Nanometer ultrasonic contrast agent targeting CD147 as well as preparation method and application of nanometer ultrasonic contrast agent

Abstract

The invention provides a CD147-targeted nano ultrasonic contrast agent as well as a preparation method and application thereof. The contrast agent comprises gas vesicles derived from microorganisms and a CD147 nano antibody connected to the surfaces of the gas vesicles through a coupling agent. The hollow nanostructure of the gas vesicle endows the contrast agent with efficient acoustic reflection capability, and the ultrasonic signal intensity is greatly improved; the CD147 nano antibody has high affinity and high specificity, and can accurately recognize a CD147 antigen on the surface of a tumor cell. The biosynthetic gas vesicles are used for replacing traditional chemical synthetic gas microvesicles, a simple and efficient coupling process is combined, the preparation difficulty and equipment dependence are remarkably reduced, the contrast agent overcomes the defects of the prior art in the aspects of imaging performance, targeting performance, preparation process, safety and multifunctional diagnosis and treatment capacity, and the contrast agent has good application prospects. And a brand new technical path is provided for precise diagnosis and treatment of tumors.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedicine and molecular imaging, and particularly relates to a nano-ultrasound contrast agent targeting CD147, a preparation method thereof, and an application thereof. Background Art

[0002] Among existing molecular imaging technologies, traditional ultrasound contrast agents are primarily made of gas microbubbles encapsulated by 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 tumor vascular spaces, preventing 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 issues such as large molecular weight, 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. It has biological functions such as inducing matrix metalloproteinase (MMP) production and promoting tumor invasion and metastasis, making it a hot topic in tumor diagnosis and treatment. However, targeting strategies based on traditional monoclonal antibodies are limited by their large molecular structure, resulting in difficulty 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 micron range, which makes it difficult to penetrate the tumor blood vessel space and has 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, complex steps and high costs. (5) Safety issues: Some contrast agents may have immunogenicity risks or non-specific binding with normal tissues, affecting clinical applications.

[0005] Therefore, providing a targeted ultrasound contrast agent with good imaging performance, strong targeting, safety and 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 application. The present invention combines microbial-derived gas vesicles with CD147 nanoantibodies to develop an ultrasound molecular imaging and diagnostic tool targeting tumor tissue.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a nano-ultrasound contrast agent targeting CD147, wherein the contrast agent comprises: a gas vesicle derived from a microorganism and a CD147 nano-antibody connected to the surface of the gas vesicle via a coupling agent.

[0009] The present invention introduces biosynthetic nanoscale gas bubbles (derived from halophilic archaea or Serratia or genetically engineered bacteria thereof) and CD147 nanoantibodies with high specificity and high affinity, combined with targeted specific molecular modifications, which can effectively overcome the bottlenecks of existing technologies and achieve precise targeted ultrasonic 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 azide polyethylene glycol active ester (N3-PEG-NHS).

[0011] Preferably, the coupling agent is maleimide polyethylene glycol active ester, 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 the present invention, the N-hydroxysuccinimide ester in the maleimide polyethylene glycol active ester is connected to the gas vesicles, wherein the NHS active ester group can react with the amino group (-NH2) to form a stable amide bond.

[0013] In the present invention, the maleimide in the maleimide polyethylene glycol active ester is connected to the CD147 nanobody, wherein the maleimide group can undergo Michael addition reaction with the sulfhydryl group to form a stable thioether bond.

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

[0015] In this study, gas vesicles (GVs) serve as carriers, their surfaces chemically modified with Mal-PEG-NHS. The CD147 nanoantibody undergoes a coupling reaction via its thiol group with the maleimide group on the Mal-PEG-NHS, forming a stable bond. The resulting CD147-targeting nanoultrasound contrast agent exhibits uniform particle size, high binding efficiency, and demonstrated excellent stability and targeting in both in vitro and in vivo experiments.

[0016] The working principles of the CD147-targeted nano-ultrasound contrast agent described in this invention include: Targeting: The CD147 nano-antibody can specifically bind to the CD147 protein, which is highly expressed on the surface of tumor cells, allowing the contrast agent to efficiently accumulate at the tumor site. Excellent imaging performance: The gas vesicles, through the properties of their internal gas, generate strong echo signals under ultrasound excitation, significantly improving imaging contrast. Combined diagnosis and treatment: 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 the polyethylene glycol in the maleimide polyethylene glycol active ester is 1-20 kDa.

[0018] In the present invention, the preferred molecular weight of polyethylene glycol is 2000-5000Da. The chain length of PEG 2000-5000 is moderate, resulting in relatively small steric hindrance, which does not excessively hinder the proximity between reactive groups, thereby ensuring the efficiency of the ligation reaction. In maleimide polyethylene glycol active ester (Mal-PEG-NHS), the chain length of PEG 2000 does not excessively wrap or shield the maleimide group or NHS active ester group, allowing them to react smoothly with sulfhydryl or amino groups. At the same time, PEG 2000-5000 can effectively reduce the nonspecific binding of the contrast agent to plasma proteins, reducing the possibility of its recognition and clearance by the immune system, thereby prolonging the residence time of the contrast agent in the blood circulation and enhancing the imaging effect.

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

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

[0021] In the present invention, the gas vesicles have a particle size of approximately 100-300 nm. This nanoscale size range gives them strong penetrating power, enabling them to pass through the interstitial spaces between vascular endothelial cells and enter interstitial spaces, achieving intravascular and extravascular imaging, thereby achieving more subtle image contrast. This property enables the gas vesicles to more effectively reach the lesion site, improving imaging resolution and sensitivity. They can also generate strong scattering signals across a wider range of ultrasonic frequencies, making them suitable for high-resolution ultrasound imaging. This allows the gas vesicles to demonstrate excellent imaging results under different ultrasound imaging equipment and parameters, enhancing their adaptability in clinical applications.

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

[0023] Existing contrast agents have limited targeting ability for tumor tissue, and traditional monoclonal antibodies suffer from large molecular weight and poor permeability. This invention utilizes high-affinity CD147 nanobodies combined with gas vesicles to achieve highly specific recognition of tumor cells that overexpress CD147, thereby improving targeting and allowing the contrast agent to more precisely accumulate at the tumor site.

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

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

[0026] Some traditional contrast agents may nonspecifically bind to normal tissues, posing a risk of immunogenicity. The CD147 nanoantibody used in this invention has a small molecular weight and low immunogenicity. Furthermore, through the loading and protective effects of gas vesicles, it significantly reduces interference with normal tissues, further improving safety and meeting clinical requirements.

[0027] In a second aspect, the present invention provides a use 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 anti-tumor drug on the surface of the CD147-targeting nano-ultrasound contrast agent described in the first aspect.

[0028] In the present invention, the surface loading method includes: utilizing the physical properties of the contrast agent surface, such as electrostatic effect or hydrophobic effect, to adsorb the anti-tumor drug on the contrast agent surface.

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

[0030] The CD147-targeted nano-ultrasound contrast agent developed by this invention not only improves ultrasound imaging performance but also has the potential to integrate 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] In a third aspect, the present invention provides a method for preparing the CD147-targeting nano-ultrasound contrast agent according to the first aspect, the method comprising:

[0032] (1) mixing a coupling agent with a gas vesicle solution to react, and performing dialysis to remove free coupling agent to obtain coupling agent-modified gas vesicles;

[0033] (2) reducing the CD147 nanoantibody; incubating the reduced CD147 nanoantibody with a coupling agent-modified gas vesicle solution to prepare a CD147-targeted nanoultrasound contrast agent.

[0034] The preparation process of existing ultrasound contrast agents is complex and relies on multiple high-precision equipment, resulting in high process costs and difficulties in large-scale production. This invention combines biosynthesized gas vesicles with the CD147 nanoantibody conjugation process to design a more efficient and economical preparation method, reducing reliance on high-end equipment and production costs while improving 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, 5 mg / mL, 10 mg / mL, 15 mg / mL or 20 mg / mL.

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

[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, 3 mg / mL, 4 mg / mL or 5 mg / mL.

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

[0041] Preferably, the reduction condition is 20-25°C for 2-3h, wherein 20-25°C can be, for example, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C.

[0042] Preferably, in step (2), the OD of the gas vesicle solution modified by the coupling agent 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, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL or 500 μg / mL, etc.

[0044] Preferably, in step (3), the incubation condition is 2-6°C for 10-20h, wherein 2-6°C can be, for example, 2°C, 3°C, 4°C, 5°C or 6°C, and 10-20h can be, for example, 10h, 15h or 20h.

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

[0046] In the present invention, the method for removing free coupling agent and unbound CD147 nanobody includes: dialysis or 250-500g centrifugation.

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

[0048] (1) First, 5-20 mg of coupling agent was mixed with 1 mL of gas vesicle solution (OD 500 2.5-3.0) react at 35-37°C for 3-5h;

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

[0050] (3) Adjust the CD147 antibody protein concentration to 3-5 mg / mL and add TCEP to a final concentration of 1-20 mM. Reduce at 20-25°C for 2-3 h.

[0051] (4) Mix 50-500 μg CD147 nanobody with 1 mL OD 500 The gas vesicle solution modified with a 2.5-3.0 coupling agent was incubated at 2-6° C. for 10-20 h to prepare a CD147-targeted nano-ultrasound contrast agent.

[0052] In the present invention, the maleimide-thiol coupling method was selected to design a contrast agent targeting CD147, rather than the traditional biotin-streptavidin chemical connection method. This choice was mainly based on the immunogenicity problem that streptavidin may cause. The maleimide-thiol coupling reaction, with its high specificity and efficiency, provides a more superior strategy for the binding of targeting antibodies to the surface of nanobubbles. The covalent bond formed by this method shows good stability both in vitro and in vivo, which is crucial for maintaining the integrity and function of the nanocapsules during storage and administration. In addition, polyethylene glycol (PEG) modification has been confirmed in many studies as an effective means to reduce the immunogenicity of biomaterials. 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 the body, thereby enhancing their performance in in vivo imaging.

[0053] In the present invention, after the coupling reaction, the step of removing free coupling agent and unbound CD147 nanobody by dialysis or low-speed centrifugation is also included.

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

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

[0056] The imaging performance and targeted aggregation ability of the CD147-targeting nano-ultrasound contrast agent of the present invention in in vitro cell experiments and animal models are significantly better than those of the prior art.

[0057] The contrast agent of the present invention can be used for ultrasound imaging of targeted diagnosis of tumors (such as liver cancer or lung cancer), and can also be extended to integrated diagnosis and treatment, by modifying the surface of nanobubbles with chemotherapy drugs to achieve drug delivery.

[0058] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

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

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

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

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

[0063] (3) Higher safety: Nanobodies have a small molecular weight and low immunogenicity, which can reduce nonspecific binding and potential immune responses. Gas vesicles are highly stable and their components are derived from organisms, making them non-toxic, thus reducing the risk of side effects of contrast agents on the body.

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

[0065] (5) Potential for integrated diagnosis and treatment: 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), giving the contrast agent therapeutic function. This contrast agent can not only be used for tumor diagnosis, but also has the potential for drug delivery, providing a technical basis for integrated diagnosis and treatment.

[0066] (6) Broad Application Prospects: The present invention can be widely used for 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 the present invention can also be used for ultrasound imaging of other diseases such as inflammation and thrombosis, showing good application scalability. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0068] Figure 2 This is the electrophoresis result of CD147 nanoantibody.

[0069] Figure 3is the particle size distribution graph.

[0070] Figure 4 It is the electric potential distribution diagram.

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

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

[0073] Figure 7 These are the in vivo imaging results of animals with targeted and non-targeted blebs (BAL B / C mice). DETAILED DESCRIPTION

[0074] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0075] 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 that can be purchased through regular channels.

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

[0077] Trace element solution: 1.32g zinc sulfate heptahydrate, 0.34g manganese sulfate, 0.82g ferrous ammonium sulfate, 0.14g copper sulfate pentahydrate, 200mL ddH2O. Prepare the appropriate amount of solution according to the above reagent ratios. Stir thoroughly on a magnetic stirrer until dissolved, then filter sterilize using a 0.22μm filter in a clean bench. Add 100μL of trace elements per 1.0L ATCC basal medium to prepare ATCC complete medium.

[0078] Halophilic Archaea culture medium: 250.0 g sodium chloride, 20.0 g magnesium sulfate heptahydrate, 3.0 g trisodium citrate, 2.0 g potassium chloride, 5.0 g tryptone, 3.0 g yeast extract, 1.0 L ddH2O. Prepare an appropriate amount of solution according to the above reagent ratios. Stir thoroughly on a magnetic stirrer until dissolved, then sterilize in an autoclave (121°C, 20 min). Cool and set aside.

[0079] TMC lysis buffer: 2.42g tris (hydroxymethyl)aminomethane hydrochloride), 1.01g magnesium chloride, 0.58g calcium chloride, 2.0L ddH2O. Prepare the appropriate amount of solution according to the above reagent ratios, adjust the pH to 7.5, and filter sterilize using a 0.22μm filter in a clean bench. TMC lysis buffer can be stored long-term at room temperature.

[0080] Example 1

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

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

[0083] 1.1 Preparation of gas vesicles (GVs)

[0084] (1) Cultivation of Halo halophilic archaea

[0085] Medium preparation and inoculation: Add 100 μL of the trace element solution to 1.0 L of Halo Halophilic Archaea medium to create a transparent, pale yellow complete medium. Thaw the frozen solution from -80°C at room temperature. Add 10 mL of the bacterial culture to a conical flask containing 1.0 L of complete medium, shake well, and seal with a breathable membrane.

[0086] Culture process: Place the conical flask in a constant temperature shaker at 37°C and 200 rpm for about 14 days. Observe daily and the culture medium will gradually change from transparent light yellow to dark red and finally to pink, indicating that the culture is complete.

[0087] (2) Extraction and purification of GVs

[0088] Static stratification 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 pink-white ring layer appears on the upper layer.

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

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

[0091] Initial centrifugation: Centrifuge at 4°C, 300g for 4 hours. The solution will separate into three layers. The upper layer contains most of the lysed GVs and a small amount of broken bacterial residue. The middle layer is the culture medium solution. The lower layer contains precipitated culture medium impurities and most of the lysed bacterial fragments. Use a syringe to aspirate the middle and lower layers, retaining 8-10 mL of the upper layer of pink-white liquid.

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

[0093] Purification and Storage: Add an appropriate amount of PBS to a 1.5 mL centrifuge tube and centrifuge multiple times at 300g at 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 parafilm, and refrigerate at 4°C until needed.

[0094] 1.2 Preparation of CD147 Nanobody

[0095] A specific, high-affinity CD147 nanobody was screened from a phage display library. The nucleotide sequence of the CD147 nanobody is shown in SEQ ID NO: 2; and the amino acid sequence of the CD147 nanobody is shown in SEQ ID NO: 1.

[0096] The screened antibody gene was inserted into an E. coli expression vector and induced to express using IPTG.

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

[0098] Figure 2 This is the electrophoresis result of CD147 nanoantibody. Figure 2 It can be seen that the target band was identified using 12% Tria-Glycine SDS-PAGEGel, indicating that the size of the purified anti-CD147 nanobody is approximately 19 kDa, where lane 1 represents: protein marker Marker; lane 2: bacterial lysate before induction; lane 3: bacterial lysate after induction; lane 4: supernatant after ultrasonic treatment; lane 5: precipitate after ultrasonic treatment; lane 6: purified anti-CD147 nanobody (19 kDa).

[0099] 1.3 Activated gas vesicles

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

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

[0102] 2. Reduce the CD147 nanoantibody; incubate the reduced CD147 nanoantibody with a coupling agent-modified gas vesicle solution to prepare a CD147-targeted nanoultrasound contrast agent.

[0103] (1) The CD147 antibody protein concentration was adjusted to 5 mg / mL, and TCEP was added to a final concentration of 1 mM. The mixture was reduced at 25°C for 2 h.

[0104] (2) The activated gas vesicles were mixed with the reduced CD147 nanobody, and approximately 1 mL of Mal-PEG-GVs was mixed with 200 μg of anti-CD147 nanobody. The reaction was allowed to proceed overnight at 4°C.

[0105] (3) removing free coupling agent and unbound CD147 nanoantibodies to obtain a CD147-targeted nanoultrasound contrast agent.

[0106] Dialysis is used to separate and purify the CD147-targeting nano-ultrasound contrast agent. The specific steps include transferring the reaction mixture to a dialysis bag with an appropriate molecular weight cutoff, sealing both ends with clamps, and placing the bag in a container filled with 2L of PBS buffer for dialysis to remove free conjugate and unbound antibody. The dialysis process lasts for at least 48 hours, with fresh PBS exchanged at 2, 14, 20, 30, and 40 hours to ensure effective dialysis.

[0107] Alternatively, low-speed centrifugation is used to separate and purify the CD147-targeting nano-ultrasound contrast agent. The specific steps include: the reaction mixture is divided into 2 mL EP tubes, each tube has a volume of about 1.6 mL, and centrifuged at 300g and 4°C for 2-3 hours. After removal, the lower layer solution is aspirated and discarded with a 2 mL syringe, and fresh PBS solution is added. Centrifugation is repeated under the same conditions for 2-3 times to obtain the final product.

[0108] Test Example 1

[0109] Finished product testing and performance evaluation

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

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

[0112] Figure 4 It is a potential distribution diagram, which shows the potential distribution of targeted bubbles and non-targeted bubbles. It can be seen from the figure that the zeta potential of CD147-hGVs and Con-hGVs are -15.03±2.09mV and -17.36±0.98mV, respectively, indicating that the surface of the particles has a negative charge. Due to the mutual repulsion of surface charges, it is not easy to deposit and aggregate, and the dispersion state can be effectively maintained with good stability.

[0113] 2. Verify its ability to bind to tumor cells through in vitro cell targeting experiments.

[0114] The experimental steps include:

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

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

[0117] (3) Cell nuclei were stained with 4% DAPI, incubated at room temperature in the dark for 10 min, and then washed three times with cold PBS, each time for 5 min.

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

[0119] Figure 5 The in vitro imaging pictures of targeted and non-targeted vesicles at different concentrations are shown in the figure. 500 =0.5-3.0) were used to evaluate the acoustic contrast imaging performance of CD147-hGVs. As can be seen from the figure, 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 results of an in vitro targeting assay comparing targeted and non-targeted follicles to tumor cells (PC-3 cells). The figure shows the results of in vitro targeting ability testing, with the vertical axis representing the target cells and the horizontal axis representing DAPI, FITC, and a superimposed merge image of the two. As can be seen, CD147-GVs showed significant binding to PC-3 cells, while Con-GVs showed minimal binding. While the ultrasound contrast agent Con-GV group showed only DAPI fluorescence labeling, CD147-GVs showed both DAPI and FITC fluorescence labeling, demonstrating that the ultrasound contrast agent CD147-GVs can effectively target prostate cancer cell lines in vitro.

[0121] 3. Conduct ultrasound imaging tests in animal models to evaluate its imaging signal intensity and targeted aggregation efficiency.

[0122] The experimental steps include:

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

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

[0125] (3) Ultrasound imaging was performed using a Mindray R9T 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 minimize deviations between injections in the same mouse, the interval between injections was at least 30 min.

[0126] Figure 7 The figure shows in vivo imaging results of animals (BAL B / C nude mice) with targeted and non-targeted follicles. The figure shows the results of in vivo ultrasound imaging tests. The vertical axis represents the test subject, and the horizontal axis represents time, increasing from left to right. It can be seen that after 1 minute, the signal intensity of the ultrasound contrast agents CD147-GVs and Con-GVs both increased rapidly, then showed a continuous downward trend. During this period, the ultrasound signal intensity of the ultrasound contrast agent CD147-GVs was consistently higher than that of the ultrasound contrast agent Con-GVs. In addition, compared with the ultrasound contrast agent Con-GVs, CD147-GVs have a longer circulation time in the tumor and a relatively slower decay curve, which facilitates more comprehensive imaging of deep tissues.

[0127] In summary, the present invention solves the shortcomings of existing technologies in imaging performance, targeting, preparation process, safety and multifunctional diagnostic and treatment capabilities through the combination of gas vesicles and CD147 nanoantibodies, providing a new technical path for the precise diagnosis and treatment of tumors.

[0128] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure 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 nano antibodies connected to the surface of the gas vesicles through a coupling agent.

2. The nano-ultrasound contrast agent targeting CD147 according to claim 1, characterized in that The coupling agent is selected from any one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, maleimide polyethylene glycol active ester or azide polyethylene glycol active ester; Preferably, the coupling agent is maleimide polyethylene glycol active ester, 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.

3. The nano-ultrasound contrast agent targeting CD147 according to claim 2, characterized in that The molecular weight of the polyethylene glycol in the maleimide polyethylene glycol active ester is 1-20k.

4. The nano-ultrasound contrast agent targeting CD147 according to any one of claims 1 to 3, characterized in that: The particle size of the gas vesicles ranges from 100 to 300 nm.

5. The CD147-targeting nano-ultrasound contrast agent according to any one of claims 1 to 4, characterized in that: The CD147 nanobody has a molecular weight of 15-19 kDa; Preferably, the amino acid sequence of the CD147 nanobody is as shown in SEQ ID NO:

1.

6. Use of the CD147-targeting nano-ultrasound contrast agent according to any one of claims 1 to 5 in the preparation of a drug, characterized in that: The drug is obtained by loading an anti-tumor drug on the surface of the CD147-targeting nano-ultrasound contrast agent according to any one of claims 1 to 5; Preferably, the anti-tumor drug is selected from chemotherapy drugs and / or nucleic acid fragments.

7. A method for preparing a nano-ultrasound contrast agent targeting CD147 according to any one of claims 1 to 5, characterized in that: The method comprises: (1) mixing a coupling agent with a gas vesicle solution to react, and performing dialysis to remove free coupling agent to obtain coupling agent-modified gas vesicles; (2) reducing the CD147 nanoantibody; incubating the reduced CD147 nanoantibody with a coupling agent-modified gas vesicle solution to prepare a CD147-targeted nanoultrasound contrast agent.

8. The preparation method according to claim 7, characterized in that In step (1), the OD of the gas vesicle solution is 500 2.5-3.0; Preferably, in step (1), the final concentration of the coupling agent in the gas vesicle solution is 5-20 mg / mL; Preferably, in step (1), the reaction conditions are 35-37° C. for 3-5 hours.

9. The preparation method according to claim 7 or 8, characterized in that In step (2), TCEP is added to the CD147 nanobody solution for reduction; Preferably, the final concentration of the CD147 nanobody solution is 3-5 mg / mL; Preferably, the final concentration of TCEP in the solution is 1-20 mM; Preferably, the reduction condition is 20-25° C. for 2-3 hours; Preferably, in step (2), the OD of the gas vesicle solution modified by the coupling agent is 500 2.5-3.0; 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; Preferably, in step (3), the incubation condition is 2-6°C for 10-20 hours. Preferably, in step (3), the incubation further includes a step of removing free coupling agent and unbound CD147 nanobody.

10. Use of the method for preparing the CD147-targeting nano-ultrasound contrast agent according to any one of claims 1 to 5 and / or the CD147-targeting nano-ultrasound contrast agent according to any one of claims 7 to 9, characterized in that: The application includes: preparing any one of products for molecular imaging, tumor diagnosis or tumor treatment, or a combination of at least two of them.

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