Preparation method of Annexin V coupling magnetic navigation ultrasonic microbubbles

Abstract

The invention relates to the field of biomedical engineering, and discloses an AnnexinV coupled magnetic navigation ultrasonic microbubble which comprises a microbubble core with a nano-liposome coating, a magnetic navigation system and an AnnexinV coupled magnetic navigation system, the AnnexinV coupling layer is used for targeting phosphatidylserine; the magnetic nanoparticles are used for magnetic navigation control; the perfluoropropane or perfluorobutane gas core is used for enhancing ultrasonic contrast; comprising the following steps: S1, preparation of a nano-liposome membrane: dissolving 1, 2-distearoyl-sn-glycerol-3-phosphorylcholine, biotinylated polyethylene glycol phospholipid and polyoxyethylene 40 stearate in an organic solvent, carrying out ultrasonic mixing, removing the solvent to form a phospholipid membrane, and carrying out hydration treatment and ultrasonic homogenization treatment to obtain the nano-liposome. The AnnexinV is coupled to the microbubble, so that the microbubble can accurately target a lesion area, the problem of insufficient targeting in a traditional method is solved, and a stronger targeting effect and an accurate imaging capability are obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and specifically provides a method for preparing Annexin V-conjugated magnetic navigation ultrasound microbubbles. Background Art

[0002] In modern medicine, the combination of targeted therapy and imaging technology has received increasing attention. Annexin V is a protein that can specifically bind to phosphatidylserine on the cell membrane and is widely used in the detection of apoptosis and targeted drug delivery. In the prior art, a variety of Annexin V-based targeted microbubbles have been developed to enhance the sensitivity of ultrasound imaging. However, the binding efficiency of existing Annexin V-targeted microbubbles to target molecules in the blood circulation is still low, which limits their effectiveness in clinical applications.

[0003] Specifically, the disadvantages include the following points: 1. Low binding efficiency: The binding efficiency of existing Annexin V-targeted microbubbles to target molecules in the blood circulation is not high, resulting in unsatisfactory targeted imaging effects. 2. Poor stability: Since microbubbles are easily ruptured in the blood, the targeted imaging effects are unstable, affecting the contrast effect. 3. Insufficient imaging sensitivity: The prior art has insufficient sensitivity to the lesion area in ultrasound imaging, resulting in a low ultrasound detection rate of lesions and affecting early diagnosis. 4. Lack of magnetic navigation technology: Most current targeted microbubbles are not combined with magnetic navigation technology and cannot achieve precise positioning of the target, limiting their application potential in complex biological environments. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for preparing Annexin V-conjugated magnetic navigation ultrasound microbubbles, which solves the problem of low binding efficiency of existing Annexin V-targeted microbubbles in the blood circulation.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: An Annexin V-conjugated magnetic navigation ultrasound microbubble, comprising:

[0006] A microbubble core with a nanoliposome coating;

[0007] An Annexin V conjugation layer for targeting phosphatidylserine;

[0008] Magnetic nanoparticles for magnetic navigation control;

[0009] A perfluoropropane or perfluorobutane gas core for enhancing ultrasound contrast.

[0010] A method for preparing Annexin V-conjugated magnetic navigation ultrasound microbubbles, comprising the following steps:

[0011] S1. Preparation of nano-liposome membrane: Dissolve 1,2-distearoyl-sn-glycero-3-phosphocholine, biotinylated polyethylene glycol phospholipid and polyoxyethylene 40 stearate in an organic solvent, ultrasonically mix, remove the solvent to form a phospholipid membrane, and obtain nano-liposomes by hydration treatment and ultrasonic homogenization treatment;

[0012] S2. Construction of enzyme-resistant degradation polymer coating: Dissolve sodium polyacrylate and polylysine in a buffer solution, and form an enzyme-resistant degradation coating on the surface of nano-liposomes under the action of a cross-linking agent;

[0013] S3. Preparation of biotinylated microbubbles: Introduce perfluoropropane or perfluorobutane gas into the nano-liposome suspension, and ultrasonically stir to form microbubbles;

[0014] S4. Preparation of avidinylated microbubbles: Mix biotinylated microbubbles with avidin solution, incubate and then centrifuge and wash to obtain avidinylated microbubbles;

[0015] S5. Coupling streptavidin magnetic beads: Mix avidinylated microbubbles with streptavidin-modified magnetic nanoparticles, incubate and then centrifuge and wash to obtain microbubbles coupled with magnetic beads;

[0016] S6. Coupling biotinylated AnnexinV: Mix microbubbles coupled with magnetic beads with biotinylated AnnexinV, incubate and then centrifuge and wash to obtain AnnexinV-coupled magnetic navigation ultrasonic microbubbles.

[0017] Preferably, the mass percentage range of the phospholipid material described in S1 is:

[0018] 1,2-distearoyl-sn-glycero-3-phosphocholine: 40–60 wt%;

[0019] Biotinylated polyethylene glycol phospholipid: 5–15 wt%;

[0020] Polyoxyethylene 40 stearate: 1–5 wt%;

[0021] The hydration treatment temperature of the nano-liposome membrane described in S1 is 40–50 °C, the stirring speed is 200–500 revolutions per minute, and the time is 30–60 minutes.

[0022] Preferably, the mass percentage range of the enzyme-resistant degradation polymer described in S2 is:

[0023] Sodium polyacrylate: 5–15 wt%;

[0024] Polylysine: 2–8 wt%.

[0025] Preferably, the covalent cross-linking in S2 is carried out by the cross-linking reaction of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide;

[0026] Among them, the cross-linking agent concentration is 5–15 mg / mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 5–10 mg / mL of N-hydroxysuccinimide, the reaction temperature is 20–37 °C, and the reaction time is 2–6 hours.

[0027] Preferably, the microbubble core gas in S3 is perfluoropropane or perfluorobutane, wherein the volume fraction of perfluoropropane is 80–99%, and the volume fraction of perfluorobutane is 1–20%.

[0028] Preferably, the concentration of the avidin solution in S4 is 1 μg / mL, 30 μg of avidin is added for every 1×10^8 microbubbles, the incubation time is 30 minutes, and the incubation temperature is 4 °C.

[0029] Preferably, the ratio of streptavidin-modified magnetic nanoparticles to microbubbles in S5 is 1×10^8:1×10^8, the incubation time is 30 minutes, and the incubation temperature is 4 °C.

[0030] Preferably, the concentration of the AnnexinV solution in S6 is 0.1–0.5 mg / mL, 30 μg of biotinylated AnnexinV is added for every 1×10^8 microbubbles, the incubation time is 30 minutes, and the incubation temperature is 4 °C.

[0031] Preferably, the centrifugal washing conditions in S4 to S6 are 1000 revolutions per minute, the centrifugation time is 1 minute, the number of washing times is 3 times, and each time phosphate buffered saline solution is used for washing.

[0032] The present invention provides a preparation method of Annexin V-conjugated magnetic navigation ultrasound microbubbles. It has the following beneficial effects:

[0033] 1. By conjugating AnnexinV to the microbubbles, the present invention enables the microbubbles to accurately target the lesion area, solves the problem of insufficient targeting in traditional methods, and thus obtains a stronger targeting effect and accurate imaging ability.

[0034] 2. By constructing an enzyme-resistant degradation polymer coating on the surface of the microbubbles, the present invention solves the problem of easy rupture of the microbubbles in the blood, realizes a longer circulation time of the microbubbles in the body, and obtains the effect of significantly improving stability and persistence.

[0035] 3. By using perfluoropropane or perfluorobutane gas core, the present invention improves the ultrasonic response of the microbubbles, solves the problem of weak signals of traditional ultrasonic microbubbles, and obtains a clearer and stronger ultrasonic imaging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a flowchart of the steps of the present invention;

[0037] Figure 2 is a schematic diagram showing the aggregation of coupled magnetic bead microbubbles attracted to one side under the attraction of a magnet in the present invention;

[0038] Figure 3 is a schematic diagram showing the dispersed distribution of coupled magnetic bead microbubbles not attracted by a magnet in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0039] The technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0040] Please refer to the attached Figure 1 , the embodiment of the present invention provides a method for preparing Annexin V-coupled magnetic navigation ultrasound microbubbles, including the following steps:

[0041] S1. Preparation of nano-liposome membrane: Dissolve 1,2-distearoyl-sn-glycero-3-phosphocholine, biotinylated polyethylene glycol phospholipid and polyoxyethylene 40 stearate in an organic solvent, ultrasonically mix, remove the solvent to form a phospholipid membrane, and obtain nano-liposomes by hydration treatment and ultrasonic homogenization treatment;

[0042] Specifically, in this embodiment, first weigh 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), biotinylated polyethylene glycol phospholipid (DSPE-PEG) and polyoxyethylene 40 stearate (PEG40) according to the mass ratio and dissolve them in an appropriate organic solvent. Generally, chloroform, methanol or a mixed solvent of chloroform / methanol is used to dissolve the phospholipids. These solvents can effectively dissolve the above phospholipid components and have good volatility, which is convenient for the subsequent solvent removal process.

[0043] As an option, the mass percentage of DSPC usually accounts for 40%–60% of the total formulation, the mass percentage of biotinylated polyethylene glycol phospholipid is 5%–15%, and the mass percentage of polyoxyethylene 40 stearate is 1%–5%. The selection of this ratio is based on the molecular arrangement during the formation of the phospholipid membrane and the performance of the membrane stability.

[0044] Specifically, the dissolved solution is ultrasonically treated to make it fully mixed, forming a uniform phospholipid solution. The role of ultrasound is to break the aggregation between molecules, ensure the uniform distribution of each component in the solution, avoid the non-uniformity of components, and thus obtain a consistent phospholipid distribution during the film formation process.

[0045] After that, the solvent is gradually removed. The solvent can be volatilized from the solution by rotary evaporation to form a thin film layer. The rotary evaporator can effectively remove the solvent at a certain temperature and pressure, enabling the phospholipid molecules in the solution to deposit into a film and remain on the flask wall. This process is usually carried out in the temperature range of 40°C - 50°C to ensure the stability of phospholipid molecules and avoid the degradation of phospholipid molecules due to excessive temperature.

[0046] After removing the solvent, the thin film is further hydrated. In this embodiment, a buffer solution containing appropriate salts (such as PBS) is used to hydrate the thin film. During the hydration process, the volume of the buffer solution is usually 50 to 100 times that of the thin film, the hydration temperature is 40°C - 50°C, the stirring speed is 200 - 500 revolutions per minute, and the time is controlled between 30 minutes and 60 minutes. This step can promote the rearrangement of phospholipid molecules to form a nano-liposome structure.

[0047] After the hydration treatment is completed, ultrasonic homogenization treatment (for example, using an ultrasonic crusher or a high-pressure homogenization device) is carried out on the nano-liposomes. Through this step, the particle size distribution of the nano-liposomes can be further refined to reach the ideal range of 1 - 5 μm. Ultrasonic homogenization treatment can improve the uniformity of the nano-liposomes, avoid the phenomenon of too large or too small particle size, and thus provide a good basis for the subsequent formation of microbubbles.

[0048] In some embodiments, by adjusting the time and power of ultrasonic homogenization, the particle size distribution of the nano-liposomes can be controlled. Usually, the power of ultrasonic treatment is 100 - 500 W, and the time is 1 - 10 minutes to ensure a suitable particle size range.

[0049] The preparation method of this step can not only ensure the stability of the nano-liposomes, but also provide strong support for the subsequent formation of microbubbles and targeted modification. Through this method of preparing the nano-liposome membrane, it is ensured that the prepared microbubbles have high stability and a controllable particle size distribution, avoiding the problems of microbubble rupture and failure caused by unstable membrane structure or inconsistent particle size.

[0050] S2. Construction of the enzyme-resistant degradation polymer coating: Dissolve sodium polyacrylate and polylysine in a buffer solution, and form an enzyme-resistant degradation coating on the surface of the nano-liposomes under the action of a cross-linking agent;

[0051] Specifically, in the aforementioned step S1, nano-liposome membranes have been obtained through ultrasonic homogenization treatment and nano-liposomes have been formed. In order to further enhance the stability of microbubbles in the blood circulation and prevent their degradation under the action of enzymes, the core task of this step is to construct an enzyme-resistant polymer coating, thereby improving the in vivo biostability of microbubbles and prolonging their circulation time.

[0052] In this embodiment, first, sodium polyacrylate (PAA) and polylysine (PLL) are dissolved in a suitable buffer solution according to a certain mass ratio. As an option, the mass percentage of sodium polyacrylate is 5%–15%, and the mass percentage of polylysine is 2%–8%. Generally, PBS buffer solution is selected as the buffer, and the pH value is controlled at about 7.4 to ensure that the two polymers can be fully dissolved and stable. In some embodiments, different types of buffer solutions can be selected according to needs to adapt to different preparation conditions.

[0053] Next, the dissolved sodium polyacrylate and polylysine solutions are added to the nano-liposome suspension obtained in step S1. Through gentle stirring, ensure that the polymer is evenly distributed on the surface of the nano-liposomes, thereby forming a stable polymer coating. Generally, the stirring speed is 200 - 500 revolutions per minute, and the stirring time is 30 minutes to 1 hour.

[0054] As an option, the stability and enzyme-resistant degradation ability of the coating are further enhanced through a covalent cross-linking reaction. Specifically, in this embodiment, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHSS) are used as cross-linking agents for covalent cross-linking. The cross-linking reaction can effectively form strong chemical bonds between the sodium polyacrylate and polylysine molecules and the phospholipid molecules in the nano-liposome membrane, thereby improving the stability of the coating.

[0055] In a possible implementation, the concentration of the cross-linking agent is 5–15 mg / mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 5–10 mg / mL of N-hydroxysuccinimide. The cross-linking reaction is usually carried out at a temperature of 20°C to 37°C, and the reaction time is 2 hours to 6 hours to ensure that the cross-linking reaction is complete.

[0056] After the cross-linking is completed, the reaction solution needs to be centrifuged and washed to remove unreacted cross-linking agents and impurities in the solution. Usually, the centrifugation conditions are 1000 revolutions per minute, and the centrifugation time is 5 minutes. After each washing, it is washed 3 times with PBS buffer solution to ensure the purity of the coating. Through this step, the stability and enzyme-resistant degradation performance of the coating can be ensured to achieve the expected effect.

[0057] S3. Preparation of biotinylated microbubbles: Introduce perfluoropropane or perfluorobutane gas into the nanoliposome suspension, and form microbubbles by ultrasonic stirring;

[0058] Specifically, in the aforementioned steps S1 and S2, the preparation of the nanoliposome membrane and the construction of the enzyme-resistant degradation polymer coating have been completed. To further enhance the ultrasound contrast effect of the microbubbles, the core task of this step is to introduce a gas core by ultrasonic stirring to form microbubbles suitable for ultrasonic imaging. The introduction of the gas core not only enhances the acoustic properties of the microbubbles but also improves their stability and imaging effect in vivo.

[0059] In this embodiment, first introduce perfluoropropane (C3HF7) or perfluorobutane (C4HF9) gas into the coated nanoliposome suspension. As an option, the proportion of the gas core can be adjusted, where the volume fraction of perfluoropropane is generally 80% - 99%, and the volume fraction of perfluorobutane is 1% - 20%. The selection of such gases is based on their excellent acoustic properties, which can generate obvious echo signals under the action of ultrasonic waves, helping to improve the ultrasound contrast effect of the microbubbles.

[0060] Specifically, mix the gas with the nanoliposome solution and form microbubbles by ultrasonic stirring. In some embodiments, an ultrasonic probe (with a frequency usually in the range of 20 - 50 kHz) is used to treat the gas and nanoliposome mixture. The action of ultrasonic waves will generate tiny bubbles in the liquid, and the bubbles combine with the nanoliposome membrane to form a stable microbubble structure. To ensure the stability of the microbubbles, the ultrasonic treatment time is generally controlled between 1 minute and 5 minutes, and the power range is 100 - 500 W. The specific power and time depend on the size and stability of the required microbubbles.

[0061] In some embodiments, during ultrasonic stirring, control the temperature of the liquid within the range of 5°C to 25°C to avoid the gas volume expanding too fast due to excessive temperature, which affects the stability of the microbubbles. To avoid excessive aggregation or non-uniformity of bubbles during ultrasonic stirring, usually use low-frequency and low-power ultrasonic waves and gradually increase the energy to ensure uniform distribution of bubbles.

[0062] The diameter of the microbubbles formed by ultrasonic stirring is generally controlled within the range of 1 - 5 μm. This range can ensure the stability of the microbubbles in blood circulation and also improve their imaging effect in ultrasonic imaging. Further, by adjusting the power, frequency, and time of ultrasonic waves, the size distribution of the microbubbles can be effectively controlled to meet the requirements of different clinical applications.

[0063] S4. Preparation of avidinylated microbubbles: Mix the biotinylated microbubbles with avidin solution, incubate, and then centrifuge and wash to obtain avidinylated microbubbles;

[0064] Specifically, in the aforementioned step S3, ultrasonic microbubbles with a stable gas core have been formed through ultrasonic agitation, and the stability of the microbubbles has been improved through the treatment of the nano-liposome membrane and the enzyme-resistant degradation coating. To achieve the targeting function, the core of this step is to bind the microbubbles with avidin solution, so that the surface of the microbubbles has avidin function, which provides a basis for subsequent targeted binding.

[0065] In this embodiment, first, the prepared biotinylated avidin solution is mixed with the microbubbles obtained in step S3. The concentration of the avidin solution is generally selected to be 1 μg / mL to 10 μg / mL to ensure that it can effectively bind to the surface of the microbubbles. Specifically, the mass ratio of avidin to microbubbles is 1×10^8:1×10^8, which can ensure that there are sufficient avidin molecules on the surface of each microbubble for targeted reactions.

[0066] In some embodiments, the addition amount of the avidin solution can be appropriately adjusted according to the concentration of the microbubbles. To ensure that avidin can be evenly distributed on the surface of the microbubbles, the microbubbles and the avidin solution are usually incubated under low-temperature conditions of 4°C. The incubation time is generally controlled between 30 minutes and 1 hour to ensure that the binding between avidin and the surface of the microbubbles is sufficient and stable.

[0067] As an option, gentle shaking or slow stirring can be performed during the incubation process to enhance the binding efficiency of avidin. In this way, avidin molecules can be evenly distributed on the surface of the microbubbles, ensuring their functionality in subsequent targeted binding.

[0068] Specifically, after the incubation is completed, the microbubbles and avidin complex need to be centrifuged and washed to remove unbound avidin. Usually, the centrifugation conditions are 1000 revolutions per minute, the centrifugation time is 1 minute, and the number of washing times is 3 times. PBS buffer is used for washing during the washing process. Through this step, unbound avidin molecules can be removed to ensure the obtained pure avidinylated microbubbles.

[0069] In a possible implementation manner, the binding efficiency of avidin can be further optimized by changing the incubation temperature, time, and avidin concentration to ensure the maximization of the targeting effect. By controlling these parameters, microbubbles with a high avidin density can be obtained, making them have a stronger targeting ability in subsequent targeted imaging or treatment processes.

[0070] S5. Coupling streptavidin magnetic beads: Mix the avidinylated microbubbles with streptavidin-modified magnetic nanoparticles, incubate, and then centrifuge and wash to obtain microbubbles coupled with magnetic beads;

[0071] Specifically, in the aforementioned step S4, the microbubbles have successfully bound to avidin, thus providing a basis for subsequent targeting functions. To further enhance the magnetic navigation ability of the microbubbles, the core task of this step is to bind the avidinylated microbubbles to streptavidin-modified magnetic nanoparticles, thereby forming magnetic navigation ultrasound microbubbles that can be directionally controlled in a magnetic field.

[0072] In this embodiment, streptavidin-modified magnetic nanoparticles are first prepared. The magnetic nanoparticles are usually Fe3O4 (magnetite) or other nanomaterials with good magnetic properties. After the formation of ultrasound microbubbles, these particles will endow the microbubbles with good magnetic response characteristics. In some embodiments, the size of the magnetic nanoparticles is usually 10–100 nanometers to ensure that they can effectively adhere to the surface of the microbubbles without affecting the stability of the microbubbles.

[0073] Specifically, the streptavidin-modified magnetic nanoparticles are mixed with the avidinylated microbubbles. The concentration of streptavidin-modified magnetic nanoparticles is generally selected to be 1×10^8 particles / μL to 1×10^10 particles / μL, and the specific concentration can be adjusted according to the number of microbubbles and the binding efficiency of the magnetic particles. At this time, the binding force between avidin and streptavidin enables the magnetic particles to firmly adhere to the surface of the microbubbles.

[0074] In some embodiments, to improve the binding efficiency, the mixed liquid can be incubated at 4°C. The incubation time is generally controlled between 30 minutes and 1 hour to ensure that the streptavidin-modified magnetic nanoparticles can fully adhere to the surface of the avidinylated microbubbles. During the incubation process, low-speed stirring or shaking can be used to promote the binding of the magnetic particles to the surface of the microbubbles.

[0075] As an option, the incubated microbubble and magnetic particle complex needs to be centrifuged to remove the unbound magnetic particles. The centrifugation conditions are generally 1000 revolutions per minute, and the centrifugation time is 1 minute. After centrifugation, the microbubbles are washed with PBS buffer to ensure the removal of excess streptavidin-modified magnetic nanoparticles. Generally, the washing is performed 3 times to ensure that the finally obtained magnetic microbubbles are pure and free of impurities.

[0076] In a possible implementation, the incubation temperature, time, and stirring speed can be adjusted according to factors such as the properties, size, and incubation conditions of the magnetic nanoparticles to optimize the binding effect between avidin and the magnetic particles. By finely adjusting these parameters, microbubbles with high magnetic response ability and targeting can be obtained, providing a stable basis for subsequent magnetic navigation and ultrasound imaging.

[0077] S6. Coupling biotinylated Annexin V: Mix the microbubbles conjugated with magnetic beads with biotinylated Annexin V, and after incubation, centrifuge and wash to obtain Annexin V-conjugated magnetic navigation ultrasound microbubbles.

[0078] Specifically, step S6 involves the final preparation of Annexin V-conjugated magnetic navigation ultrasound microbubbles. The aforementioned steps S1 to S5 have completed the preparation of the nanoliposome membrane, the construction of the enzyme-resistant degradation coating, the introduction of the biogas core, the preparation of avidinylated microbubbles, and the binding of streptavidin-modified magnetic nanoparticles. At this point, the microbubbles possess targeting, stability, and magnetic navigation capabilities. The key to this step is to conjugate the biotinylated Annexin V molecules with the processed microbubbles, endowing the microbubbles with specific targeting functions, enabling them to recognize and bind to specific targets, and further enhancing the application effect of the microbubbles in targeted therapy or ultrasound imaging.

[0079] In this embodiment, first prepare a biotinylated Annexin V solution. The concentration of biotinylated Annexin V is generally 0.1 - 0.5 milligrams per milliliter. This concentration range can not only ensure its effective binding to the microbubble surface but also avoid non-specific binding caused by too high a concentration. As an option, the mass of Annexin V can be adjusted according to the number of microbubbles. Usually, 30 micrograms of biotinylated Annexin V is added per 1×10^8 microbubbles to ensure sufficient coupling density on the surface of each microbubble.

[0080] Specifically, mix the biotinylated Annexin V solution with the magnetic bead-conjugated microbubbles obtained in step S5. At this time, biotinylated Annexin V firmly attaches to the surface of avidinylated microbubbles through the specific binding force between biotin and avidin. After mixing, the complex of microbubbles and biotinylated Annexin V needs to be incubated at 4°C, and the incubation time is generally controlled between 30 minutes and 1 hour. This incubation process helps to ensure the full binding of biotinylated Annexin V to the avidin on the microbubble surface, thereby improving the targeting recognition ability of the microbubbles.

[0081] As an option, during the incubation process, gentle stirring or shaking can be used to accelerate the coupling reaction. Through these operations, it can be ensured that biotinylated Annexin V is evenly distributed on the microbubble surface, avoiding insufficient avidin binding in local areas and affecting the targeting effect of the microbubbles.

[0082] After incubation, the microbubbles and AnnexinV complex need to be centrifuged and washed to remove unbound biotinylated AnnexinV. The centrifugation conditions are usually 1000 revolutions per minute, the centrifugation time is 1 minute, and the number of washes is 3 times. The microbubbles are washed with PBS buffer. This step can ensure the purity of the finally obtained AnnexinV-conjugated magnetic navigation ultrasound microbubbles and avoid non-specific reactions caused by excess biotinylated AnnexinV.

[0083] Through the above steps, the present invention has successfully conjugated biotinylated AnnexinV to the surface of microbubbles, endowing the microbubbles with highly specific targeting. AnnexinV can recognize and bind to phosphatidylserine (PS) on the cell membrane, and this property enables the microbubbles to accurately target tumor cells or other diseased tissues. This conjugation process provides a more efficient technical basis for the targeted therapy, targeted drug delivery, and ultrasound imaging of microbubbles. The finally prepared AnnexinV-conjugated magnetic navigation ultrasound microbubbles not only have enhanced stability and targeting, but also can be accurately positioned under the guidance of a magnetic field, greatly improving the effect of ultrasound imaging and the detection accuracy of the diseased area.

[0084] Please refer to the attached Figure 2 - attached Figure 3 , the comparison of the preparation and targeted binding efficiency of AnnexinV-conjugated magnetic navigation ultrasound microbubbles;

[0085] Prepare the microbubble solution:

[0086] Respectively prepare AnnexinV-conjugated microbubbles and AnnexinV-conjugated magnetic navigation ultrasound microbubbles to ensure that their concentrations are the same. The AnnexinV-conjugated microbubbles are prepared according to the normal preparation method, while the AnnexinV-conjugated magnetic navigation ultrasound microbubbles are modified with magnetic nanoparticles on the basis of the above technical solution.

[0087] Inject the microbubbles into an in vitro flow chamber:

[0088] Select a suitable in vitro flow chamber and coat the inner surface of the chamber with phosphatidylserine (PS). The coating of PS can be carried out by self-assembly technology to ensure that sufficient phosphatidylserine (PS) molecules are enriched on the surface of the chamber, providing conditions for the subsequent targeted binding of microbubbles.

[0089] Inject the AnnexinV-conjugated microbubbles and AnnexinV-conjugated magnetic navigation ultrasound microbubbles into the chamber at the same concentration and ensure that they are evenly distributed in the flow chamber.

[0090] Process the flow chamber:

[0091] Start the flow system of the flow chamber to simulate the blood flow environment. At a certain flow rate, observe the movement of the two groups of microbubbles in the chamber, and record the binding of the microbubbles to the surface PS through a microscope.

[0092] Microbubble quantity statistics:

[0093] Observe the binding of AnnexinV-conjugated microbubbles and AnnexinV-conjugated magnetic navigation ultrasound microbubbles on the surface of the flow chamber. Take images through a microscope and count the number of the two types of microbubbles on the surface of the flow chamber. Record the average number of bound microbubbles and make a comparison.

[0094] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An Annexin V-conjugated magnetic navigation ultrasound microbubble, characterized in that, Comprising: A microbubble core with a nano-liposome coating; An AnnexinV conjugate layer for targeting phosphatidylserine; Magnetic nanoparticles for magnetic navigation control; A perfluoropropane or perfluorobutane gas core for enhancing ultrasound contrast imaging.

2. A preparation method of Annexin V-conjugated magnetic navigation ultrasound microbubbles. According to the Annexin V-conjugated magnetic navigation ultrasound microbubbles described in claim 1, it is characterized in that Including the following steps: S1. Preparation of the nano-liposome membrane: Dissolve 1,2-distearoyl-sn-glycero-3-phosphocholine, biotinylated polyethylene glycol phospholipid, and polyoxyethylene 40 stearate in an organic solvent, mix by ultrasound, remove the solvent to form a phospholipid membrane, and obtain nano-liposomes through hydration treatment and ultrasonic homogenization; S2. Construction of the enzyme-resistant degradation polymer coating: Dissolve sodium polyacrylate and polylysine in a buffer solution, and form an enzyme-resistant degradation coating on the surface of the nano-liposomes under the action of a cross-linking agent; S3. Preparation of biotinylated microbubbles: Introduce perfluoropropane or perfluorobutane gas into the nano-liposome suspension and stir by ultrasound to form microbubbles; S4. Preparation of avidinylated microbubbles: Mix the biotinylated microbubbles with an avidin solution, incubate, and then centrifuge and wash to obtain avidinylated microbubbles; S5. Coupling streptavidin magnetic beads: Mix the avidinylated microbubbles with streptavidin-modified magnetic nanoparticles, incubate, and then centrifuge and wash to obtain microbubbles coupled with magnetic beads; S6. Coupling biotinylated AnnexinV: Mix the microbubbles coupled with magnetic beads with biotinylated AnnexinV, incubate, and then centrifuge and wash to obtain AnnexinV-coupled magnetic navigation ultrasound microbubbles.

3. The preparation method of an Annexin V-conjugated magnetic navigation ultrasonic microbubble according to claim 2, wherein The mass percentage range of the phospholipid materials described in S1 is: 1,2-distearoyl-sn-glycero-3-phosphocholine: 40–60 wt%; Biotinylated polyethylene glycol phospholipid: 5–15 wt%; Polyoxyethylene 40 stearate: 1–5 wt%; The hydration treatment temperature of the nano-liposome membrane described in S1 is 40–50 °C, the stirring speed is 200–500 revolutions per minute, and the time is 30–60 minutes.

4. The preparation method of an Annexin V-conjugated magnetic navigation ultrasonic microbubble according to claim 2, characterized in that, The mass percentage range of the enzyme-resistant degradation polymer described in S2 is: Sodium polyacrylate: 5–15 wt%; Polylysine: 2–8 wt%.

5. The preparation method of an Annexin V-conjugated magnetic navigation ultrasound microbubble according to claim 2, characterized in that, The covalent cross-linking described in S2 uses 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide cross-linking reaction; Among them, the cross-linking agent concentration is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 5–15 mg / ml, N-hydroxysuccinimide 5–10 mg / ml, the reaction temperature is 20–37 °C, and the reaction time is 2–6 hours.

6. The preparation method of an Annexin V-conjugated magnetic navigation ultrasonic microbubble according to claim 2, characterized in that, The microbubble core gas described in S3 is perfluoropropane or perfluorobutane, where the volume fraction of perfluoropropane is 80–99%, and the volume fraction of perfluorobutane is 1–20%.

7. The preparation method of an Annexin V-conjugated magnetic navigation ultrasonic microbubble according to claim 2, characterized in that, The concentration of the avidin solution described in S4 is 1 μg / ml, 30 μg of avidin is added corresponding to every 1×10^8 microbubbles, the incubation time is 30 minutes, and the incubation temperature is 4 °C.

8. The preparation method of an Annexin V-conjugated magnetic navigation ultrasonic microbubble according to claim 2, characterized in that, The ratio of the streptavidin-modified magnetic nanoparticles to the microbubbles described in S5 is 1×10^8:1×10^8, the incubation time is 30 minutes, and the incubation temperature is 4 °C.

9. The preparation method of an Annexin V-conjugated magnetic navigation ultrasonic microbubble according to claim 2, characterized in that, The concentration of the Annexin V solution described in S6 is 0.1–0.5 mg / ml. 30 μg of biotinylated Annexin V is added for every 1×10^8 microbubbles. The incubation time is 30 minutes and the incubation temperature is 4°C.

10. The preparation method of an Annexin V-conjugated magnetic navigation ultrasound microbubble according to claim 2, wherein The centrifugation and washing conditions in S4 to S6 are 1000 revolutions per minute, the centrifugation time is 1 minute, the number of washing times is 3 times, and phosphate buffered saline is used for washing each time.

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