Therapeutic ultrasonic microbubble for tumor ultrasonic treatment and preparation method thereof

Abstract

The invention relates to the technical field of tumor treatment, and discloses a treatment type ultrasonic microbubble for tumor ultrasonic treatment and a preparation method thereof.The treatment type ultrasonic microbubble is formed by combining an ultrasonic contrast agent microbubble and a novel treatment factor, a shell of the treatment type ultrasonic microbubble for tumor ultrasonic treatment is made of PCL-PEG-RGD copolymer, the RGD peptide fragment can be specifically combined with a tumor cell surface integrin receptor, so that precise targeting is realized, and the aggregation concentration of a therapeutic factor at a tumor part is improved to enhance the curative effect; the novel therapeutic factor ETV contains a tumor specific activation fragment and is only activated by enzymolysis in a tumor microenvironment, so that damage to normal tissues can be avoided, toxic and side effects can be reduced, and treatment safety can be guaranteed; the microbubbles and the ETV are covalently bound through an EDC-NHS mixed activator, the binding is stable, the falling of therapeutic factors can be prevented, the dosage delivered to a tumor site is ensured, the preparation process is simple and convenient, the reaction conditions are easy to control, the used materials are good in biocompatibility, the in-vivo use safety is further ensured, and the tumor ultrasonic treatment effect and safety are comprehensively improved.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment technology, specifically to a therapeutic ultrasonic microbubble for tumor ultrasound treatment and its preparation method. Background Technology

[0002] As one of the diseases that seriously threaten human life and health, cancer treatment has always been a key area of ​​medical research. Ultrasound therapy has received widespread attention in cancer treatment due to its advantages such as being non-invasive, having few side effects, and being repeatable. Ultrasound microbubbles are a type of ultrasound contrast agent and an important carrier for ultrasound therapy. They can not only enhance the effects of ultrasound imaging and help doctors accurately locate tumors, but also serve as a delivery carrier for drugs or therapeutic factors, enabling the targeted release of therapeutic factors at the tumor site. However, existing ultrasound microbubbles still have many shortcomings in tumor treatment applications. On the one hand, the shell material of traditional ultrasound microbubbles has poor targeting, making it difficult to accurately aggregate at the tumor site, resulting in the inability of therapeutic factors to effectively act on tumor cells and reducing the treatment effect. On the other hand, the therapeutic factors carried by existing microbubbles are mostly conventional drugs or biological factors that have not undergone specific modification. They are easily degraded non-specifically in vivo and cannot achieve specific activation in the tumor microenvironment, which not only affects the treatment efficiency but may also produce toxic side effects on normal tissues. In addition, the binding mode between some ultrasound microbubbles and therapeutic factors is unstable, and therapeutic factors are prone to detachment during delivery, further reducing the treatment effect. Therefore, developing a therapeutic ultrasound microbubble with high targeting and the ability of therapeutic factors to be specifically activated and stably bound in the tumor microenvironment is of great significance for improving the efficacy of ultrasound therapy for tumors and reducing damage to normal tissues. Summary of the Invention

[0003] The purpose of this invention is to provide a therapeutic ultrasound microbubble for tumor ultrasound therapy and its preparation method, so as to solve the problems mentioned in the background art, such as poor targeting, inability to specifically activate therapeutic factors, and unstable binding of existing ultrasound microbubbles.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a therapeutic ultrasound microbubble for tumor ultrasound therapy, which is composed of ultrasound contrast agent microbubbles and novel therapeutic factors; The outer shell of the ultrasound contrast agent microbubbles is made of polycaprolactone-polyethylene glycol-arginine-glycine-aspartic acid (PCL-PEG-RGD) copolymer, and the gas core is perfluoropentane. The novel therapeutic factor is a genetically engineered thrombin variant (ETV) containing a tumor-specific activating fragment.

[0005] As a preferred embodiment of the present invention, the molar ratio of polycaprolactone (PCL), polyethylene glycol (PEG), and arginine-glycine-aspartic acid (RGD) in the PCL-PEG-RGD copolymer is 5:3:2.

[0006] As a preferred embodiment of the present invention, the tumor-specific activation fragment is a recognition sequence for specific antigens on the surface of tumor cells, which can be specifically enzymatically activated in the tumor microenvironment.

[0007] As a preferred embodiment of the present invention, the ultrasound contrast agent microbubbles are covalently bonded to the novel therapeutic factor, and the thrombin variant (ETV) is attached to the surface of the PCL-PEG-RGD copolymer shell by an activator, wherein the activator is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0008] A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy includes the following steps: Step 1, Shell Preparation: Polycaprolactone, polyethylene glycol and arginine-glycine-aspartic acid are added to a reaction vessel and reacted with a catalyst under nitrogen protection to obtain PCL-PEG-RGD copolymer; Step 2, Microbubble Preparation: The PCL-PEG-RGD copolymer obtained in Step 1 is dissolved in an organic solvent to obtain a solution. The solution is then passed through a microfluidic chip while perfluoropentane gas is introduced. The flow rate ratio (organic phase: gas phase) is controlled at 8-12:1 to prepare ultrasound contrast agent microbubbles. Step 3, Activation: Dissolve the genetically engineered thrombin variant (ETV) in phosphate buffered saline (PBS), add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and stir to complete the activation; Step 4, Combination: Add the ultrasound contrast agent microbubbles obtained in Step 2 to the activated ETV solution in Step 3 and stir to allow ETV to be covalently bonded to the surface of the microbubble shell. Step 5, Purification: Centrifuge the mixture obtained in Step 4, remove the supernatant, and wash with PBS 2-3 times to obtain pure therapeutic ultrasound microbubbles.

[0009] As a preferred embodiment of the present invention, the catalyst in step one is stannous octoate, and its addition amount is 0.1-0.5% of the total mass of the reactants, and the reaction is carried out at 80-100°C for 6-8 hours.

[0010] As a preferred embodiment of the present invention, the organic solvent in step two is dichloromethane or chloroform, and the solution concentration is adjusted to 5-10 mg / mL.

[0011] As a preferred embodiment of the present invention, the phosphate buffer solution in step three has a pH of 7.4 and a concentration of 0.01-0.05 mol / L; the molar ratio of ETV, EDC and NHS is 1:2-3:2-3, and the mixture is stirred at room temperature for 15-30 minutes.

[0012] As a preferred embodiment of the present invention, in step four, the ultrasound contrast agent microbubbles and the activated ETV solution are stirred at 25-30°C for 2-4 hours.

[0013] As a preferred embodiment of the present invention, in step five, the centrifugal speed is controlled at 3000-4000 rpm and the time is 10-15 minutes.

[0014] Compared with the prior art, the beneficial effects of the present invention are: the therapeutic ultrasound microbubble for tumor ultrasound therapy; 1. The outer shell of the ultrasonic microbubble is made of PCL-PEG-RGD copolymer, in which the RGD peptide can specifically bind to the integrin receptor on the surface of tumor cells, so as to achieve precise targeting of the microbubble to the tumor site, increase the concentration of therapeutic factors at the tumor site, and enhance the therapeutic effect. 2. The novel therapeutic factor ETV contains tumor-specific activation fragments, which are inactive in normal tissues but are specifically activated by enzymatic hydrolysis in the tumor microenvironment. This can effectively avoid damage to normal tissues, reduce toxic side effects, and improve the safety of treatment. 3. Ultrasound contrast agent microbubbles and ETV are covalently bonded through EDC-NHS mixed activator. The binding mode is stable and can prevent the therapeutic factor from falling off during delivery, ensuring the dose of therapeutic factor reaching the tumor site and further improving the treatment effect. 4. The preparation process is simple and the reaction conditions are easy to control, enabling the large-scale production of therapeutic ultrasound microbubbles. Furthermore, the materials used in the preparation process all have good biocompatibility, ensuring the safety of the microbubbles in vivo. Attached Figure Description

[0015] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1 Please see Figure 1 The present invention provides a therapeutic ultrasound microbubble for tumor ultrasound therapy, comprising an ultrasound contrast agent microbubble and a novel therapeutic factor. The outer shell of the ultrasound contrast agent microbubble is made of a polycaprolactone-polyethylene glycol-arginine-glycine-aspartic acid (PCL-PEG-RGD) copolymer, and the gas core is perfluoropentane. The novel therapeutic factor is a genetically engineered thrombin variant (ETV) containing a tumor-specific activating fragment.

[0018] The molar ratio of polycaprolactone (PCL), polyethylene glycol (PEG), and arginine-glycine-aspartic acid (RGD) in the PCL-PEG-RGD copolymer is 5:3:2.

[0019] Tumor-specific activation fragments are recognition sequences targeting specific antigens on the surface of tumor cells, which can be specifically activated by enzymatic digestion in the tumor microenvironment.

[0020] The ultrasound contrast agent microbubbles are covalently bound to the novel therapeutic factor. Thrombin variant (ETV) is attached to the surface of the PCL-PEG-RGD copolymer shell by an activator, which is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0021] A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy includes the following steps: Step 1, Shell Preparation: Polycaprolactone, polyethylene glycol and arginine-glycine-aspartic acid are added to a reaction vessel. Under nitrogen protection, 0.1% stannous octoate is added and the reaction is carried out at 80°C for 8 hours to obtain PCL-PEG-RGD copolymer. Step 2, Microbubble Preparation: The PCL-PEG-RGD copolymer obtained in Step 1 was dissolved in dichloromethane and the concentration was adjusted to 5 mg / mL. The solution was then passed through a microfluidic chip while perfluoropentane gas was introduced. The flow rate ratio (organic phase: gas phase) was controlled at 8:1 to prepare ultrasound contrast agent microbubbles. Step 3, Activation: Dissolve the genetically engineered thrombin variant (ETV) in a phosphate buffer solution with a pH of 7.4 and a concentration of 0.01 mol / L. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and stir at room temperature for 15-30 minutes to complete the activation. The molar ratio of ETV, EDC and NHS is 1:2:2. Step 4, Combination: The ultrasound contrast agent microbubbles obtained in Step 2 are added to the ETV solution activated in Step 3 and stirred at 25°C for 4 hours to allow ETV to be covalently bonded to the surface of the microbubble shell. Step 5, Purification: Centrifuge the mixture obtained in Step 4 at 3000 rpm for 15 minutes, remove the supernatant, and wash twice with PBS to obtain pure therapeutic ultrasound microbubbles.

[0022] Example 2 Please see Figure 1 The present invention provides a therapeutic ultrasound microbubble for tumor ultrasound therapy, comprising an ultrasound contrast agent microbubble and a novel therapeutic factor. The outer shell of the ultrasound contrast agent microbubble is made of a polycaprolactone-polyethylene glycol-arginine-glycine-aspartic acid (PCL-PEG-RGD) copolymer, and the gas core is perfluoropentane. The novel therapeutic factor is a genetically engineered thrombin variant (ETV) containing a tumor-specific activating fragment.

[0023] The molar ratio of polycaprolactone (PCL), polyethylene glycol (PEG), and arginine-glycine-aspartic acid (RGD) in the PCL-PEG-RGD copolymer is 5:3:2.

[0024] Tumor-specific activation fragments are recognition sequences targeting specific antigens on the surface of tumor cells, which can be specifically activated by enzymatic digestion in the tumor microenvironment.

[0025] The ultrasound contrast agent microbubbles are covalently bound to the novel therapeutic factor. Thrombin variant (ETV) is attached to the surface of the PCL-PEG-RGD copolymer shell by an activator, which is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0026] A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy includes the following steps: Step 1, Shell Preparation: Polycaprolactone, polyethylene glycol and arginine-glycine-aspartic acid are added to a reaction vessel. Under nitrogen protection, 0.2% stannous octoate by total mass is added and the reaction is carried out at 85°C for 7.5 hours to obtain PCL-PEG-RGD copolymer. Step 2, Microbubble Preparation: The PCL-PEG-RGD copolymer obtained in Step 1 was dissolved in dichloromethane and the concentration was adjusted to 7 mg / mL. The solution was then passed through a microfluidic chip while perfluoropentane gas was introduced. The flow rate ratio (organic phase: gas phase) was controlled at 10:1 to prepare ultrasound contrast agent microbubbles. Step 3, Activation: Dissolve the genetically engineered thrombin variant (ETV) in a phosphate buffer solution with a pH of 7.4 and a concentration of 0.03 mol / L. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and stir at room temperature for 15-30 minutes to complete the activation. The molar ratio of ETV, EDC and NHS is 1:2:3. Step 4, Combination: The ultrasound contrast agent microbubbles obtained in Step 2 are added to the ETV solution activated in Step 3 and stirred at 27°C for 3.5 hours to allow ETV to be covalently bonded to the surface of the microbubble shell. Step 5, Purification: Centrifuge the mixture obtained in Step 4 at 3200 rpm for 14 minutes, remove the supernatant, and wash twice with PBS to obtain pure therapeutic ultrasound microbubbles.

[0027] Example 3 Please see Figure 1 The present invention provides a therapeutic ultrasound microbubble for tumor ultrasound therapy, comprising an ultrasound contrast agent microbubble and a novel therapeutic factor. The outer shell of the ultrasound contrast agent microbubble is made of a polycaprolactone-polyethylene glycol-arginine-glycine-aspartic acid (PCL-PEG-RGD) copolymer, and the gas core is perfluoropentane. The novel therapeutic factor is a genetically engineered thrombin variant (ETV) containing a tumor-specific activating fragment.

[0028] The molar ratio of polycaprolactone (PCL), polyethylene glycol (PEG), and arginine-glycine-aspartic acid (RGD) in the PCL-PEG-RGD copolymer is 5:3:2.

[0029] Tumor-specific activation fragments are recognition sequences targeting specific antigens on the surface of tumor cells, which can be specifically activated by enzymatic digestion in the tumor microenvironment.

[0030] The ultrasound contrast agent microbubbles are covalently bound to the novel therapeutic factor. Thrombin variant (ETV) is attached to the surface of the PCL-PEG-RGD copolymer shell by an activator, which is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0031] A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy includes the following steps: Step 1, Shell Preparation: Polycaprolactone, polyethylene glycol and arginine-glycine-aspartic acid are added to a reaction vessel. Under nitrogen protection, 0.4% stannous octoate by total mass is added and the reaction is carried out at 90°C for 7 hours to obtain PCL-PEG-RGD copolymer. Step 2, Microbubble Preparation: The PCL-PEG-RGD copolymer obtained in Step 1 was dissolved in chloroform and the concentration was adjusted to 8 mg / mL. The solution was then passed through a microfluidic chip while perfluoropentane gas was introduced. The flow rate ratio (organic phase: gas phase) was controlled at 10:1 to prepare ultrasound contrast agent microbubbles. Step 3, Activation: Dissolve the genetically engineered thrombin variant (ETV) in a phosphate buffer solution with a pH of 7.4 and a concentration of 0.05 mol / L. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and stir at room temperature for 15-30 minutes to complete the activation. The molar ratio of ETV, EDC and NHS is 1:3:2. Step 4, Combination: The ultrasound contrast agent microbubbles obtained in Step 2 are added to the ETV solution activated in Step 3 and stirred at 28°C for 2.5 hours to allow ETV to be covalently bonded to the surface of the microbubble shell. Step 5, Purification: Centrifuge the mixture obtained in Step 4 at 3600 rpm for 12 minutes, remove the supernatant, and wash 3 times with PBS to obtain pure therapeutic ultrasound microbubbles.

[0032] Example 4 Please see Figure 1 The present invention provides a therapeutic ultrasound microbubble for tumor ultrasound therapy, comprising an ultrasound contrast agent microbubble and a novel therapeutic factor. The outer shell of the ultrasound contrast agent microbubble is made of a polycaprolactone-polyethylene glycol-arginine-glycine-aspartic acid (PCL-PEG-RGD) copolymer, and the gas core is perfluoropentane. The novel therapeutic factor is a genetically engineered thrombin variant (ETV) containing a tumor-specific activating fragment.

[0033] The molar ratio of polycaprolactone (PCL), polyethylene glycol (PEG), and arginine-glycine-aspartic acid (RGD) in the PCL-PEG-RGD copolymer is 5:3:2.

[0034] Tumor-specific activation fragments are recognition sequences targeting specific antigens on the surface of tumor cells, which can be specifically activated by enzymatic digestion in the tumor microenvironment.

[0035] The ultrasound contrast agent microbubbles are covalently bound to the novel therapeutic factor. Thrombin variant (ETV) is attached to the surface of the PCL-PEG-RGD copolymer shell by an activator, which is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0036] A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy includes the following steps: Step 1, Shell Preparation: Polycaprolactone, polyethylene glycol and arginine-glycine-aspartic acid are added to a reaction vessel. Under nitrogen protection, 0.5% stannous octoate is added and the reaction is carried out at 100°C for 6 hours to obtain PCL-PEG-RGD copolymer. Step 2, Microbubble Preparation: The PCL-PEG-RGD copolymer obtained in Step 1 was dissolved in chloroform and the concentration was adjusted to 10 mg / mL. The solution was then passed through a microfluidic chip while perfluoropentane gas was introduced. The flow rate ratio (organic phase: gas phase) was controlled at 12:1 to prepare ultrasound contrast agent microbubbles. Step 3, Activation: Dissolve the genetically engineered thrombin variant (ETV) in a phosphate buffer solution with a pH of 7.4 and a concentration of 0.05 mol / L. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and stir at room temperature for 15-30 minutes to complete the activation. The molar ratio of ETV, EDC and NHS is 1:3:3. Step 4, Combination: Add the ultrasound contrast agent microbubbles obtained in Step 2 to the activated ETV solution in Step 3 and stir at 30°C for 2 hours to allow ETV to be covalently bonded to the surface of the microbubble shell. Step 5, Purification: Centrifuge the mixture obtained in Step 4 at 4000 rpm for 10 minutes, remove the supernatant, and wash 3 times with PBS to obtain pure therapeutic ultrasound microbubbles.

[0037] test To verify the efficacy and safety of the therapeutic ultrasound microbubbles prepared in this invention in tumor treatment, microbubbles prepared in Examples 1 to 4 (referred to as experimental groups 1-4) were selected. Commercially available ordinary ultrasound microbubbles (without targeted modification and specific therapeutic factors, referred to as control group 1), microbubbles containing only a PCL-PEG shell (without RGD targeting and ETV, referred to as control group 2), and microbubbles containing a PCL-PEG-RGD shell but loaded with unmodified thrombin (without tumor-specific activation fragments, referred to as control group 3) were used as controls. Animal experiments were conducted to evaluate their targeting, tumor inhibition rate, and safety.

[0038] Forty-two BALB / c nude mice aged 6-8 weeks and weighing 18-22g were selected. A human breast cancer MCF-7 subcutaneous xenograft model was constructed in all mice (the experiment was started when the tumor volume reached 80-100mm³). The mice were randomly divided into 7 groups with 6 mice in each group. Administration method: All groups received microbubble suspension (concentration 1×10⁻⁶) via tail vein injection. 8(0.2 mL / animal) After injection, the tumor site is irradiated with ultrasound at a frequency of 1 MHz and a sound pressure of 0.5 MPa for 5 minutes each time, twice a week, for 3 consecutive weeks.

[0039] detection indicators Targeting assessment: One week after treatment, the ratio of fluorescence intensity of the tumor site to that of normal muscle tissue was detected using an in vivo fluorescence imaging system (excitation wavelength 488nm, emission wavelength 520nm). The higher the ratio, the stronger the targeting. Tumor inhibition rate: The tumor volume was measured weekly, and the tumor inhibition rate was calculated after 3 weeks of treatment. The formula is: Tumor inhibition rate = (average tumor volume of control group - average tumor volume of experimental group) / average tumor volume of control group × 100%.

[0040] Safety assessment: During the treatment period, the mental state and weight changes of nude mice were observed daily. After the treatment, serum was collected to detect alanine aminotransferase (ALT, reflecting liver function) and serum creatinine (Scr, reflecting kidney function). Heart, liver and kidney tissues were also taken for HE staining to observe pathological damage.

[0041] Experimental Results and Analysis Targeted results The fluorescence intensity ratios of tumor sites to normal muscle tissue in each group are shown in the table below: As shown in the table above, the ratio of experimental groups 1-4 was significantly higher than that of control groups 1-3, and there was no significant difference between the groups. This indicates that the RGD peptide of the PCL-PEG-RGD shell can effectively mediate the targeted aggregation of microbubbles at the tumor site.

[0042] Tumor inhibition rate results The tumor inhibition rate results after 3 weeks of treatment are shown in the table below: As shown in the table above, the tumor inhibition rates of experimental groups 1-4 all exceeded 65%, which was significantly higher than that of control groups 1-3 (P<0.05). The tumor inhibition rate of control group 3 was only 32.1% due to the lack of specifically activated ETV, further demonstrating that tumor-specific activation of ETV can improve the therapeutic effect.

[0043] Security Results Weight and mental state: Nude mice in experimental groups 1-4 had good mental state and steady weight gain (0.5-1.0g per week) during treatment, with no significant difference from control groups 1-3; The specific serum biochemical indicators are shown in the table below: The ALT and Scr levels in experimental groups 1-4 were all within the normal range and showed no significant difference from those in control groups 1-3, indicating that microbubbles do not damage liver and kidney function. Histopathological observation: HE staining of heart, liver and kidney tissues in experimental groups 1-4 and control groups 1-3 showed no obvious pathological damage (such as hepatocyte necrosis, renal tubular dilation, etc.), proving that the microbubbles have good in vivo safety.

[0044] Experimental conclusions The therapeutic ultrasound microbubbles prepared in this invention achieve highly efficient tumor targeting through the RGD peptide segment of the PCL-PEG-RGD shell, with significantly better targeting performance than the control group without targeting modification. The loaded tumor-specific activated thrombin variant (ETV) can be specifically activated in the tumor microenvironment, significantly improving the tumor inhibition rate, and the effect is better than the control group loaded with unmodified thrombin; Microbubbles have no adverse effects on the body weight, liver and kidney function, or major organs of nude mice, and have good safety profile, providing a novel, efficient, and safe carrier for ultrasound therapy of tumors.

[0045] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0046] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A therapeutic ultrasonic microbubble for tumor ultrasound therapy, characterized in that, It is composed of ultrasound contrast agent microbubbles and novel therapeutic factors; The outer shell of the ultrasound contrast agent microbubbles is made of polycaprolactone-polyethylene glycol-arginine-glycine-aspartic acid (PCL-PEG-RGD) copolymer, and the gas core is perfluoropentane. The novel therapeutic factor is a genetically engineered thrombin variant (ETV) containing a tumor-specific activating fragment.

2. The therapeutic ultrasound microbubble for tumor ultrasound therapy according to claim 1, characterized in that, The molar ratio of polycaprolactone (PCL), polyethylene glycol (PEG), and arginine-glycine-aspartic acid (RGD) in the PCL-PEG-RGD copolymer is 5:3:

2.

3. The therapeutic ultrasound microbubble for tumor ultrasound therapy according to claim 1, characterized in that, The tumor-specific activation fragment is a recognition sequence for specific antigens on the surface of tumor cells, which can be specifically activated by enzymatic digestion in the tumor microenvironment.

4. The therapeutic ultrasound microbubble for tumor ultrasound therapy according to claim 1, characterized in that, The ultrasound contrast agent microbubbles are covalently bonded to the novel therapeutic factor. Thrombin variant (ETV) is attached to the surface of the PCL-PEG-RGD copolymer shell by an activator, wherein the activator is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

5. A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy, characterized in that, Includes the following steps: Step 1, Shell Preparation: Polycaprolactone, polyethylene glycol and arginine-glycine-aspartic acid are added to a reaction vessel and reacted with a catalyst under nitrogen protection to obtain PCL-PEG-RGD copolymer; Step 2, Microbubble Preparation: The PCL-PEG-RGD copolymer obtained in Step 1 is dissolved in an organic solvent to obtain a solution. The solution is then passed through a microfluidic chip while perfluoropentane gas is introduced. The flow rate ratio (organic phase: gas phase) is controlled at 8-12:1 to prepare ultrasound contrast agent microbubbles. Step 3, Activation: Dissolve the genetically engineered thrombin variant (ETV) in phosphate buffered saline (PBS), add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and stir to complete the activation; Step 4, Combination: Add the ultrasound contrast agent microbubbles obtained in Step 2 to the activated ETV solution in Step 3 and stir to allow ETV to be covalently bonded to the surface of the microbubble shell. Step 5, Purification: Centrifuge the mixture obtained in Step 4, remove the supernatant, and wash with PBS 2-3 times to obtain pure therapeutic ultrasound microbubbles.

6. A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy according to claim 5, characterized in that, In step one, the catalyst is stannous octoate, and its addition amount is 0.1-0.5% of the total mass of the reactants. The reaction is carried out at 80-100℃ for 6-8 hours.

7. A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy according to claim 5, characterized in that, In step two, the organic solvent is dichloromethane or chloroform, and the solution concentration is adjusted to 5-10 mg / mL.

8. A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy according to claim 5, characterized in that, In step three, the phosphate buffer solution has a pH of 7.4 and a concentration of 0.01-0.05 mol / L; the molar ratio of ETV, EDC and NHS is 1:2-3:2-3, and the mixture is stirred at room temperature for 15-30 minutes.

9. A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy according to claim 5, characterized in that, In step four, the ultrasound contrast agent microbubbles and the activated ETV solution are stirred at 25-30°C for 2-4 hours.

10. A method for preparing therapeutic ultrasound microbubbles for tumor ultrasound therapy according to claim 5, characterized in that, In step five, the centrifugal speed is controlled at 3000-4000 rpm, and the time is 10-15 minutes.

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