2.9 or 3.0, etc.

[0043] Preferably, in step (3), the final concentration of the reduced CD147 nanobody in the coupling agent-modified gas vesicle solution is 50-500 μg / mL, for example, it can be 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL or 500 μg / mL.

[0044] Preferably, in step (3), the incubation conditions are 2-6℃ for 10-20h, where 2-6℃ can be, for example, 2℃, 3℃, 4℃, 5℃ or 6℃, and 10-20h can be, for example, 10h, 15h or 20h.

[0045] Preferably, step (3) further includes a step of removing the free coupling agent and unbound CD147 nanoantibody after incubation.

[0046] The methods for removing free coupling agent and unbound CD147 nanoantibody in this invention include: dialysis or centrifugation at 250-500g.

[0047] Preferably, the preparation method of the CD147-targeting nano-ultrasound contrast agent includes:

[0048] (1) First, mix 5-20 mg of coupling agent with 1 mL of gas vesicle solution (OD). 500 2.5-3.0) react at 35-37℃ for 3-5 hours;

[0049] (2) Dialyze with PBS solution (MWCO: 1000kDa, Solarbio) for 10-24h to remove free coupling agent and obtain coupling agent modified gas vesicles;

[0050] (3) After adjusting the CD147 antibody protein concentration to 3-5 mg / mL, add TCEP to a final concentration of 1-20 mM and reduce at 20-25℃ for 2-3 h.

[0051] (4) Mix 50-500 μg of CD147 nanobody with 1 mL of OD 500 A CD147-targeting nano-ultrasound contrast agent was prepared by incubating a gas vesicle solution modified with a coupling agent of 2.5-3.0 at 2-6°C for 10-20 h.

[0052] In this invention, a maleimide-thiol coupling method was chosen to design contrast agents targeting CD147, rather than the traditional biotin-streptavidin chemical linkage method. This choice was primarily based on the potential immunogenicity issues posed by streptavidin. The maleimide-thiol coupling reaction, with its high specificity and efficiency, provides a superior strategy for the binding of targeting antibodies to the surface of nanobubbles. The covalent bonds formed through this method exhibit good stability both in vitro and in vivo, which is crucial for maintaining the integrity and function of the nanobubbles during storage and drug delivery. Furthermore, polyethylene glycol (PEG) modification, as an effective means of reducing the immunogenicity of biomaterials, has been demonstrated in numerous studies. In this study, we used MAL-PEG2000-NHS for modification, fully utilizing the advantages of PEGylation. This modification not only significantly improves the biocompatibility of GVs but also prolongs their circulation time in vivo, thereby enhancing their performance in in vivo imaging.

[0053] In this invention, the coupling reaction is followed by a step of removing the free coupling agent and unbound CD147 nanoantibody by dialysis or low-speed centrifugation.

[0054] This invention uses Mal-PEG-NHS as a coupling agent to efficiently and stably couple gas vesicles with CD147 nanoantibodies, ensuring the uniformity and binding efficiency of the contrast agent while avoiding damage to active molecules during the coupling process.

[0055] Fourthly, the present invention provides the application of the method for preparing the CD147-targeting nano-ultrasound contrast agent described in the first aspect and / or the CD147-targeting nano-ultrasound contrast agent described in the third aspect, the application including: preparing any one or a combination of at least two of the products for molecular imaging, tumor diagnosis or tumor treatment.

[0056] The CD147-targeting nano-ultrasound contrast agent described in this invention exhibits significantly superior imaging performance and targeting aggregation ability compared to existing technologies in in vitro cell experiments and animal models.

[0057] The contrast agent in this invention can be used for ultrasound imaging in targeted diagnosis of tumors (such as liver cancer or lung cancer), and can also be extended to therapeutic integration, achieving drug delivery by modifying the surface of nanobubbles with chemotherapeutic drugs.

[0058] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] This invention constructs an ultrasound contrast agent with strong targeting, excellent imaging performance, and efficient preparation process by conjugating gas vesicles (GVs) with CD147 nanobodies. The advantages of this ultrasound contrast agent are as follows:

[0061] (1) Significantly improved imaging performance: The hollow nanostructure of the gas vesicles endows the contrast agent with efficient acoustic reflection capabilities, greatly improving the intensity of the ultrasound signal. In in vitro and in vivo experiments, the contrast agent exhibited a longer signal decay time and higher signal contrast, making tumor tissue clearer in ultrasound imaging.

[0062] (2) Strong targeting: CD147 nanobodies have high affinity and high specificity, enabling them to accurately recognize the CD147 antigen on the surface of tumor cells. Animal experimental images show that the CD147-targeting nano-ultrasound contrast agent has significant signal enhancement at the tumor site, forming a sharp contrast with normal tissue.

[0063] (3) Enhanced safety: Nanobodies have small molecular weights and low immunogenicity, reducing non-specific binding and potential immune responses. Gas vesicles are highly stable and derived from biological sources, making them non-toxic and reducing the risk of contrast agents causing side effects on the body.

[0064] (4) Simplified preparation process: Using gas vesicles instead of traditional gas microbubbles, combined with a simple and efficient Mal-PEG-NHS coupling process, significantly reduces the difficulty of preparation and equipment dependence. Compared with traditional contrast agents, the preparation process of this invention is more efficient, with lower production costs and stronger large-scale production capabilities.

[0065] (5) Potential for Therapeutic Integration: The hollow structure and surface properties of gas vesicles can be further loaded with anti-tumor drugs (such as chemotherapy drugs or nucleic acid molecules), endowing contrast agents with therapeutic functions. This contrast agent can not only be used for tumor diagnosis, but also has potential drug delivery capabilities, providing a technological foundation for therapeutic integration.

[0066] (6) Broad application prospects: This invention can be widely used in the precise diagnosis of various tumors (such as liver cancer, lung cancer, or breast cancer), and is particularly suitable for tumors that highly express CD147. Through technical optimization, the contrast agent of this invention can also be used in ultrasound imaging of other diseases such as inflammation and thrombosis, and has good application extensibility. Attached Figure Description

[0067] Figure 1 This is the preparation process of a CD147-targeted nano-ultrasound contrast agent.

[0068] Figure 2 These are the electrophoresis results of the CD147 nanobody.

[0069] Figure 3This is a particle size distribution diagram.

[0070] Figure 4 It is a potential distribution diagram.

[0071] Figure 5 These are in vitro imaging images of targeted and non-targeted vesicles at different concentrations.

[0072] Figure 6 These are the results of adhesion experiments between targeted and non-targeted vesicles and tumor cells (PC-3 cells).

[0073] Figure 7 These are in vivo imaging results in animals (BAL B / C mice) with targeted and non-targeted vesicles. Detailed Implementation

[0074] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0075] 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.

[0076] The sources of experimental materials in the following specific implementation methods are as follows:

[0077] Trace element solution: Prepare an appropriate amount of solution according to the above reagent ratio: 1.32g zinc sulfate heptahydrate, 0.34g manganese sulfate, 0.82g ferrous ammonium sulfate, 0.14g copper sulfate pentahydrate, and 200mL ddH2O. After thorough dissolution by stirring on a magnetic stirrer, filter the solution through a 0.22μm filter in a clean bench for sterilization. Add 100μL of trace elements to every 1.0L LATCC basal medium to prepare ATCC complete medium.

[0078] Halophilic archaea culture medium: Sodium chloride 250.0g, magnesium sulfate heptahydrate 20.0g, trisodium citrate 3.0g, potassium chloride 2.0g, tryptone 5.0g, yeast extract 3.0g, ddH2O 1.0L. Prepare an appropriate amount of solution according to the above reagent ratio, stir thoroughly on a magnetic stirrer to dissolve, then autoclave (121℃, 20min), cool and remove for later use.

[0079] TMC lysis buffer: 2.42 g tris(hydroxymethyl)aminomethane hydrochloride, 1.01 g magnesium chloride, 0.58 g calcium chloride, 2.0 L ddH₂O. Prepare an appropriate amount of solution according to the above reagent ratio, adjust the pH to 7.5, and then filter it through a 0.22 μm filter in a clean bench for sterilization. The TMC lysis buffer can be stored for a long time at room temperature.

[0080] Example 1

[0081] This embodiment provides a method for preparing a CD147-targeted nano-ultrasound contrast agent. The preparation process is as follows: Figure 1 As shown, the preparation method includes:

[0082] 1. The coupling agent is mixed with the gas vesicle solution and reacted. The free coupling agent is then removed by dialysis to obtain the coupling agent-modified gas vesicles.

[0083] 1.1 Preparation of gas vesicles (GVs)

[0084] (1) Cultivation of Halo archaea

[0085] Culture medium preparation and inoculation: Take 100 μL of trace element solution and add it to 1.0 L of Halo halophilic archaea medium to prepare a transparent pale yellow complete culture medium. After thawing the frozen solution at room temperature from a -80℃ freezer, take 10 mL of bacterial solution and add it to an Erlenmeyer flask containing 1.0 L of complete culture medium, shake well, and seal with a breathable membrane.

[0086] Culture process: Place the conical flask in a constant temperature shaker at 37℃ and 200rpm for about 14 days. Observe it every day and you can see the culture medium gradually change from transparent light yellow to dark red, and finally turn pink, indicating that the culture is complete.

[0087] (2) Extraction and purification of GVs

[0088] Static layering treatment: After the Halo halophilic archaea culture is completed, pour the culture medium into a separatory funnel and let it stand at room temperature for about 7 days until a pinkish-white ring layer appears on the upper layer.

[0089] Collect bubbly bacteria: Remove the lower layer of culture medium and transfer the upper layer of about 20 mL of bubbly bacterial solution to a 50 mL centrifuge tube.

[0090] Lysis process: Pour an equal volume of TMC lysis buffer to the collected bubbly bacteria into the separatory funnel, rinse the inside of the funnel thoroughly, and then collect the lysis buffer into a centrifuge tube. Place the centrifuge tube on a horizontal decolorizing shaker and mix thoroughly at 50 rpm overnight.

[0091] Initial centrifugation: Centrifuge at 300g for 4 hours at 4℃. The solution separates into three layers: the upper layer contains most of the lysed GVs and a small amount of bacterial debris; the middle layer is the culture medium solution; and the lower layer contains precipitated culture medium impurities and most of the lysed bacterial fragments. Aspirate the middle and lower layers with a syringe, retaining 8-10 mL of the upper layer of off-white liquid.

[0092] Secondary lysis and centrifugation: Add an equal volume of TMC lysis buffer again, aliquot into 1.5 mL centrifuge tubes, and centrifuge at 300 g for 4 h at 4 °C. The upper layer turns milky white, and the lower layer is a light red lysis product. Aspirate the lower layer and retain the preliminarily extracted GVs gas vesicles in the upper layer.

[0093] Purification and preservation: Add an appropriate amount of PBS to a 1.5 mL centrifuge tube, centrifuge multiple times at 300 g and 4°C until the upper layer is pure milky white and the lower layer is clear and transparent. Finally, add an appropriate amount of sterile PBS, seal with sealing film, and store at 4°C for later use.

[0094] 1.2 Preparation of CD147 nanobodies

[0095] CD147 nanobodies with specific high affinity were screened from a phage display library. The nucleotide sequence of the CD147 nanobodies is shown in SEQ ID NO:2; the amino acid sequence of the CD147 nanobodies is shown in SEQ ID NO:1.

[0096] The selected antibody genes were inserted into an E. coli expression vector and expressed using IPTG.

[0097] The antibody was purified using a Ni-NTA affinity chromatography column and washed with PBS buffer.

[0098] Figure 2 These are the electrophoresis results of the CD147 nanobody. Figure 2 The target band was identified using 12% Tria-Glycine SDS-PAGEGel, indicating that the purified anti-cd147 nanobody was approximately 19 kDa in size. Lane 1 represents the protein marker; lane 2 represents the bacterial lysate before induction; lane 3 represents the bacterial lysate after induction; lane 4 represents the supernatant after sonication; lane 5 represents the precipitate after sonication; and lane 6 represents the purified anti-cd147 nanobody (19 kDa).

[0099] 1.3 Activation of gas vesicles

[0100] (1) First, 10 mg Mal-PEG2000-NHS was reacted with 1 mL GVs (OD500 3.0) at 37 °C for 4 h.

[0101] (2) Dialyze with PBS solution (MWCO: 1000kDa, Solarbio) for 10-24h to remove free Mal-PEG-NHS and obtain MAL-PEG-GVs.

[0102] 2. The CD147 nanobody was reduced; the reduced CD147 nanobody was incubated with a gas vesicle solution modified with a coupling agent to prepare a CD147-targeted nano-ultrasound contrast agent.

[0103] (1) After adjusting the CD147 antibody protein concentration to 5 mg / mL, add TCEP to a final concentration of 1 mM and reduce at 25°C for 2 h.

[0104] (2) Mix the activated gas vesicles with the reduced CD147 nanobody, and mix approximately 1 mL of Mal-PEG-GVs with 200 μg of anti-CD147 nanobody. React overnight at 4°C.

[0105] (3) Remove the free coupling agent and unbound CD147 nanobody to obtain a CD147-targeted nano-ultrasound contrast agent.

[0106] The CD147-targeting nano-ultrasound contrast agent was separated and purified using dialysis. The specific steps included: transferring the reaction mixture to a dialysis bag with an appropriate molecular weight cutoff, sealing both ends with clips, placing the dialysis bag in a container containing 2L of PBS buffer, and performing dialysis to remove free conjugates and unbound antibodies. Dialysis time was at least 48 hours, with fresh PBS buffer replaced at 2h, 14h, 20h, 30h, and 40h to ensure dialysis effectiveness.

[0107] Alternatively, low-speed centrifugation can be used to separate and purify the CD147-targeted nano-ultrasound contrast agent. The specific steps include: dividing the reaction mixture into 2mL EP tubes, each with a volume of about 1.6mL, centrifuging at 300g and 4℃ for 2-3 hours, removing the lower layer solution with a 2mL syringe, adding fresh PBS solution, and centrifuging again under the same conditions. Repeating this process 2-3 times will yield the final product.

[0108] Test Example 1

[0109] Finished product testing and performance evaluation

[0110] 1. The particle size distribution, potential stability and other physical properties of the coupled CD147-targeting nano-ultrasound contrast agent were detected.

[0111] Figure 3The figure shows the particle size distribution of the targeted bubbles (nano-ultrasound contrast agents targeting CD147) and the non-targeted bubbles. As can be seen from the figure, the particle size distribution of CD147-hGVs and Con-hGVs is relatively uniform, with sizes of 236.04±9.81 nm and 222.66±12.28 nm, respectively, and there is no significant difference between the two.

[0112] Figure 4 The figure shows the potential distribution of targeted and non-targeted bubbles. As can be seen from the figure, the zeta potentials of CD147-hGVs and Con-hGVs are -15.03±2.09mV and -17.36±0.98mV, respectively. This indicates that the particle surface has a negative charge. Due to the mutual repulsion of surface charges, it is not easy to deposit and aggregate, and it can effectively maintain the dispersed state and has good stability.

[0113] 2. Its binding ability to tumor cells was verified through in vitro cell targeting experiments.

[0114] The experimental steps include:

[0115] (1) 1×10 4 One PC-3 cell was seeded in a 24-well plate and incubated overnight in a cell culture incubator. When the cell density reached about 60%-70%, the cells were fixed with 4% paraformaldehyde.

[0116] (2) Add 100 μL of FITC-CD147-GVs or FITC-Con-GVs (OD) to each well. 500 (1.5). All samples were incubated at room temperature in the dark for 10 min, then washed three times with cold PBS for 5 min each time.

[0117] (3) Stain cell nuclei with 4% DAPI, incubate at room temperature in the dark for 10 min, and then wash with cold PBS 3 times for 5 min each time.

[0118] (4) Images were recorded using a confocal microscope (A1R, Nikon, Japan).

[0119] Figure 5 These are in vitro imaging images of targeted and non-targeted vesicles at different concentrations. The images show different concentrations (OD). 500 Representative ultrasound contrast images of CD147-GVs and Con-GVs at concentrations of 0.5-3.0 were used to evaluate the acoustic contrast imaging performance of CD147-hGVs. As can be seen from the images, CD147-GVs and Con-GVs have significantly enhanced contrast signals in vitro, and the higher the concentration, the stronger the contrast signal.

[0120] Figure 6The figure shows the adhesion assay results of targeted and non-targeted vesicles with tumor cells (PC-3 cells). The results of the in vitro targeting ability test are displayed, with the vertical axis representing the target cell and the horizontal axis indicating DAPI, FITC cell staining, and the Merge image of both superimposed on the horizontal axis. The figure shows that CD147-GVs bind significantly to PC-3 cells, while Con-GVs bind very little. The Con-GVs group showed only DAPI I fluorescent labeling, while CD147-GVs showed both DAPI and FITC fluorescent labeling, indicating that the CD147-GVs can effectively target prostate cancer cell lines in vitro.

[0121] 3. Ultrasound imaging tests were conducted in animal models to evaluate the imaging signal intensity and targeting aggregation efficiency.

[0122] The experimental steps include:

[0123] (1) Human prostate cancer PC-3 cells (2×10⁻⁶) 7 The tumor was inoculated into the right back of BALB / c nude mice. After the tumor-bearing mice developed palpable tumors (approximately 5-10 mm in diameter), ultrasound imaging was performed.

[0124] (2) During ultrasound imaging, BALB / c Nude mice were anesthetized by inhaling oxygen containing 1% isoflurane at a rate of 2 L / min on a heated pad. CD147-GVs and Con-GVs (100 μL, OD) were administered. 500 The drug (3.0%) was randomly injected into mice via the tail vein.

[0125] (3) Ultrasound imaging was performed using a Mindray R9T transducer equipped with a linear array transducer. All parameters (acoustic power 5.13%, mechanical index 0.145, contrast gain 70 dB) were kept constant during imaging, and the entire imaging video was recorded. To reduce the bias of two injections in the same mouse, the injection interval was at least 30 min.

[0126] Figure 7 This figure shows in vivo imaging results in animals (BAL B / C nude mice) with and without targeted vesicles. The results of in vivo ultrasound imaging tests are displayed, with the vertical axis representing the object being examined and the horizontal axis representing time, increasing sequentially from left to right. It can be observed that after 1 minute, the signal intensity of both the contrast agents CD147-GVs and Con-GVs increased rapidly, and then showed a continuous decreasing trend. During this period, the ultrasound signal intensity of CD147-GVs was consistently higher than that of Con-GVs. Furthermore, compared to Con-GVs, CD147-GVs had a longer circulation time in tumors, and its decay curve was relatively slower, which is beneficial for more comprehensive imaging of deep tissues.

[0127] In summary, this invention, through the combination of gas vesicles and CD147 nanobodies, overcomes the shortcomings of existing technologies in terms of imaging performance, targeting, preparation process, safety, and multifunctional diagnostic and therapeutic capabilities, providing a novel technical approach for the precise diagnosis and treatment of tumors.

[0128] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A nano-ultrasound contrast agent targeting CD147, characterized in that, The contrast agent comprises: gas vesicles derived from microorganisms and CD147 nanoantibodies attached to the surface of the gas vesicles by a coupling agent; The coupling agent is maleimide polyethylene glycol active ester, in which N-hydroxysuccinimide ester is linked to gas vesicles; and in which maleimide in maleimide polyethylene glycol active ester is linked to the CD147 nanobody. The molecular weight of the polyethylene glycol in the maleimide polyethylene glycol active ester is 2000-5000 Da; The amino acid sequence of the CD147 nanobody is shown in SEQ ID NO:

1.

2. The CD147-targeting nano-ultrasound contrast agent according to claim 1, characterized in that, The particle size range of the gas vesicles is 100-300 nm.

3. The CD147-targeting nano-ultrasound contrast agent according to claim 1, characterized in that, The CD147 nanobody has a molecular weight of 15-19 kDa.

4. A method for preparing a CD147-targeting nano-ultrasound contrast agent according to any one of claims 1-3, characterized in that, The method includes: (1) The coupling agent is mixed with the gas vesicle solution and reacted, and the free coupling agent is removed by dialysis to obtain the coupling agent modified gas vesicles; (2) The CD147 nanobody was reduced; the reduced CD147 nanobody was incubated with a gas vesicle solution modified with a coupling agent to prepare a nano-ultrasound contrast agent targeting CD147.

5. The preparation method according to claim 4, characterized in that, In step (1), the OD of the gas vesicle solution 500 It is 2.5-3.

0.

6. The preparation method according to claim 4, characterized in that, In step (1), the final concentration of the coupling agent in the gas vesicle solution is 5-20 mg / mL.

7. The preparation method according to claim 4, characterized in that, In step (1), the reaction conditions are 35-37℃ for 3-5 h.

8. The preparation method according to claim 4, characterized in that, In step (2), TCEP is added to the CD147 nanobody solution for reduction.

9. The preparation method according to claim 4, characterized in that, The final concentration of the CD147 nanobody solution is 3-5 mg / mL.

10. The preparation method according to claim 8, characterized in that, The final concentration of TCEP in the solution is 1-20 mM.

11. The preparation method according to claim 4, characterized in that, The reduction conditions are 20-25℃ for 2-3 hours.

12. The preparation method according to claim 4, characterized in that, In step (2), the OD of the coupling agent-modified gas vesicle solution 500 It is 2.5-3.

0.

13. The preparation method according to claim 4, characterized in that, In step (2), the final concentration of the reduced CD147 nanobody in the coupling agent-modified gas vesicle solution is 50-500 μg / mL.

14. The preparation method according to claim 4, characterized in that, In step (2), the incubation conditions are 2-6℃ for 10-20 h.

15. The preparation method according to claim 4, characterized in that, In step (2), the incubation process also includes the removal of the free coupling agent and the unbound CD147 nanoantibody.

16. The use of the CD147-targeting nano-ultrasound contrast agent according to any one of claims 1-3 in the preparation of products for molecular imaging, diagnosis or treatment of prostate cancer.

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