Nanometer ultrasonic contrast agent for enhancing immunotherapy as well as preparation method and application of nanometer ultrasonic contrast agent

Abstract

The invention belongs to the technical field of ultrasonic molecular imaging and biomedical engineering, and particularly relates to a nano ultrasonic contrast agent for enhancing immunotherapy as well as a preparation method and application of the nano ultrasonic contrast agent. The nano ultrasonic contrast agent for enhancing immunotherapy, provided by the invention, is a novel ultrasonic nano vesicle formed by adopting a nano carrier with special composition to load a protein drug for blocking an immune checkpoint, is high in biological safety, small and uniform in particle size, and capable of long-acting circulation in vivo; the tumor extravascular field imaging effect is good, the penetrating power is good, the targeting performance is high, the medicine utilization rate is high, the tumor immunotherapy medicine delivery efficiency and the immune effect can be improved, and meanwhile the integration of tumor diagnosis and immunotherapy under ultrasonic guidance can be achieved; the problems that tumor ultrasonic diagnosis is difficult at present, and immunotherapy drugs are low in tumor responsiveness, weak in targeting performance, incapable of being regulated in vitro and the like are effectively solved.

Description

Technical Field

[0001] The present invention belongs to the field of ultrasonic molecular imaging and biomedical engineering technology, and in particular relates to a nano ultrasonic contrast agent for enhancing immunotherapy, and a preparation method and application thereof. Background Art

[0002] Cancer, as one of the leading causes of human death, has become a major threat to human life and public health. At present, the global cancer rate is increasing year by year. It has become a major problem that needs to be solved globally to detect cancer as early and accurately as possible and to treat it in time to reduce cancer mortality. However, at this stage, the diagnosis process of clinical tumors is often separated from the treatment process, and due to the constraints of some technologies and means, the diagnosis and treatment of tumors are greatly restricted, thereby delaying the best treatment time for patients and reducing the treatment effect. Therefore, organically combining the diagnosis or monitoring of tumors with treatment to form a unified treatment system and realizing integrated diagnosis and treatment can greatly improve the survival rate of tumor patients.

[0003] At present, the commonly used medical imaging diagnostic methods in clinical practice mainly include ultrasound, CT, X-ray and magnetic resonance imaging. Among them, ultrasound technology has become the primary means of cancer diagnosis and examination due to its advantages such as low price, non-invasiveness, no radiation, and real-time imaging. However, the low-resolution traditional ultrasound imaging technology limits further detection of tissues and organs. The emergence of ultrasound contrast agents (UCA) as a major advancement in the field of ultrasound diagnosis in recent years has provided a very favorable basis for the identification and diagnosis of various diseases, especially tumor diseases. UCA ultrasound diagnostic technology uses blood perfusion information to distinguish normal tissues from diseased tissues by intravenous injection of ultrasound contrast agents, effectively enhancing the sensitivity and specificity of disease diagnosis. The ultrasound contrast agents currently used in clinical practice are artificially synthesized gas-encapsulated microbubbles, which are mostly 1-10um in size, uneven in particle size, poor in stability, lack of targeting, lack of specificity for tissues, and cannot penetrate the human reticuloendothelial system to enter the tissues. They can only perform blood pool angiography. Therefore, this type of contrast agent is limited in the application of ultrasound molecular imaging. In addition, high-intensity focused ultrasound is used to focus the sound beam to generate instantaneous high temperature to cause irreversible necrosis of tumor cells in the focal area, which can achieve the effect of ablating tumors. Therefore, ultrasound treatment technology has gradually been applied in clinical practice. However, it is difficult for a single imaging technology to meet the needs of clinical diagnosis and treatment of major diseases such as tumors. In order to solve the problem that micron-level ultrasound contrast agents can only be used for imaging or treatment within tumor blood vessels, and the existing nano-level ultrasound contrast agents have poor imaging effects and cannot be used for treatment. The present invention attempts to develop a new type of targeted drug-loaded nano-ultrasound contrast agent, which can not only actively target imaging, but also use its concentrated imaging performance to produce ultra-strong echo characteristics to achieve efficient tumor treatment.

[0004] With the gradual development of tumor immunology, immunotherapy that uses immune pathways to treat cancer is rapidly becoming a recognized cancer treatment method after chemotherapy, radiotherapy and surgery. Among them, the success of immune checkpoint blockade therapy in clinical practice has made tumor immunotherapy more and more widely concerned. 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 has attracted widespread attention. In the tumor microenvironment, the PD-1 / PD-L1 interaction can inhibit lymphocyte proliferation and activation, induce antigen-specific T cell apoptosis, etc., and has the effect of negative immune regulation, leading to T cell dysfunction and mediating immunosuppression. PD-L1 monoclonal antibody drugs can be applied to a variety of solid tumors, and have the advantages of inhibiting tumor recurrence and metastasis, being able to be used to treat advanced tumors and having relatively few side effects. However, the single-drug administration of PD-L1 monoclonal antibodies has the disadvantages of low utilization rate, poor targeting, and short in vivo circulation time, which limits its further application.

[0005] In summary, the development of a new type of nano-ultrasound contrast agent with high biosafety, good imaging effect in the extravascular area of ​​tumors, good penetration, strong targeting, high drug utilization rate, and the ability to enhance the effect of immunotherapy is of great significance for improving the efficiency of tumor immunotherapy drug delivery and realizing the integration of tumor diagnosis and immunotherapy under ultrasound guidance. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a nano-ultrasound contrast agent for enhancing immunotherapy, which is a new type of ultrasonic nanovesicle formed by a specially composed nano-carrier loaded with a protein drug that blocks the immune checkpoint. It not only has high biosafety, small and uniform particle size, and long-term circulation in the body, but also has good imaging effect in the extravascular area of ​​the tumor, good penetration, strong targeting, and high drug utilization rate. It can improve the delivery efficiency of tumor immunotherapy drugs and the immune effect while realizing the integration of tumor diagnosis and immunotherapy under ultrasound guidance, effectively solving the current difficulties in ultrasonic diagnosis of tumors, low tumor responsiveness of immunotherapy drugs, weak targeting, and inability to be regulated in vitro.

[0007] The second purpose of the present invention is to provide a method for preparing the above-mentioned nano-ultrasound contrast agent for enhancing immunotherapy. The process is simple and the obtained contrast agent has a high drug loading capacity. It can achieve rapid and accurate drug delivery to targeted tumor areas under the guidance of external ultrasonic imaging, effectively solving the current problems of complex preparation of ultrasonic contrast agents, difficulty in ultrasonic diagnosis of tumors, low tumor responsiveness of immunotherapy drugs, slow release and inability to be regulated in vitro, and realizing the integration of tumor diagnosis and immunotherapy under ultrasound guidance.

[0008] The third object of the present invention is to provide the application of the above-mentioned nano-ultrasound contrast agent for enhancing immunotherapy.

[0009] One of the purposes of the present invention is achieved by the following technical solution:

[0010] A nano-ultrasound contrast agent for enhancing immunotherapy, the nano-ultrasound contrast agent comprising: a nano-carrier and a protein drug for blocking immune checkpoints loaded by the nano-carrier;

[0011] Wherein, 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-polyethylene imine high molecular weight 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 polyethylene imine is 1700-1850 g / mol.

[0013] The nano-ultrasound contrast agent provided by the present invention adopts polycaprolactone-polyethylene glycol-polyethyleneimine macromolecular polymer (PCL-PEG-PEI) as a drug carrier and is loaded with protein drugs that block immune checkpoints.

[0014] Among them, in the drug carrier used in the present invention, polycaprolactone (PCL) belongs to polymeric polyester, has excellent properties such as good biocompatibility, biodegradability, and high safety, and is a widely used biodegradable slow and controlled release carrier. Polyhexylene glycol (PEG) is a hydrophilic polymer, non-toxic and non-immunogenic in vivo, and its modified nanoparticles have good stability, and can also avoid being recognized by the reticuloendothelial system in the human body, prolonging the circulation time in the body. The present invention further combines cationic-rich polyethyleneimine (PEI) to form a polycaprolactone-polyethylene glycol-polyethyleneimine ternary polymer based on the good biocompatibility, biodegradability, and high safety of PCL and the long-circulating non-immunogenic polymer material PEG. In this process, the present invention greatly guarantees the safety of nanocarriers by controlling the structural composition and molecular weight of the polymer material. Moreover, the prepared nanocarrier has a long-acting circulation performance in the body, and its non-immunogenic performance ensures the efficient performance of the drug efficacy carried by the drug, and the drug can be quickly and safely degraded in the body after the drug functions, which not only ensures the safe and efficient delivery of the drug by the nanocarrier, but also ensures safe degradation in the body. Therefore, the nanocarrier used in the present invention can enhance the interaction between the drug molecules and the polymer molecules, improve the drug stability, and reduce the toxicity in the body, so as to achieve the effect of sustained release in the tumor.

[0015] Further, the drug loaded on the drug carrier of the present invention is a protein drug (such as PD-L1 monoclonal antibody, etc.) that blocks immune checkpoints. By loading the above-mentioned protein drug that blocks immune checkpoints, after tail vein injection, a certain amount of energy of ultrasonic irradiation is given to the treatment site or the tumor locally through the body surface, so that the vesicles produce compression and expansion phenomena under ultrasound, triggering ultrasonic cavitation effect, causing the vesicles to rupture and release the protein drugs carried around the tumor cells, and the released protein drugs can enter the vascular wall or even the interstitial space, specifically targeting the PD-L1 protein on the surface of the tumor cells and combining with it, so that the nano-ultrasound contrast agent gathers around the tumor tissue, blocking the PD-1 / PD-L1 immune checkpoints, so as to improve the internal immune microenvironment of the tumor tissue, and achieve the purpose of killing tumor cells. At the same time, the ultrasonic irradiation formed during the ultrasound contrast diagnosis process can cause mechanical vibration to destroy the structure of the cancer cell membrane, resulting in an increase in the probability of exposure of the PD-1 antigen on the surface of the cancer cell membrane, thereby changing 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 to 120 nm. Further preferably, the average particle size of the nano-ultrasound contrast agent is 90 to 100 nm.

[0019] The second object of the present invention is achieved by adopting the following technical solution:

[0020] The method for preparing the nano-ultrasound contrast agent for enhancing immunotherapy comprises the following steps:

[0021] (1) stirring and dissolving a polycaprolactone-polyethylene glycol-polyethyleneimine polymer in a first solvent, and then evaporating the first solvent; adding a PBS solution to the evaporated system and stirring and dissolving it, adding the first solvent again and stirring, and evaporating the first solvent again;

[0022] (2) adding a second solvent to the system after evaporation again in step (1), and then ultrasonic emulsifying to obtain polymer nanovesicles; the second solvent is one of perfluorohexane, perfluoropentane, decafluoropentane, and perfluoropropane;

[0023] (3) adding a protein drug that blocks immune checkpoints to the polymer nanovesicles obtained in step (2), followed by incubation, centrifugation, and resuspending the precipitate to obtain the nano-ultrasound contrast agent for enhancing immunotherapy.

[0024] The preparation method of the nano ultrasonic contrast agent provided by the present invention is to construct a nano carrier in the form of a cationic polymer by solvent evaporation and ultrasonic emulsification of a polycaprolactone-polyethylene glycol-polyethyleneimine polymer, and then load a protein drug that blocks the immune checkpoint by electrostatic layer-by-layer self-assembly technology, thereby preparing a nano ultrasonic contrast agent. The preparation method of the present invention not only has a simple preparation process and a high drug loading capacity, but also has high biological safety, small and uniform particle size, long-term circulation in the body, good imaging effect in the field outside the tumor blood vessels, good penetration, strong targeting, and high drug utilization. It can achieve rapid and accurate drug delivery to the targeted tumor area under the guidance of external ultrasonic imaging, enhance the immunotherapy effect, and realize the integrated diagnosis and treatment of tumors.

[0025] Furthermore, the nano-ultrasound contrast agent prepared by the above method of the present invention, due to the low boiling point of the second solvent, will change from liquid to gas after ultrasonic emulsification, becoming micron-sized bubbles, and the change in particle size will enhance the intensity of its ultrasonic echo, achieving the effect of contrast imaging inside the tumor. In addition, the nano-ultrasound contrast agent provided by the present invention can not only be imaged by ultrasound machine before rupture, but also rupture after entering the tumor tissue and being irradiated by ultrasound, so as to efficiently target tumor cells and enrich drugs at the lesion, thereby enhancing the immunotherapy effect. As a result, the present invention successfully combines ultrasound contrast agents with tumor immunotherapy, realizes the integrated diagnosis and treatment of tumors, and solves the problems of 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. Further preferably, the first solvent is acetone; the second solvent is perfluorohexane. In view of the large size and uneven particle size of ultrasound contrast agents currently on the market, the present invention utilizes the second solvent to change from liquid to nano-scale gas after ultrasonic emulsification to construct a new type of nano-ultrasound contrast agent with uniform size, small particle size, strong stability and high safety, and it can load various protein drugs that block immune checkpoints to meet the needs of ultrasound diagnosis and / or immunotherapy of various tumors.

[0027] Preferably, the usage ratio of polycaprolactone-polyethylene glycol-polyethyleneimine polymer, the first solvent, the 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 may be a conventional solvent removal process in the art, and the present invention does not specifically limit it. Preferably, the first solvent is evaporated by 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 wave used in the ultrasonic emulsification is 100 to 150W.

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

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

[0033] The third object of the present invention is achieved by adopting the following technical solution:

[0034] The above-mentioned nano-ultrasound contrast agent for enhancing immunotherapy is used in the preparation of diagnostic reagents for tumor ultrasound diagnosis and / or pharmaceutical preparations for tumor immunotherapy.

[0035] The comprehensive beneficial effects of the present invention are:

[0036] The nano ultrasonic contrast agent provided by the present invention is a droplet-type contrast agent, which has a small particle size and a long circulation time in the body. It can pass through the vascular endothelial cells to reach the target outside the blood vessel, target the tumor tissue through high permeability and retention effect, and enter the gap of the tumor tissue. After the second solvent is ultrasonically emulsified, a nano droplet is formed, which has good phase change performance, and changes from liquid to gas under ultrasonic irradiation to form gas-containing vesicles. Due to the small size of the nano contrast agent, it is difficult to achieve a good imaging effect when dispersed in the blood. When the nano contrast agent enters the tumor tissue through the blood vessel, under the action of ultrasonic irradiation outside the tumor tissue, the nano contrast agent is enriched at the tumor tissue, and the imaging effect is effectively exerted through the aggregation of the contrast agent, which is more suitable for imaging of disease tissues that require targeted imaging, and can also monitor the internal situation of the tumor tissue in real time under in vitro ultrasound conditions, providing strong evidence for tumor diagnosis. In addition, the nano ultrasonic contrast agent prepared by the present invention can perform fixed-point imaging and targeted drug delivery to the lesion site under the guidance of ultrasonic imaging, and can be regulated in vitro to enrich the drug at the lesion site to achieve precise delivery, thereby achieving maximum drug utilization. By allowing the contrast agent provided by the present invention to penetrate into the tumor tissue through the vascular system under ultrasound conditions, rapid and accurate drug delivery to the targeted tumor area can be achieved under the guidance of external ultrasound contrast imaging conditions, 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 conditions. Therefore, the nano-ultrasound contrast agent provided by the present invention has broad application prospects in the field of ultrasonic molecular imaging and / or tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is an image of the nano-ultrasound contrast agent for enhancing immunotherapy in Example 1 of the present invention under a white light microscope;

[0038] Figure 2 This is a transmission electron microscope image of the nano-ultrasound contrast agent for enhancing immunotherapy in Example 2 of the present invention;

[0039] Figure 3 is the OD value corresponding to different time and concentration after the non-drug-loaded nano-ultrasound contrast agent and SW480 cells were co-incubated in Experimental Example 2 of the present invention;

[0040] Figure 4 This is a study on the anti-tumor effect of the nano-ultrasound contrast agent for enhancing immunotherapy in Experimental Example 3 of the present invention;

[0041] Figure 5 This is the biosafety evaluation result of the non-drug-loaded nano-ultrasound contrast agent in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0042] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only further explanations of the present invention, rather than limitations of the present invention. Unless otherwise specified, the raw materials and reagents used in the following examples can be obtained from commercial channels.

[0043] Among them, in the polycaprolactone-polyethylene glycol-polyethyleneimine polymer used in the following examples, 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. The polymer is denoted as PCL2K-PEG2K-PEI1.8K, provided by Xi'an Ruixi Biotechnology Co., Ltd., item number R-350K-62.

[0044] Example 1

[0045] The present embodiment provides a nano-ultrasound contrast agent for enhancing immunotherapy, which includes: a nano-carrier and a protein drug for blocking immune checkpoints loaded on the nano-carrier; wherein the nano-carrier 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 method for preparing the nano-ultrasound contrast agent of this embodiment comprises the following steps:

[0047] Take 5mg of PCL2K-PEG2K-PEI1.8K polymer in a sterile enzyme-free round-bottom flask, add 0.5mL of acetone, stir for 15min and then perform rotary evaporation until the acetone in the flask is completely volatilized, then add 1mL of calcium-magnesium-free PBS solution (pH=6.4) to dissolve and then stir for 15min, and add 0.5mL of acetone while stirring during this stirring process. After stirring, perform rotary evaporation again until the acetone is completely volatilized, add 20uL of perfluorohexane to the solution after rotary evaporation, and then emulsify for 1s under the condition of ultrasonic power of 100W to obtain polymer nanovesicles without drug loading. 2 mg of protein drugs (PD-L1 monoclonal antibody) that block immune checkpoints were added to the unloaded polymer nanovesicles, and the loaded nanovesicles were placed on ice (-5 to 5°C) for 30 minutes, and then centrifuged at 3400 rpm and 1°C for 4 minutes, and then the precipitate was 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 the nano-ultrasound contrast agent is shown in the figure below. Figure 1 As shown. 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, which includes: a nano-carrier and a protein drug that blocks immune checkpoints loaded on the nano-carrier; wherein the nano-carrier 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 of this embodiment is basically the same as that of Example 1, except that CTLA-4 monoclonal antibody is used instead of PD-L1 monoclonal antibody. The transmission electron microscope (TEM) image of the nano-ultrasound contrast agent of this embodiment is as follows: Figure 2 As shown. 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 is successfully loaded with the CTLA-4 protein.

[0050] Experimental Example 1: Particle Size and Particle Concentration Test of Nano-ultrasound Contrast Agents for Immunotherapy Enhancement

[0051] The particle size and concentration of the nano-ultrasound contrast agent prepared in Example 1 were tested using an NTA (Zetaview X30) nanoparticle tracking analyzer. The test 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 10 Pieces / mL.

[0052] Experimental Example 2: Cytotoxicity Evaluation Experiment of Non-drug-loaded Nano-ultrasound Contrast Agent

[0053] Referring to the preparation method of Example 1, different concentrations of non-drug-loaded nano-ultrasound contrast agents were prepared. The cytotoxicity of different concentrations of non-drug-loaded nano-ultrasound contrast agents (0, 50, 100, 200, 400, 600 ug / mL) after incubation with SW480 cells for different time periods (0, 12, 24, 36, 48 h) was evaluated using the CCK8 method, and the absorbance changes at 450 nm at different time points and different concentrations were recorded using an ELISA instrument. The experimental results are shown in Figure 2. Figure 3 shown. Figure 3 In the figure, the bar groups corresponding to the same incubation time are the test results at control, 50, 100, 200, 400, and 600ug / mL concentrations from left to right.

[0054] Depend on Figure 3It can be seen that with the increase of drug concentration during the same incubation time, the OD value of each concentration remains basically unchanged, indicating that the nano-ultrasound contrast agent provided by the present invention has no cytotoxicity. In addition, at the same concentration, with the increase of incubation time, the OD value gradually increases, and the cytotoxicity gradually decreases and is below the toxicity of the control group, proving that the nano-ultrasound contrast agent carrier without drug loading has no cytotoxicity.

[0055] Experimental Example 3: Study on the anti-tumor effect of nano-ultrasound contrast agents for enhancing immunotherapy

[0056] Male Balb / c mice inoculated with SW480 cells and successfully bearing tumors were randomly divided into 4 groups, with 6 mice in each group. The experimental groups were: (1) normal saline group; (2) free PD-L1 monoclonal antibody (Anti-PDL1) group; (3) ultrasound + unloaded nano-ultrasound contrast agent (UTMD) group; (4) ultrasound + drug-loaded nano-ultrasound contrast agent of Example 1 (UTMD+Anti-PDL1) group. Each group was given 100uL of different preparations with a concentration of 5ug / mL (the amount of PD-L1 monoclonal antibody) through tail vein injection, once every 3 days, and the treatment time was 16 days. At the same time, for the groups that require ultrasonic stimulation, 1h after the administration of the preparation, a power of 1W / cm 2 The tumor site was stimulated by ultrasound for 10 minutes. The volume of the mouse tumor was measured every two days. Figure 4 shown.

[0057] Depend on Figure 4 The test results show that there is no significant difference between the ultrasound plus unloaded nano-ultrasound contrast agent (UTMD) group and the saline group, indicating that the ultrasound plus unloaded nano-ultrasound contrast agent cannot effectively inhibit tumor growth. The free PD-L1 monoclonal antibody (Anti-PDL1) group also has a certain tumor treatment effect, proving that the PD-L1 monoclonal antibody can effectively block the PD-1 / PD-L1 signaling pathway and inhibit the proliferation of tumor cells. However, the tumor volume of the experimental group of the ultrasound-loaded nano-ultrasound contrast agent (UTMD+Anti-PDL1) of Example 1 of the present invention has the slowest growth and a decreasing trend, indicating that the ultrasound-loaded nano-ultrasound contrast agent (UTMD+Anti-PDL1) of Example 1 has the best anti-tumor effect. This phenomenon indicates that after ultrasonic irradiation, the second solvent vaporizes to produce ultrasonic cavitation effect, releasing the carried PD-L1 monoclonal antibody around the tumor cells. The released PD-L1 monoclonal antibody can enter the blood vessel wall and even the tissue gap, specifically targeting the PD-L1 protein on the surface of tumor cells and binding to it, blocking the PD-1 / PD-L1 immune checkpoints to improve the immune microenvironment inside the tumor tissue, and ultimately enhancing the inhibitory effect on the tumor.

[0058] Experimental Example 4: Study on the biosafety of non-drug-loaded nano-ultrasound contrast agents

[0059] After the treatment in the saline group and the ultrasound plus un-drug-loaded nano-ultrasound contrast agent (UTMD) group in Experimental Example 3, blood was collected from the eye sockets of the mice. The blood was subjected to routine blood tests and biochemical analyses. The results are shown in Tables 1 and 2.

[0060] Table 1 Analysis of blood routine test results of mice

[0061]

[0062] Table 2 Analysis of mouse blood biochemical results

[0063]

[0064]

[0065] From the experimental results in Table 1 and Table 2, it can be seen that the indicators in the blood of each group of mice are within the normal range, proving that the mice treated with ultrasound and non-drug-loaded nano-ultrasound contrast agents have good biosafety. This also proves that the nano-ultrasound contrast agent of the present invention has good biosafety.

[0066] The mice in the ultrasound plus unloaded nano-ultrasound contrast agent (UTMD) group were further sacrificed after blood was drawn, and heart, liver, spleen, lung, and kidney tissue sections were taken for HE observation, with a scale of 50 μm. Figure 5 As shown. Figure 5 It can be seen that there were no abnormalities in the cell structure, morphology and histology of the mouse organs, which proves that the drug-free nano-ultrasound contrast agent has good in vivo safety and can be further used in in vivo treatment.

[0067] In summary, the nano-ultrasound contrast agent for enhancing immunotherapy provided by the present invention is a new type of ultrasonic nanovesicle formed by a specially composed nano-carrier loaded with a protein drug that blocks the immune checkpoint. It not only has high biosafety, small and uniform particle size, and can circulate in the body for a long time, but also has good imaging effect in the extravascular area of ​​the tumor, good penetration, strong targeting, and high drug utilization rate. It can improve the delivery efficiency of tumor immunotherapy drugs and the immune effect while realizing the integration of tumor diagnosis and immunotherapy under ultrasound guidance, effectively solving the current problems of difficulty in ultrasonic diagnosis of tumors, low tumor responsiveness of immunotherapy drugs, weak targeting, and inability to be regulated in vitro.

Claims

1. A nano-ultrasound contrast agent for enhancing immunotherapy, characterized in that: The nano-ultrasound contrast agent comprises: a nano-carrier and a protein drug loaded by the nano-carrier and blocking immune checkpoints; Wherein, 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; In the polycaprolactone-polyethylene glycol-polyethylene imine high molecular weight 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 polyethylene imine is 1700-1850 g / mol.

2. The nano-ultrasound contrast agent for enhancing immunotherapy according to claim 1, characterized in that: In the polycaprolactone-polyethylene glycol-polyethylene imine high molecular weight 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 polyethylene imine is 1800 g / mol.

3. The nano-ultrasound contrast agent for enhancing immunotherapy according to claim 1, characterized in that: The protein drug 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 claimed in claim 1, characterized in that: The following steps are involved: (1) stirring and dissolving a polycaprolactone-polyethylene glycol-polyethyleneimine polymer in a first solvent, and then evaporating the first solvent; adding a PBS solution to the evaporated system and stirring and dissolving it, adding the first solvent again and stirring, and evaporating the first solvent again; (2) adding a second solvent to the system after evaporation again in step (1), and then ultrasonic emulsifying to obtain polymer nanovesicles; the second solvent is one of perfluorohexane, perfluoropentane, decafluoropentane, and perfluoropropane; (3) adding a protein drug that blocks immune checkpoints to the polymer nanovesicles obtained in step (2), followed by incubation, centrifugation, and resuspending the precipitate to obtain the nano-ultrasound contrast agent for enhancing immunotherapy.

6. The method for preparing a nano-ultrasound contrast agent for enhancing immunotherapy according to claim 5, characterized in that: The first solvent is one or more of acetone, methanol, ethanol, and dimethyl sulfoxide.

7. The method for preparing a nano-ultrasound contrast agent for enhancing immunotherapy according to claim 5, characterized in that: The usage ratio of polycaprolactone-polyethylene glycol-polyethyleneimine high molecular 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.

8. The method for preparing a 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 wave used in the ultrasonic emulsification is 100 to 150W.

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

10. A use of the nano-ultrasound contrast agent for enhancing immunotherapy as claimed in any one of claims 1 to 4, characterized in that: Application in the preparation of diagnostic reagents for tumor ultrasound diagnosis and / or pharmaceutical preparations for tumor immunotherapy.

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