Nanometer ultrasound contrast agent for enhancing immunotherapy and preparation method and application thereof

Abstract

The present application belongs to the technical field of ultrasonic molecular imaging and biomedical engineering, and particularly relates to a nano-ultrasound contrast agent for enhancing immunotherapy and a preparation method and application thereof. The nano-ultrasound contrast agent for enhancing immunotherapy provided by the present application is a novel ultrasonic nano-vesicle formed by loading a protein drug for blocking an immune checkpoint into a nano-carrier with a special composition. The nano-ultrasound contrast agent has high biological safety, small and uniform particle size, can be long-circulated in vivo, has good imaging effect on the extravascular field of a tumor, has good penetration, is highly targeted, has high drug utilization rate, can improve the delivery efficiency of a tumor immunotherapy drug and the immunotherapy effect, can realize tumor diagnosis and immunotherapy integration under ultrasonic guidance, and effectively solves the current problems of difficult tumor ultrasonic diagnosis, low tumor responsiveness of an immunotherapy drug, weak targeting, and inability to be regulated in vitro.

Description

Technical Field

[0001] This invention belongs to the fields of ultrasound molecular imaging and biomedical engineering technology, specifically relating to nano-ultrasound contrast agents for enhancing immunotherapy, their preparation methods, and applications. Background Technology

[0002] Cancer, as one of the leading causes of death worldwide, has become a major threat to human life and public health. Currently, the global cancer rate is increasing year by year, and early and accurate detection and timely treatment to reduce cancer mortality have become a major global challenge. However, at present, the clinical diagnosis and treatment of tumors are often separated, and limitations in technology and methods significantly restrict both diagnosis and management, thus delaying optimal treatment and reducing treatment effectiveness. Therefore, organically combining tumor diagnosis and monitoring with treatment to form a unified treatment system and achieve integrated diagnosis and treatment can greatly improve the survival rate of cancer patients.

[0003] Currently, commonly used medical imaging diagnostic methods in clinical practice include ultrasound, CT, X-ray, and MRI. Among them, ultrasound technology, with its advantages of low cost, non-invasiveness, no radiation, and real-time imaging, has become the primary means of cancer diagnosis and examination. However, the relatively low resolution of traditional ultrasound imaging technology limits further detection of tissues and organs. The advent of ultrasound contrast agents (UCA) is a major advancement in the field of ultrasound diagnostics in recent years, providing highly valuable evidence for the differentiation and diagnosis of various diseases, especially tumors. UCA ultrasound diagnostic technology uses intravenous injection of ultrasound contrast agents to differentiate normal tissues from diseased tissues based on blood perfusion information, effectively enhancing the sensitivity and specificity of disease diagnosis. Currently, the ultrasound contrast agents used clinically are artificially synthesized gas-encapsulated microbubbles, mostly ranging from 1-10 μm in size, with uneven particle size, poor stability, lack of targeting, and lack of tissue specificity. Furthermore, they cannot penetrate the human reticuloendothelial system to enter the tissue interior, and can only be used for blood pool imaging. Therefore, this type of contrast agent is limited in the application of ultrasound molecular imaging. Furthermore, high-intensity focused ultrasound (HIFU) generates instantaneous high temperatures by focusing the sound beam, causing irreversible necrosis of tumor cells in the focal area, thus ablating the tumor. Therefore, ultrasound therapy is increasingly being applied clinically. However, single imaging techniques are insufficient to meet the clinical diagnostic and treatment needs of major diseases such as tumors. To address the limitations of micron-sized ultrasound contrast agents, which can only perform intravascular imaging or treatment of tumors, and the poor imaging effects and inability to treat tumors with existing nano-sized ultrasound contrast agents, this invention attempts to develop a novel targeted drug-loaded nano-ultrasound contrast agent. This agent can actively target imaging and utilize its focusing imaging properties to generate ultra-strong echo characteristics, achieving highly efficient tumor treatment.

[0004] With the gradual development of tumor immunology, immunotherapy, which utilizes the immune pathway to treat cancer, is rapidly becoming a recognized cancer treatment method after chemotherapy, radiotherapy, and surgery. Among these, the clinical success of immune checkpoint blockade therapy has brought increasing attention to tumor immunotherapy. Since the first PD-1 / PD-L1 monoclonal antibody drug (PD-1 is anti-programmed death protein 1; PD-L1 is programmed death ligand 1) was approved for marketing in China in 2018, its application and development in tumor immunotherapy have attracted widespread attention. In the tumor microenvironment, the interaction between PD-1 and PD-L1 can inhibit lymphocyte proliferation and activation, induce antigen-specific T cell apoptosis, and exert a negative immunomodulatory effect, leading to T cell dysfunction and mediating immunosuppression. PD-L1 monoclonal antibody drugs are applicable to various solid tumors and have advantages such as inhibiting tumor recurrence and metastasis, being used to treat advanced tumors, and having relatively few side effects. However, PD-L1 monoclonal antibody monotherapy has disadvantages such as low utilization rate, poor targeting, and short circulation time in vivo, which limits its further application.

[0005] In summary, developing a novel nano-ultrasound contrast agent with high biocompatibility, excellent extravascular imaging of tumors, good penetration, strong targeting, high drug utilization, and the ability to enhance the efficacy of immunotherapy is of great significance for improving the delivery efficiency of tumor immunotherapy drugs and realizing the integration of ultrasound-guided tumor diagnosis and immunotherapy. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the primary objective of this invention is to provide a nano-ultrasound contrast agent for enhancing immunotherapy. This nano-ultrasound contrast agent is a novel type of ultrasound nanovesicle formed by loading protein drugs that block immune checkpoints onto a specially composed nanocarrier. It not only has high biosafety, small and uniform particle size, and long-term circulation in vivo, but also provides good imaging effects in the extravascular domain of tumors, good penetration, strong targeting, and high drug utilization. It can improve the delivery efficiency of tumor immunotherapy drugs and the immune effect while realizing the integration of ultrasound-guided tumor diagnosis and immunotherapy, effectively solving the current problems such as the difficulty of tumor ultrasound diagnosis, low tumor response of immunotherapy drugs, weak targeting, and inability to regulate in vitro.

[0007] The second objective of this invention is to provide a method for preparing the above-mentioned nano-ultrasound contrast agent for enhancing immunotherapy. The process is simple, and the resulting contrast agent has a high drug loading capacity. It can achieve rapid and precise drug delivery to the tumor region under the guidance of external ultrasound imaging, effectively solving the current problems such as the complexity of ultrasound contrast agent preparation, difficulty in tumor ultrasound diagnosis, low tumor responsiveness of immunotherapy drugs, slow release, and inability to be controlled in vitro. This realizes the integration of ultrasound-guided tumor diagnosis and immunotherapy.

[0008] A third objective of this invention is to provide the application of the above-mentioned nano-ultrasound contrast agent for enhancing immunotherapy.

[0009] One of the objectives of this invention is achieved through the following technical solution:

[0010] A nano-ultrasound contrast agent for enhancing immunotherapy, the nano-ultrasound contrast agent comprising: a nanocarrier and a protein drug loading the nanocarrier to block immune checkpoints;

[0011] The nanocarrier is a polycaprolactone-polyethylene glycol-polyethyleneimine polymer; the protein drug is one or more of PD-1 monoclonal antibody, PD-L1 monoclonal antibody, and CTLA-4 monoclonal antibody.

[0012] In the polycaprolactone-polyethylene glycol-polyethyleneimine polymer, the average molecular weight of polycaprolactone is 1900-2200 g / mol, the average molecular weight of polyethylene glycol is 1900-2200 g / mol, and the average molecular weight of polyethyleneimine is 1700-1850 g / mol.

[0013] The nano-ultrasound contrast agent provided by this invention uses polycaprolactone-polyethylene glycol-polyethyleneimine polymer (PCL-PEG-PEI) as a drug carrier and loads protein drugs that block immune checkpoints.

[0014] In this invention, polycaprolactone (PCL), a polymeric polyester, is a widely used biodegradable sustained-release and controlled-release carrier, possessing excellent properties such as good biocompatibility, biodegradability, and high safety. Polyhexanediol (PEG), a hydrophilic polymer, is non-toxic and non-immunogenic in vivo. Its modified nanoparticles exhibit good stability and can avoid recognition by the reticuloendothelial system, thus prolonging in vivo circulation time. This invention, based on the excellent biocompatibility, biodegradability, and high safety properties of PCL, and the long-circulating, non-immunogenic polymer PEG, further combines it with cationic-rich polyethyleneimine (PEI) to form a polycaprolactone-polyethylene glycol-polyethyleneimine ternary polymer. In this process, this invention greatly ensures the safety of the nanocarrier by controlling the structural composition and molecular weight of the polymer materials. Furthermore, the prepared nanocarrier exhibits long-lasting circulating properties in vivo, and its non-immunogenicity ensures the efficient efficacy of the loaded drug. After the drug exerts its function, it can rapidly and safely degrade in vivo, ensuring both safe and efficient drug delivery and safe degradation within the body. Therefore, the nanocarrier used in this invention can enhance the interaction between drug molecules and polymer molecules, improve drug stability, and reduce in vivo toxicity, achieving sustained release within tumors.

[0015] Furthermore, the drug loaded onto the drug carrier in this invention is a protein drug that blocks immune checkpoints (such as a PD-L1 monoclonal antibody). By loading the aforementioned protein drug that blocks immune checkpoints, after intravenous injection via the tail vein, ultrasound irradiation of a certain energy is applied to the treatment site or tumor surface. This causes the vesicles to compress and expand under ultrasound, triggering an ultrasound cavitation effect, leading to vesicle rupture and releasing the loaded protein drug around the tumor cells. The released protein drug can enter the blood vessel wall and even the interstitial space, specifically targeting and binding to the PD-L1 protein on the surface of tumor cells. This causes the nano-ultrasound contrast agent to accumulate around the tumor tissue, blocking the PD-1 / PD-L1 immune checkpoint, thereby enhancing the immune microenvironment within the tumor tissue and achieving the goal of killing tumor cells. Simultaneously, the ultrasound irradiation generated during ultrasound contrast imaging diagnosis can cause mechanical vibration, thereby disrupting the structure of the cancer cell membrane, increasing the probability of PD-1 antigen exposure on the cancer cell membrane surface, thus altering the immunogenicity of the tumor tissue and enhancing the body's immune response to the tumor tissue.

[0016] Preferably, in the polycaprolactone-polyethylene glycol-polyethyleneimine polymer, the average molecular weight of polycaprolactone is 2000 g / mol, the average molecular weight of polyethylene glycol is 2000 g / mol, and the average molecular weight of polyethyleneimine is 1800 g / mol.

[0017] Preferably, the protein drug is a PD-L1 monoclonal antibody.

[0018] Preferably, the average particle size of the nano-ultrasound contrast agent is 80–120 nm. More preferably, the average particle size of the nano-ultrasound contrast agent is 90–100 nm.

[0019] The second objective of this invention is achieved by the following technical solution:

[0020] The preparation method of the above-mentioned nano-ultrasound contrast agent for enhancing immunotherapy includes the following steps:

[0021] (1) Dissolve the polycaprolactone-polyethylene glycol-polyethyleneimine polymer in the first solvent by stirring, and then evaporate the first solvent to dryness; add PBS solution to the evaporated system by stirring and dissolving, add the first solvent again by stirring, and evaporate the first solvent to dryness again;

[0022] (2) Add a second solvent to the system after evaporation in step (1), and then emulsify with ultrasound to obtain polymer nanovesicles; the second solvent is one of perfluorohexane, perfluoropentane, decafluoropentane, and perfluoropropane;

[0023] (3) Add protein drugs that block immune checkpoints to the polymer nanovesicles obtained in step (2), then incubate, centrifuge, and resuspend the precipitate to obtain the nano-ultrasound contrast agent for enhancing immunotherapy.

[0024] The present invention provides a method for preparing a nano-ultrasound contrast agent. A cationic polymer nanocarrier is constructed by solvent evaporation and ultrasonic emulsification of a polycaprolactone-polyethylene glycol-polyethyleneimine polymer. Then, a protein drug that blocks immune checkpoints is loaded using electrostatic layer-by-layer self-assembly technology, thereby preparing the nano-ultrasound contrast agent. The preparation method of the present invention is not only simple and has a high drug loading capacity, but also results in an ultrasound contrast agent with high biocompatibility, small and uniform particle size, long-lasting in vivo circulation, good imaging effect in the extravascular region of tumors, good penetration, strong targeting, and high drug utilization. It can achieve rapid and precise drug delivery to the tumor region under external ultrasound imaging guidance, enhance the effect of immunotherapy, and realize integrated diagnosis and treatment of tumors.

[0025] Furthermore, the nano-ultrasound contrast agent prepared by the above method in this invention, due to the low boiling point of the second solvent, transforms from a liquid to a gaseous state after ultrasonic emulsification, becoming micron-sized bubbles. This change in particle size enhances the ultrasonic echo intensity, achieving the effect of contrast imaging within the tumor. Moreover, the nano-ultrasound contrast agent provided by this invention not only allows for ultrasound imaging before rupture, but also ruptures after entering tumor tissue and undergoing ultrasound irradiation, thereby efficiently targeting tumor cells and enriching the drug at the lesion site, enhancing the immunotherapy effect. Thus, this invention successfully combines ultrasound contrast agents with tumor immunotherapy, achieving integrated diagnosis and treatment of tumors and solving problems such as low tumor responsiveness, slow release, low targeted release rate, and poor in vitro controllability.

[0026] Preferably, the first solvent is one or more of acetone, methanol, ethanol, and dimethyl sulfoxide. More preferably, the first solvent is acetone; and the second solvent is perfluorohexane. Addressing the issues of large size and uneven particle size in currently available ultrasound contrast agents, this invention utilizes the property of the second solvent to transform from a liquid to a nanoscale gaseous state after ultrasonic emulsification, constructing a novel nano-ultrasound contrast agent with uniform size, small particle size, high stability, and high safety. Furthermore, it can load various protein drugs that block immune checkpoints, meeting the needs of various tumor ultrasound diagnosis and / or immunotherapy.

[0027] Preferably, the ratio of polycaprolactone-polyethylene glycol-polyethyleneimine polymer, the first solvent, PBS solution, and the second solvent is (4-6) mg:(0.4-0.6) mL:(1-1.2) mL:(15-25) μL.

[0028] Preferably, in step (1), the stirring and dissolving time is 10 to 30 minutes.

[0029] The method used to evaporate the first solvent can be any conventional solvent removal process in the art, and this invention does not impose any particular limitation. Preferably, the first solvent is evaporated using a rotary evaporation process, and the rotary evaporation time is 2 to 5 minutes.

[0030] Preferably, in step (2), the ultrasonic emulsification time is 0.5 to 2 seconds; the power of the ultrasonic waves used in ultrasonic emulsification is 100 to 150 W.

[0031] Preferably, in step (3), the concentration of the protein drug that blocks immune checkpoints added to the polymer nanovesicles is 2-5 mg / mL; the incubation temperature is -5-5°C and the incubation time is 20-60 min; the resuspension is performed using PBS solution.

[0032] Preferably, in step (3), the centrifugation process conditions are: centrifugation speed 3000-5000 rpm, centrifugation temperature 1-4℃, and centrifugation time 4-10 min.

[0033] The third objective of this invention is achieved by the following technical solution:

[0034] The above-mentioned applications of nano-ultrasound contrast agents for enhancing immunotherapy include their use in the preparation of diagnostic reagents for tumor ultrasound diagnosis and / or pharmaceutical formulations for tumor immunotherapy.

[0035] The overall beneficial effects of this invention are as follows:

[0036] The nano-ultrasound contrast agent provided by this invention is a droplet-type contrast agent with a small particle size and long in vivo circulation time. It can penetrate vascular endothelial cells to reach extravascular targets, targeting tumor tissue and entering the interstitial space through high permeability and retention effects. The second solvent, after ultrasonic emulsification, forms nanodroplets with excellent phase transition properties, changing from liquid to gaseous state under ultrasound irradiation to form gas-containing vesicles. Due to the small size of the nano-contrast agent, it is difficult to achieve good imaging results when dispersed in blood. However, when the nano-contrast agent enters the tumor tissue through blood vessels, it accumulates in the tumor tissue under the action of ultrasound irradiation outside the tumor. This accumulation effectively enhances the imaging effect, making it more suitable for imaging disease tissues requiring targeted imaging. It also allows for real-time monitoring of the internal condition of tumor tissue under in vitro ultrasound conditions, providing strong evidence for tumor diagnosis. Furthermore, the nano-ultrasound contrast agent prepared by this invention can perform targeted imaging and drug delivery at lesion sites under ultrasound guidance. It can be controlled in vitro to enrich the drug at the lesion site for precise delivery, maximizing drug utilization. By allowing the contrast agent provided by this invention to penetrate the vascular system and infiltrate tumor tissue under ultrasound conditions, rapid and precise drug delivery to the targeted tumor region can be achieved under the guidance of external ultrasound contrast imaging, significantly improving the anti-tumor effect of immunotherapy drugs and realizing the integration of tumor diagnosis and treatment under the guidance of in vitro ultrasound contrast imaging. Therefore, the nano-ultrasound contrast agent provided by this invention has broad application prospects in the field of ultrasound molecular imaging and / or tumor immunotherapy. Attached Figure Description

[0037] Figure 1 These are images of the nano-ultrasound contrast agent used to enhance immunotherapy in Embodiment 1 of this invention under a white light microscope;

[0038] Figure 2 These are transmission electron microscope images of the nano-ultrasound contrast agent used to enhance immunotherapy in Embodiment 2 of the present invention;

[0039] Figure 3 These are the OD values ​​corresponding to different times and concentrations of the drug-free nano-ultrasound contrast agent after co-incubation with SW480 cells in Experimental Example 2 of this invention;

[0040] Figure 4 This is a study on the antitumor effect of the nano-ultrasound contrast agent that enhances immunotherapy in Experimental Example 3 of this invention;

[0041] Figure 5 This is the biosafety evaluation result of the drug-free nano-ultrasound contrast agent in Experimental Example 4 of this invention. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are merely further illustrative of the present invention and not intended to limit the invention. Unless otherwise specified, the raw materials and reagents used in the following embodiments are commercially available.

[0043] In the following examples, the polycaprolactone-polyethylene glycol-polyethyleneimine polymer used has an average molecular weight of 2000 g / mol for polycaprolactone, an average molecular weight of 2000 g / mol for polyethylene glycol, and an average molecular weight of 1800 g / mol for polyethyleneimine. This polymer is designated PCL2K-PEG2K-PEI1.8K and was provided by Xi'an Ruixi Biotechnology Co., Ltd., with catalog number R-350K-62.

[0044] Example 1

[0045] This embodiment provides a nano-ultrasound contrast agent for enhancing immunotherapy, comprising: a nanocarrier and a protein drug loading the nanocarrier to block immune checkpoints; wherein, the nanocarrier is a polycaprolactone-polyethylene glycol-polyethyleneimine polymer (PCL2K-PEG2K-PEI1.8K); the protein drug is a PD-L1 monoclonal antibody; and the average particle size of the nano-ultrasound contrast agent is 95.6 nm.

[0046] The preparation method of the nano-ultrasound contrast agent in this embodiment includes the following steps:

[0047] 5 mg of PCL2K-PEG2K-PEI1.8K polymer was placed in a sterile, enzyme-free round-bottom flask. 0.5 mL of acetone was added dropwise, and the mixture was stirred for 15 min. Then, rotary evaporation was performed until the acetone was completely evaporated. Next, 1 mL of calcium- and magnesium-free PBS solution (pH = 6.4) was added to dissolve the acetone, and the mixture was stirred for another 15 min, with 0.5 mL of acetone added dropwise while stirring. After stirring, rotary evaporation was performed again until the acetone was completely evaporated. 20 μL of perfluorohexane was added to the evaporated solution, and the mixture was then emulsified for 1 second under ultrasonic power of 100 W to obtain unloaded polymer nanovesicles. 2 mg of a protein drug (PD-L1 monoclonal antibody) that blocks immune checkpoints was added to unloaded polymer nanovesicles. The loaded nanovesicles were then incubated on ice (-5–5°C) for 30 min, followed by centrifugation at 3400 rpm and 1°C for 4 min. The precipitate was then resuspended in 1 mL of calcium- and magnesium-free PBS solution (pH = 6.4) to obtain the nano-ultrasound contrast agent for enhancing immunotherapy in this embodiment. The white light microscope structure of this nano-ultrasound contrast agent is shown below. Figure 1 As shown. By Figure 1 It can be seen that the prepared nano-ultrasound contrast agent has a uniform spherical morphology.

[0048] Example 2

[0049] This embodiment provides a nano-ultrasound contrast agent for enhancing immunotherapy, comprising: a nanocarrier and a protein drug that blocks immune checkpoints loaded on the nanocarrier; wherein the nanocarrier is a polycaprolactone-polyethylene glycol-polyethyleneimine polymer (PCL2K-PEG2K-PEI1.8K); and the protein drug is a CTLA-4 monoclonal antibody. The preparation method of the nano-ultrasound contrast agent in this embodiment is basically the same as in Example 1, except that a CTLA-4 monoclonal antibody is used instead of a PD-L1 monoclonal antibody. A transmission electron microscope (TEM) image of the nano-ultrasound contrast agent in this embodiment is shown below. Figure 2 As shown. By Figure 2 It can be seen that the CTLA-4 protein is adsorbed on the surface of the nanovesicles, indicating that the prepared nano-ultrasound contrast agent was successfully loaded with the CTLA-4 protein.

[0050] Experiment Example 1: Particle Size and Particle Concentration Testing of Nano-Ultrasound Contrast Agents for Enhancing Immunotherapy

[0051] The particle size and concentration of the nano-ultrasound contrast agent in Example 1 were measured using an NTA (Zetaview X30) nanoparticle tracking analyzer. The results showed that the average particle size of the nano-ultrasound contrast agent prepared in Example 1 was 95.6 nm, and the average particle concentration was 6.6 × 10⁻⁶. 10 per mL.

[0052] Experiment Example 2: Cytotoxicity Assessment Experiment of Drug-Free Nano-Ultrasound Contrast Agent

[0053] Following the preparation method in Example 1, drug-free nano-ultrasound contrast agents of different concentrations were prepared. The cytotoxicity of different concentrations of drug-free nano-ultrasound contrast agents (0, 50, 100, 200, 400, 600 μg / mL) co-incubated with SW480 cells for different times (0, 12, 24, 36, 48 h) was evaluated using the CCK8 assay. The absorbance changes at 450 nm at different time points and at different concentrations were recorded using a microplate reader. The experimental results are as follows: Figure 3 As shown. Figure 3 In the middle, the bar groups corresponding to the same incubation time, from left to right, are the test results at concentrations of control, 50, 100, 200, 400, and 600 ug / mL.

[0054] Depend on Figure 3It can be seen that, within the same incubation time, the OD values ​​of various drug concentrations remain essentially unchanged with increasing drug concentration, indicating that the nano-ultrasound contrast agent provided by this invention has no cytotoxicity. Furthermore, at the same concentration, the OD value gradually increases with increasing incubation time, and the cytotoxicity gradually decreases and remains below that of the control group, proving that the drug-free nano-ultrasound contrast agent carrier has no cytotoxicity.

[0055] Experimental Example 3: Study on the Antitumor Effect of Nano-Ultrasound Contrast Agents Enhancing Immunotherapy

[0056] Male Balb / c mice that had successfully inoculated with SW480 cells and bore tumors were randomly divided into four groups of six mice each: (1) saline group; (2) free PD-L1 monoclonal antibody (Anti-PDL1) group; (3) ultrasound + unloaded nano-ultrasound contrast agent (UTMD) group; and (4) ultrasound + drug-loaded nano-ultrasound contrast agent (UTMD+Anti-PDL1) group (Example 1). Each group received 100 μL of the different formulation at a concentration of 5 μg / mL (the amount of PD-L1 monoclonal antibody) via tail vein injection, administered every three days for 16 days. Simultaneously, for groups requiring ultrasound stimulation, an ultrasound pulse was applied 1 W / cm² 1 hour after administration. 2 The tumor site was stimulated with ultrasound for 10 minutes. The tumor volume in mice was measured every two days, and the results were as follows: Figure 4 As shown.

[0057] Depend on Figure 4 The test results showed no significant difference between the ultrasound-enhanced unloaded nano-ultrasound contrast agent (UTMD) group and the saline group, indicating that ultrasound-enhanced unloaded nano-ultrasound contrast agent could not effectively inhibit tumor growth. The free PD-L1 monoclonal antibody (Anti-PDL1) group also showed some tumor therapeutic effect, proving that PD-L1 monoclonal antibodies can effectively block the PD-1 / PD-L1 signaling pathway and inhibit tumor cell proliferation. However, the tumor volume proliferation in the ultrasound-enhanced drug-loaded nano-ultrasound contrast agent (UTMD+Anti-PDL1) experimental group of Example 1 was the slowest and showed a decreasing trend, indicating that the ultrasound-enhanced drug-loaded nano-ultrasound contrast agent (UTMD+Anti-PDL1) of Example 1 had the best anti-tumor effect. This phenomenon indicates that the vaporization of the second solvent after ultrasonic irradiation produces an ultrasonic cavitation effect, releasing the loaded PD-L1 monoclonal antibody around the tumor cells. The released PD-L1 monoclonal antibody can enter the blood vessel wall and even the interstitial space, specifically target and bind to the PD-L1 protein on the surface of tumor cells, block the PD-1 / PD-L1 immune checkpoint, improve the immune microenvironment inside the tumor tissue, and ultimately enhance the inhibitory effect on the tumor.

[0058] Experimental Example 4: Biosafety Study of Drug-Free Nano-Ultrasound Contrast Agents

[0059] After the mice in Experiment 3 were treated with saline and ultrasound plus drug-free nano-ultrasound contrast agent (UTMD), blood was collected from their orbits. The blood samples were subjected to routine blood tests and biochemical analysis. The results are shown in Tables 1 and 2.

[0060] Table 1. Analysis of mouse blood routine results

[0061]

[0062] Table 2 Analysis of mouse blood biochemical results

[0063]

[0064]

[0065] The experimental results in Tables 1 and 2 show that all indicators in the blood of mice in each group were within the normal range, demonstrating that the mice treated with ultrasound and the drug-free nano-ultrasound contrast agent had good biocompatibility. This also confirms that the nano-ultrasound contrast agent of this invention has good biocompatibility.

[0066] Mice in the ultrasound plus drug-free nano-ultrasound contrast agent (UTMD) group were sacrificed after blood collection, and tissue sections of the heart, liver, spleen, lung, and kidney were collected for HE observation (scale bar: 50 μm). Results are as follows: Figure 5 As shown. By Figure 5 It was found that no abnormalities in cell structure, morphology, or histology were observed in any of the mouse organs, proving that the drug-free nano-ultrasound contrast agent has good in vivo safety and can be further applied to in vivo treatment.

[0067] In summary, the nano-ultrasound contrast agent for enhancing immunotherapy provided by this invention is a novel type of ultrasound nanovesicle formed by loading protein drugs that block immune checkpoints onto a nanocarrier with a special composition. It not only has high biocompatibility, small and uniform particle size, and long-term circulation in vivo, but also has good imaging effect in the extravascular domain of tumors, good penetration, strong targeting, and high drug utilization. It can realize the integration of ultrasound-guided tumor diagnosis and immunotherapy while improving the delivery efficiency and immune effect of tumor immunotherapy drugs, effectively solving the current problems such as difficulty in tumor ultrasound diagnosis, low tumor response of immunotherapy drugs, weak targeting, and inability to regulate in vitro.

Claims

1. A nano-ultrasound contrast agent for enhancing immunotherapy, characterized in that, The nano-ultrasound contrast agent comprises: a nanocarrier, a second solvent, and a protein drug loaded on the nanocarrier to block immune checkpoints; The nanocarrier is a polycaprolactone-polyethylene glycol-polyethyleneimine polymer; the second solvent is one of perfluorohexane, perfluoropentane, decafluoropentane, and perfluoropropane; the protein drug is one or more of PD-1 monoclonal antibody, PD-L1 monoclonal antibody, and CTLA-4 monoclonal antibody. In the polycaprolactone-polyethylene glycol-polyethyleneimine polymer, the average molecular weight of polycaprolactone is 1900-2200 g / mol, the average molecular weight of polyethylene glycol is 1900-2200 g / mol, and the average molecular weight of polyethyleneimine is 1700-1850 g / mol. The nano-ultrasound contrast agent used to enhance immunotherapy is a droplet-type contrast agent; the protein drug that blocks immune checkpoints is loaded onto the surface of the nanocarrier using electrostatic layer-by-layer self-assembly technology.

2. The nano-ultrasound contrast agent for enhancing immunotherapy according to claim 1, characterized in that, In the polycaprolactone-polyethylene glycol-polyethyleneimine polymer, the average molecular weight of polycaprolactone is 2000 g / mol, the average molecular weight of polyethylene glycol is 2000 g / mol, and the average molecular weight of polyethyleneimine is 1800 g / mol.

3. The nano-ultrasound contrast agent for enhancing immunotherapy according to claim 1, characterized in that, The protein drug in question is a PD-L1 monoclonal antibody.

4. The nano-ultrasound contrast agent for enhancing immunotherapy according to claim 1, characterized in that, The average particle size of the nano-ultrasound contrast agent is 80–120 nm.

5. A method for preparing a nano-ultrasound contrast agent for enhancing immunotherapy as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve the polycaprolactone-polyethylene glycol-polyethyleneimine polymer in the first solvent by stirring, and then evaporate the first solvent to dryness; add PBS solution to the evaporated system by stirring and dissolving, add the first solvent again by stirring, and evaporate the first solvent to dryness again; the first solvent is one or more of acetone, methanol, ethanol, and dimethyl sulfoxide; (2) Add a second solvent to the system after evaporation in step (1), and then emulsify with ultrasound to obtain polymer nanovesicles; the second solvent is one of perfluorohexane, perfluoropentane, decafluoropentane, and perfluoropropane; (3) Add protein drugs that block immune checkpoints to the polymer nanovesicles obtained in step (2), then incubate, centrifuge, and resuspend the precipitate to obtain the nano-ultrasound contrast agent for enhancing immunotherapy.

6. The method for preparing the nano-ultrasound contrast agent for enhancing immunotherapy according to claim 5, characterized in that, The ratio of polycaprolactone-polyethylene glycol-polyethyleneimine polymer, first solvent, PBS solution, and second solvent is (4-6) mg:(0.4-0.6) mL:(1-1.2) mL:(15-25) μL.

7. The method for preparing the nano-ultrasound contrast agent for enhancing immunotherapy according to claim 5, characterized in that, In step (2), the ultrasonic emulsification time is 0.5 to 2 seconds; the power of the ultrasonic waves used in ultrasonic emulsification is 100 to 150 W.

8. The method for preparing the nano-ultrasound contrast agent for enhancing immunotherapy according to claim 5, characterized in that, In step (3), the concentration of the protein drug that blocks immune checkpoints added to the polymer nanovesicles is 2-5 mg / mL; the incubation temperature is -5-5℃ and the incubation time is 20-60 min; the resuspension is performed using PBS solution.

9. The application of a nano-ultrasound contrast agent for enhancing immunotherapy as described in any one of claims 1 to 4, characterized in that, Applications in the preparation of diagnostic reagents for tumor ultrasound diagnosis and / or pharmaceutical formulations for tumor immunotherapy.

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