Drug-loaded microbubble as well as preparation method and application thereof

Abstract

The invention discloses a drug-loaded microbubble and a preparation method and application thereof, the drug-loaded microbubble is of a shell membrane-cavity hollow microbubble structure, the shell membrane comprises a biodegradable polymer and lipid, the cavity contains a drug active component, and the drug active component is a biodegradable polymer. The active pharmaceutical ingredient comprises a part combined with the shell membrane and a part free in the cavity, and the part combined with the shell membrane accounts for 50% or less of the total mass of the active pharmaceutical ingredient. When the drug-loaded microbubbles release drugs, the parts dissociating in the cavities can be released in a short time, the local drug concentration is improved, and the treatment purpose is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a drug-loaded microbubble and its preparation method and application. Background Art

[0002] An ultrasound contrast agent is a kind of microbubble contrast agent, usually encapsulated by a phospholipid shell and containing inert gases such as sulfur hexafluoride or perfluorobutane. These microbubbles have a diameter of about 2 to 5 micrometers and can enter the human venous system through peripheral intravenous injection and then reach the target lesion with blood circulation. The main function of the ultrasound contrast agent is to enhance the scattering signal of blood flow in ultrasound imaging, thereby improving the resolution, sensitivity and specificity of ultrasound diagnosis. The ultrasound contrast agent is very safe because it has no nephrotoxicity or cardiotoxicity, and the incidence of adverse reactions is extremely low, and the probability of allergic or pseudo-allergic events is much lower than that of X-ray or MRI contrast agents.

[0003] The microbubble contrast agent is also called a microbubble, which can rupture and generate inertial cavitation under the action of high-energy ultrasound, so that the loaded drug is detonated and released at the target area, playing a role in targeted drug delivery. At the same time, when the microbubble cavitates, microjets and shear forces are generated locally, stimulating the pores of the endothelial cell membrane and the channels between cells to open, and promoting the delivery of therapeutic drugs into cells. This makes the ultrasound-targeted microbubble receive more and more attention as a non-invasive drug delivery technology with broad development prospects.

[0004] However, the drug concentration released by microbubbles in a short time currently far from meets the clinical needs. Traditionally, the microbubble shell layer is very important, which can not only prevent the escape of gas, but also endow them with elasticity to generate acoustic properties for imaging and ultrasound-mediated bubble disruption or cavitation. When using microbubbles as drug delivery carriers, the drugs need to be coupled to the surface of the microbubble shell through electrostatic interactions or encapsulated into the microbubble shell through hydrophobic interactions. Encapsulating drugs into microbubbles is a process greatly affected by the lipophilic or hydrophilic properties of the drugs. It is necessary to optimize the shell composition according to the properties of the drugs to bind drug molecules. Generally, the outer shell of microbubbles is mainly composed of proteins, polymers or lipids. Although lipid-based and protein-based microbubbles have achieved great success in clinical imaging applications, their drug encapsulation efficiency as carriers of drug delivery systems is low. For example, lipid microbubbles have a short duration in the blood and low drug loading capacity due to their single-layer phospholipids. Protein microbubbles are more stable, but have limitations in binding drug molecules, especially for some hydrophilic small chemical drugs. In recent years, some polymers such as polylactic acid, poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(vinyl alcohol) have been used to prepare polymer microbubbles. These polymer microbubbles have higher drug encapsulation efficiency and longer circulation time than lipid microbubbles, but these bubbles are difficult to release enough drugs in response to ultrasonic blasting. Therefore, in order to increase the drug enrichment concentration at the target and improve the drug utilization rate, it is necessary to enhance the cavitation effect of microbubbles. Summary of the Invention

[0005] The present invention aims to at least solve one of the above technical problems existing in the prior art. For this reason, the object of the present invention is to provide a drug-loaded microbubble and its preparation method and application.

[0006] In order to achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] In the first aspect of the present invention, there is provided a drug-loaded microbubble, which is a hollow microbubble structure of a shell membrane - cavity. The shell membrane comprises a biodegradable polymer and a lipid. There is a drug active ingredient in the cavity. The drug active ingredient comprises a part bound to the shell membrane and a part free in the cavity. The part bound to the shell membrane accounts for less than 50% of the total mass of the drug active ingredient.

[0008] In the present invention, the part of the drug active ingredient free in the cavity refers to the remaining drug active part except the part bound to the shell membrane. The part bound to the shell membrane can be physically bound or chemically bound. Physical binding includes, for example, adsorption, partial chimerism, complete embedding, etc. The less the part bound to the shell membrane, when the drug-loaded microbubble releases drugs, it can release the part free in the cavity in a short time, increase the local drug concentration, and achieve the therapeutic purpose.

[0009] In some embodiments of the present invention, the portion bound to the shell membrane accounts for less than 45% of the total mass of the active ingredient of the drug, such as less than 40%, less than 30%, less than 20%, less than 18%, less than 15%, less than 13%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, etc.

[0010] In some embodiments of the present invention, the portion free in the cavity accounts for more than 50% of the total mass of the active pharmaceutical ingredient, such as more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, etc.

[0011] In some embodiments of the present invention, the cumulative drug release rate of the drug-loaded microbubbles within 3 to 8 minutes is at least 30%, such as at least 35%, at least 38%, at least 40%, at least 45%, at least 48%, at least 50%, at least 55%, at least 60%, etc. The cumulative drug release rate is an in vitro cumulative drug release rate, which is obtained by ultrasonicating in water or 5 wt% glucose solution at 37°C at a frequency of 1 MHz and an intensity of 2 W / cm 2 , measured by continuous ultrasound for 5 minutes.

[0012] In some embodiments of the present invention, the shell membrane has a thickness of 50 to 300 nm, such as 50 to 250 nm, 50 to 200 nm, 50 to 150 nm, 50 to 120 nm, 50 to 100 nm, 60 nm, 70 nm, 80 nm, or 90 nm. In the present invention, the shell membrane thickness of the hollow drug-loaded microbubbles is controlled to be 30 to 120 nm. When the drug-loaded microbubbles are subjected to continuous ultrasound irradiation, cavitation occurs under low-intensity ultrasound, releasing the drug at the ultrasound site through inertial cavitation. This strong cavitation effect can increase the drug concentration at the lesion site, thereby achieving the therapeutic purpose.

[0013] In some embodiments of the present invention, the particle size of the drug-loaded microbubbles is 0.6-4 μm, such as 0.6-2 μm, 0.8-1.5 μm, 0.9-1.3 μm, or 1.0-1.3 μm.

[0014] In some embodiments of the present invention, the drug loading amount of the active pharmaceutical ingredient in the drug-loaded microbubbles is 0.5% to 10% of the total weight of the drug-loaded microbubbles, such as 0.5% to 5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, and 4.5%.

[0015] In some embodiments of the present invention, the mass ratio of the biodegradable polymer to the lipid is 3 to 20:1, such as 5 to 20:1, 8 to 18:1, 9 to 15:1, 10 to 13:1, etc. By controlling the mass ratio of the two, the modification effect of the lipid on the biodegradable polymer is improved, thereby improving the flexibility, elasticity and stability of the shell, thereby improving the ultrasonic responsiveness and ultrasonic contrast effect of the ultrasonic contrast agent and the effect of microbubble targeted rupture, and reducing the irreversible mechanical damage caused by the rupture of microvascular endothelial cells due to the too strong cavitation effect of traditional drug-loaded microbubbles.

[0016] In some embodiments of the present invention, the biodegradable polymer is an aliphatic polyester. The aliphatic polyester is a homopolymer or copolymer of aliphatic polylactone, including one or more of polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), poly(lactide-glycolide) copolymer (PLGA), poly(lactide-caprolactone) copolymer (PLC), poly(glycolide-caprolactone) copolymer (PGC), poly(glycolide-lactide-caprolactone) copolymer (PGLC). The aliphatic polyester can be a carboxyl-terminated aliphatic polyester; the molecular weight of the aliphatic polyester is 10,000 to 200,000; such as 20,000, 50,000, 80,000, 100,000, 150,000, etc.

[0017] In some embodiments of the present invention, the lipid includes at least one of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine; the lipid includes phospholipids selected from the group consisting of hydrogenated soy phosphatidylcholine (HSPC); 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC); 1,2-dierucoyl-sn-glycero-3-phosphate (DEPA); 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC); 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE); 1,2-dielaidoyl-phosphatidylglycerol (DEPG); 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC); 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA); 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-dilauroyl-sn-glycero-3-phosphatidylglycerol (DLPG); 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS); 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG); 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS); 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG); 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS); 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG); 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS); 1,2-distearoyl-sn-glycero-3-phosphate (DSPA); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE);1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG); 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS); 1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (MPPC); 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC); 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG); 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC); 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC); and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC); at least one of 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC);

[0018] In some embodiments of the present invention, the lipid comprises a derivatized phospholipid; the derivatized phospholipid comprises a polyethylene glycol-modified phospholipid.

[0019] In some embodiments of the present invention, in the derivatized phospholipid, polyethylene glycol serves as a linker for the phospholipid; the molecular weight of the polyethylene glycol is from about 350 g / mol to about 30,000 g / mol. For example, the polyethylene glycol may have a molecular weight selected from the group consisting of: 350 g / mol, 550 g / mol, 750 g / mol, 1000 g / mol, 2000 g / mol, 3000 g / mol, 5000 g / mol, 10,000 g / mol, 20,000 g / mol or 30,000 g / mol.

[0020] In some embodiments of the present invention, the derivatized phospholipid comprises DSPE-PEG 1000 、DSPE-PEG 1500 、DSPE-PEG 2000 、DSPE-PEG 2500 、DSPE-PEG 3000 、DSPE-PEG 5000 .

[0021] In some embodiments of the present invention, the phospholipids include one or more of DSPS, DSPC, DSPE, and / or the derivatized phospholipids; for example, any one of DSPS, DSPC, or DSPE and the derivatized phospholipids are in a mass ratio of (1 to 10):1; such as (6 to 9):1.

[0022] In some embodiments of the present invention, the pharmaceutically active ingredient includes at least one of paclitaxel, docetaxel, hydroxycamptothecin, doxorubicin, mitomycin, tamoxifen, 5-fluorouracil, methotrexate, cytarabine, cyclophosphamide, or platinum drugs (cisplatin, carboplatin, or oxaliplatin).

[0023] In a second aspect of the present invention, there is provided an ultrasound contrast agent comprising the drug-loaded microbubbles described above.

[0024] In some embodiments of the present invention, the ultrasound contrast agent further includes gas microbubbles.

[0025] In some embodiments of the present invention, the gas in the gas microbubbles is an inert gas, which can be a gas conventionally used in microbubble ultrasound contrast agents in the art, for example, selected from one or more of perfluoropropane, perfluorobutane, and sulfur hexafluoride.

[0026] In some embodiments of the present invention, the particle size of the gas microbubbles is 0.1 to 3 μm, such as 0.5 to 2 μm, 0.8 to 1.5 μm, 0.9 to 1.3 μm, 1.0 to 1.3 μm.

[0027] In some embodiments of the present invention, the content of the gas in the ultrasound contrast agent is 0.2 to 6.0 μL / mg, such as 0.5 to 3.0 μL / mg, 0.5 to 2.8 μL / mg.

[0028] In some embodiments of the present invention, the particle size of the ultrasound contrast agent is 0.6 to 4 μm, such as 0.8 to 3 μm, 0.8 to 2 μm, 1 to 2 μm, etc.

[0029] In some embodiments of the present invention, the method for preparing the ultrasound contrast agent includes filling the drug-loaded microbubbles with the gas.

[0030] In a third aspect of the present invention, there is provided a method for preparing drug-loaded microbubbles, comprising the following steps:

[0031] S1: Placing a solution containing a pharmaceutically active ingredient and phospholipids in an organic solution of a biodegradable polymer and a lipid for emulsification treatment to obtain a water-in-oil first primary emulsion;

[0032] S2: Introduce gaseous fluorocarbon into the hydrated phospholipid solution, and prepare a phospholipid vesicle solution after ultrasonic treatment. Then place it in an organic solution of a biodegradable polymer and lipid for emulsification treatment to obtain a water-in-oil secondary primary emulsion.

[0033] S3: Mix the first primary emulsion and the second primary emulsion, add a polyvinyl alcohol solution for emulsification treatment to prepare a water-in-oil-in-water multiple emulsion.

[0034] S4: Remove the organic solvent from the multiple emulsion, collect the precipitate after purification, and perform freeze-drying treatment on the precipitate to prepare the drug-loaded microbubbles.

[0035] In the present invention, the drug active ingredient and phospholipid are placed in an aqueous solution, and an oil-in-water emulsion is formed with an organic solution of a biodegradable polymer and lipid, which can control the drug active ingredient in the aqueous phase, maintain the hydrophilicity of the drug active ingredient, and effectively avoid the migration of the drug active ingredient to the lipid shell membrane and its combination with the shell membrane during the subsequent formation of drug-loaded microbubbles. Thus, the content of the drug active ingredient free in the cavity of the drug-loaded microspheres is increased, and the release of this part of the drug active ingredient can be achieved within a short time during the drug release process, thereby significantly increasing the local drug concentration. On the other hand, different from the traditional preparation process of directly charging gas into the oil phase or directly wrapping the aqueous phase containing gas with the oil phase, in the present invention, gaseous fluorocarbon is first introduced into the hydrated phospholipid solution to form an aqueous phase, and then the oil phase is wrapped to form a second primary emulsion, which can avoid introducing too much ammonium bicarbonate, reduce the migration of the drug active ingredient, and obtain a multiple emulsion with sufficient internal cavity space, so that the final product can better respond to low-frequency ultrasound and release sufficient drugs.

[0036] In some embodiments of the present invention, in S1, the biodegradable polymer and lipid form an oil phase to wrap the aqueous phase of the solution containing the drug active ingredient and phospholipid. The ratio of the biodegradable polymer to lipid in the oil phase to the drug active ingredient and phospholipid in the aqueous phase is 5-15:1 (w / w), such as 5-10:1 (w / w), 6-10:1 (w / w), 7-10:1 (w / w), 8-10:1 (w / w), etc.

[0037] In some embodiments of the present invention, in S2, the biodegradable polymer and lipid form an oil phase to wrap the aqueous phase formed by the hydrated phospholipid solution containing gaseous fluorocarbon. The ratio of the biodegradable polymer to lipid in the oil phase to the phospholipid in the aqueous phase is 18-25:1 (w / w), such as 18-23:1 (w / w), 19-22:1 (w / w), etc.

[0038] In some embodiments of the present invention, in S3, the mass ratio of the first primary emulsion, the second primary emulsion to the organic solute (i.e., the part other than the solvent) in the polyvinyl alcohol solution is 1:(0.6 - 1.8):(5 - 20), such as 1:(0.8 - 1.6):(9 - 15), 1:(0.9 - 1.1):(9 - 11), 1:(0.9 - 1.1):(5 - 8).

[0039] In some embodiments of the present invention, the organic solvents used in the organic solution include at least one of dichloromethane, chloroform, ethyl acetate, and acetone.

[0040] In some embodiments of the present invention, the gaseous fluorocarbon includes at least one of perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, and perfluoroheptane.

[0041] In some embodiments of the present invention, in S1, the mass ratio of the drug active ingredient to the phospholipid is 1:0.8 - 2, such as 1:1 - 1.8, 1:1 - 1.5, 1:1 - 1.2, etc.

[0042] In some embodiments of the present invention, in S2, the volume - mass ratio of the gaseous fluorocarbon to the phospholipid is 30 - 60:1, such as 40 - 50:1.

[0043] In some embodiments of the present invention, in S2, the mass ratio of the phospholipid to the hydration liquid in the hydration liquid of the phospholipid is 1:180 - 220.

[0044] In some embodiments of the present invention, in S2, the hydration liquid of the phospholipid includes propylene glycol, glycerol, and water; the mass ratio of the three is 1:2 - 5:70 - 100, such as 1:2 - 5:70 - 80, 1:3 - 4:73 - 76.

[0045] In some embodiments of the present invention, the emulsification treatment in S1 includes treating at a stirring rate of 500 - 8000 rpm for 1 - 5 min (such as 1 - 3 min), and then performing ultrasonic treatment. The ultrasonic power is 40 - 280 W, and it works for 1 - 3 s and pauses for 1 - 3 s alternately for 1 - 5 min (such as 1 - 3 min); the ultrasonic treatment is carried out at 15 - 22 °C.

[0046] In some embodiments of the present invention, S1 further includes adding a stabilizer for continued emulsification treatment after the emulsification treatment; the treatment time is 0.1 to 3 min, such as 0.1 to 1 min. In the present invention, after the aqueous phase containing the drug active ingredient and the oil phase are emulsified, an oil-in-water emulsion is initially formed. At this time, adding a stabilizer and then performing emulsification treatment, the outer oil phase of the aforementioned emulsion can still be emulsified with the stabilizer, so that the stabilizer is wrapped in the outer oil phase, further controlling a relatively short emulsification treatment time (0.1 to 3 min), avoiding over-emulsification, effectively isolating the drug active ingredient in the inner aqueous phase and the stabilizer, and preventing the drug active ingredient from prematurely binding to the stabilizer, thereby causing the drug active ingredient to tend to be lipophilic, which is not conducive to the product controlling the drug active ingredient in the free inner cavity of the drug-loaded microbubble.

[0047] In some embodiments of the present invention, the stabilizer includes at least one of ammonium bicarbonate, sodium bicarbonate, and sodium carbonate; the mass ratio of the stabilizer to the drug active ingredient is 1:(1 to 50), such as 1:(1 to 30), 1:(2 to 20), 1:(3 to 15), 1:(4 to 8), 1:(5 to 7).

[0048] In some embodiments of the present invention, the ultrasonic power of the ultrasonic treatment in S2 is 40 to 280 W, and it is carried out alternately for 1 to 5 min (such as 1 to 3 min) with a working time of 1 to 3 s and a pause time of 1 to 3 s.

[0049] In some embodiments of the present invention, the emulsification treatment in S2 includes treating at 500 to 8,000 rpm for 0.5 to 3 min, and then treating at 10,000 to 40,000 rpm for 0.5 to 3 min.

[0050] In some embodiments of the present invention, the emulsification treatment in S3 includes treating at 3,000 to 15,000 rpm (such as 3,000 to 10,000 rpm) for 2 to 5 min.

[0051] In some embodiments of the present invention, the emulsification treatment in S3 includes stirring and membrane-passing treatment at 100 rpm to 600 rpm (such as 100 rpm to 200 rpm) for 10 to 600 s, such as 10 s to 60 s; the membrane includes a 2 to 20 μm (such as 2 to 15 μm) SPG membrane.

[0052] In some embodiments of the present invention, the method for removing the organic solvent from the multiple emulsion in S4 includes stirring to remove the organic solvent (such as stirring to remove the organic solvent while blowing air), the stirring rate is 80 to 500 rpm (such as 80 to 150 rpm), and the time is 15 to 25 h.

[0053] In some embodiments of the present invention, the purification in S4 includes adding water and centrifuging at 800 - 3,000 rpm (such as 800 - 1500 rpm) for 5 - 20 min (such as 10 - 20 min).

[0054] In some embodiments of the present invention, the lyophilization treatment in S4 includes pre-cooling under vacuum for 2 - 6 h and then sublimation drying for 15 - 25 h.

[0055] In the fourth aspect of the present invention, there is provided an application of the drug-loaded microbubbles and / or the ultrasound contrast agent as described above in the preparation of drugs for tumor diagnosis and treatment.

[0056] In some embodiments of the present invention, the tumor diagnosis includes ultrasound contrast imaging technology.

[0057] In some embodiments of the present invention, the tumor type can be determined according to the active ingredient of the loaded drug. Common ones include breast cancer, gastric cancer, lung cancer, ovarian cancer, liver cancer, kidney cancer, colorectal cancer, lymphoma, multiple myeloma, etc.

[0058] In some embodiments of the present invention, the drug-loaded microbubbles and / or the ultrasound contrast agent can be delivered into the detected part of the body by intravenous injection, vibrate under the action of ultrasound, and change the ultrasonic characteristics of the tissue, such as backscattering coefficient, attenuation coefficient, sound velocity, and nonlinear effect; or complete the sustained release, ultrasonic imaging, and ultrasonic controlled release of the active ingredient in the body.

[0059] In some embodiments of the present invention, the intensity of the ultrasonic action is 0.5 - 5 W / cm 2 , and the time is 1 - 10 min.

[0060] The beneficial effects of the present invention are as follows:

[0061] In the drug-loaded microbubbles of the present invention, the less the part combined with the shell membrane, when the drug-loaded microbubbles release the drug, the part free in the cavity can be released in a short time, increasing the local drug concentration and achieving the treatment purpose.

[0062] The preparation method of the present invention can control the active ingredient of the drug in the aqueous phase, maintain the hydrophilicity of the active ingredient of the drug, effectively avoid the migration of the active ingredient of the drug to the lipid shell membrane and combination with the shell membrane when forming the drug-loaded microbubbles subsequently, thereby increasing the content of the active ingredient of the drug free in the cavity of the drug-loaded microspheres, realizing the release of this part of the active ingredient of the drug in a short time during the drug release process, and thus significantly increasing the local drug concentration. On the other hand, different from the traditional preparation process of directly filling gas into the oil phase or directly wrapping the aqueous phase containing gas with the oil phase, the preparation method of the present invention can avoid introducing too much ammonium bicarbonate, reduce the migration of the active ingredient of the drug, and obtain a multiple emulsion with enough internal cavity space, so that the final product can better respond to low-frequency ultrasound and release enough drugs. Brief Description of the Drawings

[0063] Figure 1 This is the electron microscope scanning image of the ultrasound contrast agent in Example 1 of the present invention.

[0064] Figure 2 This is the electron microscope scanning image of the ultrasound contrast agent in Example 2 of the present invention.

[0065] Figure 3 This is the electron microscope scanning image of the ultrasound contrast agent in Example 3 of the present invention.

[0066] Figure 4 This is the electron microscope scanning image of the ultrasound contrast agent in Comparative Example 1 of the present invention.

[0067] Figure 5 This is the electron microscope scanning image of the ultrasound contrast agent in Comparative Example 2 of the present invention.

[0068] Figure 6 This is the physical image of the in vitro drug release of the ultrasound contrast agent in Example 1 of the present invention.

[0069] Figure 7 This is the physical image of the in vitro drug release of the ultrasound contrast agent in Example 2 of the present invention.

[0070] Figure 8 This is the physical image of the in vitro drug release of the ultrasound contrast agent in Example 3 of the present invention.

[0071] Figure 9 This is the tumor volume curve graph of treating tumor-bearing mice in Example 4 of the present invention.

[0072] Figure 10 This is the relative tumor proliferation rate curve of treating tumor-bearing mice in Example 4 of the present invention.

[0073] Figure 11 This is the bar graph of tumor weight at the experimental end point of treating tumor-bearing mice in Example 4 of the present invention. Detailed Description of the Invention

[0074] The content of the present invention will be further described in detail through specific examples below. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or testing methods are all conventional methods in the art.

[0075] Ultrasonic cell disruptor, Shanghai HuXi Industrial Co., Ltd., model JY92-IIN;

[0076] High-speed homogenizer, Shanghai Specimen Model Factory, JRJ-300-I.

[0077] Example 1

[0078] In this example, a contrast agent for ultrasound was prepared. The specific process is as follows: The raw materials for preparation include

[0079] The oil phase 1 includes 478 mg of PLGA, 47.8 mg of DSPC, and 15 mL of dichloromethane;

[0080] The inner aqueous phase 1 includes 30 mg of DOX, 32.5 mg of mPEG2000 DSPE, and 3 mL of ultrapure water;

[0081] The oil phase 2 includes 478 mg of PLGA, 47.8 mg of DSPC, and 15 mL of dichloromethane;

[0082] The inner aqueous phase 2 includes 6 mL of DSPS phospholipid vesicle solution; The DSPS phospholipid solution includes 225.2 mg of DSPS, 625 mg of propylene glycol, 2.075 g of glycerol, and 47.3 g of ultrapure water;

[0083] The stabilizer for the inner aqueous phase 1 includes 5 mg of sodium bicarbonate and 0.25 mL of ultrapure water;

[0084] The outer aqueous phase includes 300 mL of a 2 wt% PVA solution.

[0085] Preparation of the DSPS phospholipid solution:

[0086] Step 1, Dissolution of materials: Weigh each raw and auxiliary material according to the prescription composition, heat in a water bath at 80°C, and continuously shake for 5 min;

[0087] Step 2, Recovery to room temperature: Place the dissolved phospholipid solution obtained above in a room temperature environment, mix well with magnetic stirring, and the phospholipid solution is restored to room temperature for standby.

[0088] Preparation of drug-loaded microbubbles:

[0089] Step 1, Dissolution of materials: Weigh each raw and auxiliary material according to the prescription composition, and dissolve with magnetic stirring;

[0090] Step 2, Preparation of primary emulsion 1: Aspirate the inner aqueous phase 1 solution into the oil phase 1, homogenize and mix well in a water bath with a high-speed homogenizer (3,000 rpm, 1.5 min), add ammonium bicarbonate aqueous solution while homogenizing and continue to homogenize at 3,000 rpm for 0.5 min; Transfer the homogenized solution (in a 20°C water bath) to an ultrasonic cell disruptor, with a 12 - gauge probe, a power of 130 W, ultrasonic for 2 s, turn off for 3 s, and work for a total of 1.5 min to obtain a water-in-oil primary emulsion 1;

[0091] Step 3, Preparation of phospholipid vesicles: Place the DSPS phospholipid solution in a container, insert a hose under the liquid surface and introduce perfluoropropane gas, and use an ultrasonic cell disruptor to ultrasonicate for 1.5 min to prepare DSPS phospholipid micro-nanobubbles (probe No. 12, power 97.5 W, ultrasonication for 2 s, off for 3 s);

[0092] Step 4, Preparation of primary emulsion 2: Aspirate the lower-layer DSPS phospholipid micro-nanobubble solution, place it in oil phase 2, and homogenize and mix it with a high-speed homogenizer (3,000 rpm, 1 min; 15,000 rpm, 1 min) to obtain a water-in-oil primary emulsion 2;

[0093] Step 5, Preparation of multiple emulsion: Magnetically stir and mix the above two primary emulsions, add the primary emulsion under the liquid surface of a 2% PVA solution (10 - 15 °C), magnetically stir and mix, transfer to a high-speed homogenizer, and homogenize at 5000 rpm in a water bath for 3 min to obtain a water-in-oil-in-water multiple emulsion;

[0094] Step 6, Removal of organic solvents: Place the multiple emulsion in a double-layer three-necked flask for temperature control, and remove the organic solvents while blowing air and stirring with a paddle (stirring rate 100 rpm). Keep the blowing port 9 cm away from the liquid surface in the first 2 h, add 200 mL of ultrapure water at 2 h and keep the blowing port 5 cm away from the liquid surface, and stir for a total of 20 - 22 h;

[0095] Step 7, Purification: Transfer the above solution to a 500 mL centrifuge bottle, add an appropriate amount of ultrapure water, centrifuge at 1,000 rpm for 15 minutes, collect the precipitate, and disperse the precipitate with a small amount of ultrapure water;

[0096] Step 8, Freeze-drying: Aliquot the dispersed solution into vials, pre-cool in a vacuum freeze-dryer for about 4 hours, sublime and dry for about 20 hours, and fill with perfluoropropane gas until the freeze-dryer is full.

[0097] According to the preparation method of Example 1, prepare a group of empty microbubbles without loading doxorubicin.

[0098] Example 2

[0099] In this example, a contrast agent for ultrasound is prepared. The difference in the preparation materials from Example 1 lies in the differences in the inner aqueous phase 2 and the preparation method of the multiple emulsion. Specifically:

[0100] The inner aqueous phase 2 includes 6 mL of DSPC / DSPE phospholipid vesicle solution, including 10 mg of DSPC, 75 mg of mPEG2000 DSPE, 625 mg of propylene glycol, 2.075 g of glycerol, and 47.3 g of ultrapure water;

[0101] Double emulsion preparation: Mix the two primary emulsions with a magnetic stirrer at 120 rpm. Add the primary emulsion mixture below the liquid surface of a 2% PVA solution (10 - 15°C), mix well with a magnetic stirrer at 120 rpm for 30 s, and then pass through a 6 - micron SPG membrane; 21.4 mg of freeze - dried powder is obtained.

[0102] Example 3

[0103] In this example, an ultrasound contrast agent was prepared. The difference in the preparation raw materials from Example 1 lies in the inner aqueous phase 2, the primary emulsion 1, the phospholipid vesicles, and the double emulsion preparation method. Specifically:

[0104] The inner aqueous phase 2 includes 5 mL of DSPS phospholipid vesicle solution; the DSPS phospholipid solution includes 240 mg of DSPS, 500 mg of propylene glycol, 1.8 g of glycerol, and 48.2 g of ultrapure water; Dissolution of the DSPS phospholipid solution materials: According to the prescription composition, weigh the excipients separately, heat in a water bath at 60°C for 30 min, and set aside. Place the DSPS phospholipid solution in a container, insert a hose below the liquid surface and introduce perfluoropropane gas, and use an ultrasonic cell disruptor to ultrasonicate for 1 min to prepare DSPS phospholipid micro - nano vesicles (No. 12 probe, power 130 W, ultrasonication for 2 s, turn off for 3 s).

[0105] Preparation of primary emulsion 1: Absorb the inner aqueous phase 1 solution into the oil phase 1, homogenize and mix well with a high - speed homogenizer in a water bath (3000 rpm, 1.5 min), add ammonium bicarbonate aqueous solution while homogenizing and continue to homogenize at 3000 rpm for 0.5 min; Transfer the homogenized solution (20°C ice - water bath) to an ultrasonic cell disruptor, No. 12 probe, power 130 W, ultrasonication for 2 s, turn off for 3 s, and work for a total of 2 min to obtain the water - in - oil primary emulsion 1.

[0106] Double emulsion preparation: Mix the two primary emulsions with a magnetic stirrer at 120 rpm. Add the primary emulsion mixture below the liquid surface of a 2% PVA solution (10 - 15°C), mix well with a magnetic stirrer at 120 rpm for 30 s, and then pass through a 6 - micron SPG membrane; 23.1 mg of freeze - dried powder is obtained.

[0107] Comparative Example 1

[0108] In this comparative example, an ultrasound contrast agent was prepared. The preparation raw materials include:

[0109] The oil phase 1 includes 478 mg of PLGA, 47.8 mg of DSPC, and 15 mL of dichloromethane;

[0110] The oil phase 2 includes 478 mg of PLGA, 47.8 mg of DSPC, and 15 mL of dichloromethane;

[0111] The inner aqueous phase 1 includes 6 mL of DSPS phospholipid solution; the composition of the DSPS phospholipid solution is 225.2 mg of DSPS, 625 mg of propylene glycol, 2.075 g of glycerol, and 47.3 g of ultrapure water. 6 mL is taken out for standby;

[0112] The inner aqueous phase 2 includes 30 mg of doxorubicin hydrochloride, 60 mg of mPEG2000 DSPE, and 3 mL of ultrapure water;

[0113] The stabilizer 2 includes 10 mg of sodium bicarbonate and 0.25 mL of ultrapure water;

[0114] The outer aqueous phase includes 300 mL of 2 wt% PVA solution.

[0115] The specific preparation process includes:

[0116] Step 1: Add the inner aqueous phase 1 to the oil phase 1, and homogenize and mix well with a high-speed homogenizer at room temperature (3,000 rpm, 1 min; 15,000 rpm, 1 min) to obtain the primary emulsion 1;

[0117] Step 2: Slowly add the inner aqueous phase 2 to the pre-sheared oil phase 2, and homogenize and mix well with a high-speed homogenizer at room temperature (3000 rpm, 1.5 min). While homogenizing, add the stabilizer 2 and continue to homogenize at 3000 rpm for 0.5 min. Transfer it to a cell disruptor, and under a 20°C water bath, use a 12# ultrasonic probe and a power of 97.5 W to perform ultrasonic pulse for 2 min to obtain the primary emulsion 2;

[0118] Step 3: Magnetically stir and mix the above two primary emulsions, and then add them to the outer aqueous phase (10 - 15°C) under the liquid surface, and magnetically stir evenly for 30 s. While stirring, pass through a 6-micron SPG membrane to obtain the double emulsion;

[0119] Step 4: Place the double emulsion in a double-layer three-necked flask for temperature control, and while blowing air (8 - 10 L / min), use a stirring paddle (100 rpm) to remove the organic solvent. In the first 2 h, keep the blowing port 9 cm away from the liquid surface. At 2 h, add 400 mL of ultrapure water and keep the blowing port 5 cm away from the liquid surface, and solidify for a total of 20 h;

[0120] Step 5: Purification: Transfer the above solution to a 500 mL centrifuge bottle, add an appropriate amount of ultrapure water, centrifuge at 1000 rpm for 15 minutes, collect the precipitate, and disperse the precipitate with a small amount of ultrapure water;

[0121] Step 6: Freeze-dry: Aliquot the dispersed solution into vials, place it in a vacuum freeze-dryer for pre-cooling for about 4 hours, perform sublimation drying for about 20 hours, and fill with perfluoropropane gas.

[0122] Comparative Example 2

[0123] This comparative example prepared an ultrasonic contrast agent, and the preparation raw materials included:

[0124] The oil phase 1 included 478 mg of PLGA, 47.8 mg of DSPC, and 15 mL of dichloromethane;

[0125] The oil phase 2 included 478 mg of PLGA, 47.8 mg of DSPC, and 15 mL of dichloromethane;

[0126] The inner aqueous phase 2 included 30 mg of doxorubicin hydrochloride, 60 mg of mPEG2000 DSPE, and 3 mL of ultrapure water;

[0127] The stabilizer 2 included 10 mg of sodium bicarbonate and 0.25 mL of ultrapure water;

[0128] The outer aqueous phase included 300 mL of 2 wt% PVA solution.

[0129] The specific preparation process included:

[0130] Step 1: Perfluoropropane gas was introduced into the oil phase 1 for 1 min, and it was homogenized and mixed evenly by a high-speed homogenizer at room temperature (3000 rpm, 1 min; 15,000 rpm, 1 min) to obtain the primary emulsion 1;

[0131] Step 2: The inner aqueous phase 2 was slowly added to the pre-sheared oil phase 2, and it was homogenized and mixed evenly by a high-speed homogenizer at room temperature (3000 rpm, 1.5 min). While homogenizing, the stabilizer 2 was added and then homogenized at 3000 rpm for another 0.5 min. It was transferred to a cell disruptor and ultrasonically pulsed for 2 min at 20°C in a water bath with a 12# probe and a power of 97.5 W to obtain the primary emulsion 2;

[0132] Step 3: The above two primary emulsions were magnetically stirred and mixed evenly, and then added to the outer aqueous phase (10 - 15°C) under the liquid surface, and magnetically stirred evenly for 30 s. It was passed through a 6-micron SPG membrane under stirring to obtain the multiple emulsion;

[0133] Step 4: The multiple emulsion was placed in a double-layer three-necked flask for temperature control, and the organic solvent was removed while blowing air and using a stirring paddle (100 rpm). In the first 2 h, the air blowing port was 9 cm away from the liquid surface. At 2 h, 200 mL of ultrapure water was added and the air blowing port was 5 cm away from the liquid surface, and it was solidified for a total of 24 h;

[0134] Step 5: Purification: The above solution was transferred to a 500 mL centrifuge bottle, an appropriate amount of ultrapure water was added, centrifuged at 1000 rpm for 15 minutes, the precipitate was collected, and the precipitate was dispersed with a small amount of ultrapure water;

[0135] Step 6: Freeze-drying: The dispersed solution was dispensed into vials, placed in a vacuum freeze-dryer and pre-cooled for about 4 hours, sublimation-dried for about 20 hours, and perfluoropropane gas was filled.

[0136] Test Example 1

[0137] The electron microscope scanning images of the ultrasound contrast agents prepared in the examples and comparative examples are as Figures 1 - 5 shown.

[0138] It can be seen that the drug-loaded microbubble shell layer in the examples is thinner, and the middle part is a cavity structure, while the comparative example is an obvious solid sphere structure.

[0139] The PSS particle size distribution of the ultrasound contrast agents prepared in the examples and comparative examples was detected. The specific process was as follows:

[0140] 1. Inject the freeze-dried powder of the drug-loaded microbubbles into ultrapure water and re-dissolve and disperse it into 10 mg / mL;

[0141] 2. Pipette 10 μL and place it in a 50 mL centrifuge tube, add ultrapure water (filtered through a 0.2 μm filter membrane) to the scale line, and gently shake and mix;

[0142] 3. Use an A9000 Accu-sizeR FX nano particle size analyzer to measure the particle size distribution and concentration of the drug-loaded microbubbles.

[0143] The particle sizes of the ultrasound contrast agents prepared in the examples and comparative examples are shown in Table 1:

[0144] Table 1

[0145]

[0146] It can be seen that the average particle size of the drug-loaded microbubbles prepared in the examples is closer to 1 μm, and its drug release level is higher.

[0147] Test Example 2

[0148] The in vitro drug release test of the ultrasound contrast agents prepared in the examples and comparative examples was carried out. The specific process was as follows:

[0149] The freeze-dried powder of the drug-loaded microbubbles was added to ultrapure water for dispersion and dissolution. After making a 10 mg / mL dispersion, it was mixed. Pipette 0.325 mL of the dispersion and place it in a supramolecular membrane set, add 0.325 mL of ultrapure water to dilute it into a 5 mg / mL dispersion. Under a 37 °C water bath circulation, use a Sibai ultrasonic device at 1 MHz, continuous mode, 2 W ultrasonic for 5 min. After the ultrasonic cavitation ended, it was transferred to a 2 mL centrifuge tube as the ultrasonic cavitation sample; another 0.325 mL of the dispersion was pipetted and placed in a 2 mL centrifuge tube, and 0.325 mL of ultrapure water was added to dilute it into a 5 mg / mL dispersion as the blank sample. The cavitation and blank samples were centrifuged at 15000 rpm for 2 min; the supernatant was removed for liquid phase analysis of the DOX content, and three parallel tests were carried out for each example or comparative example of the ultrasound contrast agent. The drug release diagram is as Figures 6 - 8 shown.

[0150] The drug release results of the ultrasound contrast agents prepared in the examples and comparative examples are shown in Table 2:

[0151] Table 2

[0152] Group Drug loading (%) Drug release 1 (%) Drug release 2 (%)) Drug release 3 (%) Average value (%) Example 1 0.828 43.99 44.93 47.16 45.361 Example 2 0.915 37.36 33.43 35.70 35.50 Example 3 1.30 30.61 34.57 30.45 31.88 Comparative Example 1 1.75 11.26 13.85 15.04 13.38 Comparative Example 2 1.35 6.75 7.45 5.37 6.53

[0153] It can be seen that the drug-loaded microbubbles prepared in the examples have significantly increased drug release compared with the comparative examples.

[0154] Test Example 3

[0155] The gas content in the ultrasound contrast agents prepared in the examples and comparative examples was tested. The specific process was as follows:

[0156] Instrument: Agilent 7890B gas chromatograph;

[0157] Chromatographic column: Agilent A12O3 / KC1 (0.53mm * 25m * 10μm, KC-GC-035), column flow rate: 6.7 mL / min, column temperature 100 °C, maintained for 5 minutes;

[0158] Injection port temperature: 130 °C, split ratio: 10:1, carrier gas: helium;

[0159] Detector: TCD, detector temperature: 180 °C;

[0160] Injection method: headspace injection, headspace equilibrium temperature: 80 °C, equilibrium time: 20 min, quantitative loop temperature: 90 °C, transfer line temperature: 100 °C;

[0161] Injection duration: 0.5 min, GC cycle: 7 min, sample bottle filling pressure: 15 Psi;

[0162] Preparation of gas phase samples:

[0163] 1.1 Test sample

[0164] The freeze-dried powder of drug-loaded microbubbles was reconstituted and dispersed with 2 mL of ultrapure water. Then, 1.5 mL of the reconstituted and dispersed solution was placed in a 10 mL headspace bottle, 1.5 mL of NaOH was added, immediately capped and sealed, and sonicated in an ultrasonic cleaner for 1 min to break the emulsion, and thus obtained.

[0165] 1.2 Reference substance

[0166] 1.5 mL of ultrapure water was measured and placed in a 10 mL headspace bottle, 1.5 mL of NaOH was added, capped and sealed, and 250 μL of 99% octafluoropropane gas was injected with a gastight syringe and mixed evenly, and thus obtained.

[0167] 1.3 Blank sample

[0168] Measure 1.5 mL of ultrapure water and place it in a 10 mL headspace vial. Add 1.5 mL of NaOH, press on the cap to seal, and mix well to obtain the sample.

[0169] After the system stabilizes, inject the above gas-phase sample for detection and record the chromatogram.

[0170] The gas contents of the ultrasound contrast agents prepared in the examples and comparative examples are shown in Table 3:

[0171] Table 3

[0172] Group Gas content 1 (μL / mg) Gas content 2 (μL / mg) Gas content 3 (μL / mg) Average value (μL / mg) Example 1 0.39 0.34 0.42 0.39 Example 2 1.45 1.48 1.52 1.48 Example 3 1.41 1.44 2.03 1.63 Comparative Example 1 \ 0.60 0.55 0.58 Comparative Example 2 0.63 0.67 0.66 0.65

[0173] Example 4

[0174] The anti-tumor effect of the ultrasound contrast agent prepared in Example 3 was tested in mice. The specific process was as follows:

[0175] Experimental animals: SPF-grade BALB / c-nu mice, female, 4 - 5 weeks old, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.

[0176] Tumor cells: 4T1 mouse breast cancer cells, purchased from Beijing Na Biotechnology Co., Ltd.

[0177] Drug information:

[0178] DOX liposome, Shijiazhuang Pharmaceutical Group Ouyi Pharmaceutical Co., Ltd., specification 10 mL: 20 mg;

[0179] Doxorubicin hydrochloride, Zhejiang Hisun Pharmaceutical Co., Ltd.;

[0180] DOX-loaded microbubbles, Example.

[0181] 1. Experimental method

[0182] 1.1. Establishment of subcutaneous tumor-bearing model

[0183] Resuscitate 4T1 mouse breast cancer cells. When the cells grow to a sufficient number, collect the cells and inoculate them subcutaneously into BALB / c-nu mice at a dose of 2.0×10 6 cells / mouse. After inoculating the tumor cells, regularly measure the tumor volume and body weight.

[0184] 1.2. Grouping and drug administration

[0185] After the mice developed tumors, 30 animals with an average tumor volume in the range of 100 - 150 mm 3 were randomly selected and grouped. The specific grouping and drug administration plan are shown in Table 4 below. The grouping day was set as D1:

[0186] Table 4

[0187]

[0188] After the drug was injected into the tail vein, the tumor was immediately treated with ultrasound therapy using an ultrasound therapy device. The coupling agent was applied to the tumor site of the mouse, and the ultrasound probe was fixed at the tumor site at a distance of 1 cm. The ultrasound frequency was set to 1 MHz and the power was set to 2 W / cm. 2 , time 5min, continuous output mode.

[0189] 1.3 Monitoring indicators

[0190] General clinical observation: observe whether the mice are moving normally, whether there are any injuries to the body, and whether the animals have died.

[0191] Body weight and tumor volume: During the experiment, they were measured every 2 to 3 days.

[0192] Tumor weight: At the end of the experiment, the mouse tumors were dissected and weighed.

[0193] 1.4 Data Processing

[0194] The tumor growth curve and relative tumor proliferation rate curve of the mice were statistically analyzed and plotted based on the measurement data. After the experiment, the tumors of the mice were dissected and weighed, and the corresponding bar graphs were drawn.

[0195] Tumor volume (TsV) = a*b*b / 2, where a and b represent the tumor length and width, respectively.

[0196] Relative tumor volume (RTV) = V t / V0, where V0 is the tumor volume when mice were grouped, V t is the tumor volume measured each time after drug administration.

[0197] Relative tumor growth rate T / C (%) = T RTV / C RTV *100%. Of which C RTV RTV was the negative control group, T RTV The drug treatment group was RTV.

[0198] Evaluation criteria: T / C (%)>40% is invalid, T / C (%)<40% is effective.

[0199] The results are as follows Figures 9 - 11As shown, it can be seen that in the pharmacodynamic experiment with drug administration for two weeks, at the same drug administration dose, the relative tumor growth rate T / C of the drug-loaded microbubble group is less than 40%, that is, it has a significant inhibitory effect on subcutaneous tumors. While the T / C values of the groups with only ultrasound, free doxorubicin, and liposomes are all above 40%, indicating that ultrasound alone has no therapeutic effect on tumors, the therapeutic effect of free doxorubicin is also minimal, and liposomes have a certain therapeutic effect but it is also limited. From the tumor weight chart at the end of the experiment, it can also be found that the pharmacodynamic effect of the microbubble group is significantly better than that of the liposome group. Therefore, the drug-loaded microbubbles prepared in this example have obvious drug advantages in the mouse tumor experiment.

[0200] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A drug-loaded microbubble, characterized in that: The drug-loaded microbubble has a hollow microbubble structure of a shell membrane - cavity. The shell membrane includes a biodegradable polymer and a lipid. There is a drug active ingredient in the cavity. The drug active ingredient includes a part bound to the shell membrane and a part free in the cavity. The part bound to the shell membrane accounts for less than 50% of the total mass of the drug active ingredient.

2. The drug-loaded microbubble according to claim 1, wherein: The lipid includes a derivatized phospholipid; the derivatized phospholipid includes a polyethylene glycol-modified phospholipid.

3. The drug-loaded microbubble according to claim 1, wherein: The drug-loaded microbubble satisfies at least one of the following conditions: (I) The mass ratio of the biodegradable polymer to the lipid is 3 - 20:1; (II) The drug loading of the drug active ingredient in the drug-loaded microbubble is 0.5% - 10% of the total weight of the drug-loaded microbubble; (III) The particle size of the drug-loaded microbubble is 0.1 - 6 μm.

4. The drug-loaded microbubble according to claim 1, wherein: The drug-loaded microbubble satisfies at least one of the following conditions: (I) The biodegradable polymer includes one or more of polyglycolide, polylactide, polycaprolactone, poly(lactide - glycolide) copolymer, poly(lactide - caprolactone) copolymer, poly(glycolide - caprolactone) copolymer, poly(glycolide - lactide - caprolactone) copolymer; (II) The lipid includes at least one of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine; (III) The drug active ingredient includes at least one of paclitaxel, docetaxel, hydroxycamptothecin, doxorubicin, mitomycin, tamoxifen, 5-fluorouracil, methotrexate, cytarabine, cyclophosphamide or platinum drugs.

5. The drug-loaded microbubble according to claim 1, characterized in that: The cumulative drug release rate of the drug-loaded microbubble within 3 - 8 min is at least 30%.

6. An ultrasound contrast agent, characterized in that: It includes the drug-loaded microbubble according to any one of claims 1 - 5.

7. The ultrasound contrast agent according to claim 6, wherein: The ultrasound contrast agent further includes gas microbubbles.

8. A preparation method of drug-loaded microbubbles, characterized in that: It includes the following steps: S1: Place a solution containing a drug active ingredient and a phospholipid in an organic solution of a biodegradable polymer and a lipid for emulsification treatment to obtain a water-in-oil first primary emulsion; S2: Pass gaseous fluorocarbon into the hydrated solution of the phospholipid, and after ultrasonic treatment, prepare a phospholipid bubble solution, and place it in an organic solution of a biodegradable polymer and a lipid for emulsification treatment to obtain a water-in-oil second primary emulsion; S3: Mix the first primary emulsion and the second primary emulsion, add a polyvinyl alcohol solution for emulsification treatment to prepare a water-in-oil-in-water multiple emulsion; S4: Remove the organic solvent in the multiple emulsion, purify and collect the precipitate, and perform freeze-drying treatment on the precipitate to prepare the drug-loaded microbubble according to any one of claims 1 - 5.

9. The preparation method of the drug-loaded microbubbles according to claim 8, wherein: In S1, it also includes adding a stabilizer for continuous emulsification treatment after the emulsification treatment; the treatment time is 0.1 - 3 min.

10. Use of the drug-loaded microbubble according to any one of claims 1 - 5 and / or the ultrasound contrast agent according to claim 6 or 7 in the preparation of a tumor diagnosis and treatment drug.

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