Microenvironment response-ultrasonic mediation type drug-loading composite liposome as well as preparation method and application thereof

Abstract

The invention provides a microenvironment response-ultrasonic mediation type drug-loading composite liposome as well as a preparation method and application thereof, and belongs to the technical field of medicines. The microenvironment response-ultrasonic mediation type drug-loading composite liposome provided by the invention comprises a first liposome unit and a second liposome unit, wherein the first liposome unit and the second liposome unit are combined through charge attraction and hydrogen-bond interaction; the first liposome unit comprises a first shell layer and filling gas (one or more of air, O2, CO2 and fluorine-containing gas) wrapped in the first shell layer; the second liposome unit comprises a second shell layer, a transition phase wrapped in the second shell layer and a brain tumor treatment medicine wrapped in the transition phase. The microenvironment response-ultrasound mediation type drug-loading composite liposome has high blood brain barrier penetration efficiency, can circulate in blood for a long time, can be applied together with ultrasound and microbubbles, can more effectively deliver brain tumor treatment drugs to brain tissues, and has an excellent killing effect on brain tumor cells.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a microenvironment response-ultrasound-mediated drug-loaded composite liposome and a preparation method and application thereof. Background Art

[0002] With the increasing incidence of brain tumors, brain infections caused by bacteria or viruses, and neurological diseases, the demand for technologies that can accurately deliver drugs to the brain is also growing. In particular, the most commonly used treatment for brain tumors is surgical treatment, which involves opening the skull and removing the tumor. However, opening the skull not only puts pressure on the patient, but also causes concerns about the side effects of nerve cell damage during surgery. Nevertheless, surgical treatment is still needed at this stage, because even if drugs targeting the brain are used, drug delivery will be inhibited by the blood-brain barrier (blood-brainbarrier, BBB). Therefore, it is urgent to develop a technology that can accurately deliver drugs to brain tissue.

[0003] At present, relevant research on the technology of using ultrasound to deliver drugs to the brain has been carried out worldwide. For example, drugs are prepared using microbubble technology, which can utilize its sensitivity to ultrasound, easily produce cavitation, and realize the function of ultrasound microbubble contrast agent. The specific principle is that cavitation caused by ultrasound and microbubbles can temporarily open the blood-brain barrier, and is applied to the treatment of brain diseases as a drug delivery method. For this reason, after injecting microbubbles and 3D focused ultrasound, ultrasound is accurately emitted to the brain tumor site, so that the microbubbles located near the blood-brain barrier locally open the blood-brain barrier, thereby delivering drugs to brain tissue and triggering the mechanism of treating brain diseases. However, the anticancer drugs currently used are usually small molecule chemotherapeutic drugs, such as selenoprotein P, vincristine, levodopa, valrubicin, belotecan and doxorubicin, etc., which are rapidly excreted into the body after administration and rapidly absorbed by normal tissues, thus showing limitations in the delivery effect to the brain. Summary of the invention

[0004] The purpose of the present invention is to provide a microenvironment responsive-ultrasound mediated drug-loaded composite liposome and its preparation method and application. The microenvironment responsive-ultrasound mediated drug-loaded composite liposome provided by the present invention has a high blood-brain barrier penetration efficiency and can circulate in the blood for a long time. It can be co-administered with ultrasound and microbubbles, and can more effectively deliver brain tumor therapeutic drugs to brain tissue, and has an excellent killing effect on brain tumor cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a microenvironment responsive-ultrasound mediated drug-loaded composite liposome, comprising a first liposome unit and a second liposome unit, wherein the first liposome unit and the second liposome unit are combined through charge attraction and hydrogen bonding; the first liposome unit comprises a first shell layer and a filling gas encapsulated in the first shell layer, wherein the filling gas is one or more of air, O2, CO2 and fluorine-containing gas; the second liposome unit comprises a second shell layer, a transition phase encapsulated in the second shell layer and a brain tumor therapeutic drug encapsulated in the transition phase.

[0007] Preferably, the particle sizes of the first liposome unit and the second liposome unit are independently 1 to 500 μm; and the particle size ratio of the first liposome unit to the second liposome unit in the microenvironment responsive-ultrasound-mediated drug-loaded composite liposome is 0.2 to 2:1.

[0008] Preferably, the brain tumor therapeutic drug includes one or more of selenoprotein P, vincristine, levodopa, valrubicin, belotecan and doxorubicin.

[0009] Preferably, the transition phase comprises one or more of air, O2, CO2, fluorine-containing gas and water.

[0010] Preferably, the raw materials for preparing the first shell layer and the second shell layer independently include phospholipid materials, emulsifiers, stabilizers and antioxidants.

[0011] Preferably, the phospholipid material includes glycerophospholipids and / or sphingomyelin; the emulsifier includes polysorbate and / or stearic acid monoglyceride; the stabilizer includes one or more of cholesterol, oligosaccharides, chitosan, alginate and whey protein; the antioxidant includes one or more of vitamin E, hydroxydecylubiquinone, kinetin, coenzyme Q10 and α-lipoic acid.

[0012] Preferably, the molar ratio of the phospholipid material, the emulsifier, the stabilizer and the antioxidant is 1-50:0.1-20:1-20:0.1-20.

[0013] The present invention provides a method for preparing the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes described in the above technical solution, comprising the following steps:

[0014] pre-treating a dispersion containing a first liposome unit so that the zeta potential value of the first liposome unit is negative, thereby obtaining a first pre-treated dispersion;

[0015] The second liposome unit is mixed with the first pre-treated dispersion and homogenized to obtain the microenvironment responsive-ultrasound-mediated drug-loaded composite liposome.

[0016] The present invention provides a method for preparing the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes described in the above technical solution, comprising the following steps:

[0017] pre-treating the dispersion containing the second liposome unit so that the zeta potential value of the second liposome unit is negative, thereby obtaining a second pre-treated dispersion;

[0018] The first liposome unit is mixed with the second pre-treated dispersion liquid, and homogenized to obtain the microenvironment responsive-ultrasound-mediated drug-loaded composite liposome.

[0019] The present invention provides the use of the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes of the above technical scheme or the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes prepared by the preparation method described in the above technical scheme in the preparation of ultrasound-mediated blood-brain barrier penetrating brain drug delivery preparations.

[0020] The present invention provides a microenvironment responsive-ultrasound mediated drug-loaded composite liposome, comprising a first liposome unit and a second liposome unit, wherein the first liposome unit and the second liposome unit are combined by charge attraction and hydrogen bonding; the first liposome unit comprises a first shell layer and a filling gas encapsulated in the first shell layer, wherein the filling gas is one or more of air, O2, CO2 and fluorine-containing gas; the second liposome unit comprises a second shell layer, a transition phase encapsulated in the second shell layer, and a brain tumor therapeutic drug encapsulated in the transition phase. The microenvironment responsive-ultrasound mediated drug-loaded composite liposome provided by the present invention has a high blood-brain barrier penetration efficiency and can circulate in the blood for a long time, can be co-administered with ultrasound and microbubbles, and can more effectively deliver brain tumor therapeutic drugs to brain tissue. Specifically, traditional anticancer drugs (such as selenoprotein P, vincristine, levodopa, valrubicin, belotecan and doxorubicin, etc.) are rapidly excreted out of the body and rapidly absorbed by normal tissues after administration, and the blood circulation time is usually only 24 to 36 hours. The microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes provided by the present invention are micron-sized drugs. Compared with the above-mentioned traditional anticancer drugs, the blood circulation time of micron-sized drugs can be extended to 48 to 72 hours. At the same time, in the microenvironment response-ultrasound-mediated drug-loaded composite liposome provided by the present invention, the first liposome unit is a directional pressure boosting end, and the second liposome unit is a brain tumor therapeutic drug delivery end. In the process of entering the blood-brain barrier, under the action of ultrasound, high-frequency vibration will destroy the stability of the first shell structure in the first liposome unit and cause it to rupture. In addition, since ultrasound has an obvious "cavitation" effect, the first liposome unit mainly contains filling gas, and the gas pressure increases under the influence of the "cavitation" effect. When it is greater than the external environmental pressure, due to the obvious pressure gradient (≤10kPa), the filling gas will break through the first shell and be released. Under the influence of the pressure gradient, a driving force with instantaneous high speed (≤10μm / s) is formed, which pushes the second liposome unit in a directional manner and accelerates it through the blood-brain barrier into the neuronal tissue to release the brain tumor therapeutic drug for treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposome in an embodiment of the present invention, in which 1 is the first shell layer, 2 is the filling gas, 3 is the brain tumor therapeutic drug, 4 is the second shell layer, and 5 is the transition phase;

[0022] Figure 2Flow chart of preparing the second liposome unit in an embodiment of the present invention, wherein 3 is a brain tumor therapeutic drug, 4 is a second shell layer, 5 is a transition phase, 6 is a T-type micromixer (the transition phase is a gas) or a high shear homogenizer (the transition phase is a liquid), 7 is a phospholipid material, 8 is an emulsifier, 9 is a stabilizer, 10 is an antioxidant, 11 is a microemulsification reactor, and 12 is a second liposome unit. DETAILED DESCRIPTION

[0023] The present invention provides a microenvironment responsive-ultrasound mediated drug-loaded composite liposome, comprising a first liposome unit and a second liposome unit, wherein the first liposome unit and the second liposome unit are combined through charge attraction and hydrogen bonding; the first liposome unit comprises a first shell layer and a filling gas encapsulated in the first shell layer, wherein the filling gas is one or more of air, O2, CO2 and fluorine-containing gas; the second liposome unit comprises a second shell layer, a transition phase encapsulated in the second shell layer and a brain tumor therapeutic drug encapsulated in the transition phase.

[0024] The microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes provided by the present invention have excellent drug loading efficiency and ultrasound-induced drug release effect, and can effectively penetrate the blood-brain barrier during ultrasound stimulation; in particular, based on the environmentally sensitive characteristics of the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes, it can ensure that while having better blood-brain barrier penetration efficiency, it can directly and accurately deliver drugs to neurons, avoid physical damage to cells and blood vessels by focused ultrasound, and have an excellent killing effect on brain tumor cells.

[0025] The microenvironment-responsive ultrasound-mediated drug-loaded composite liposome of the present invention (schematic diagram as shown in Figure 1 As shown) includes a first liposome unit and a second liposome unit, wherein the first liposome unit and the second liposome unit are combined by charge attraction and hydrogen bonding (specifically, charge attraction is mainly combined with hydrogen bonding as a supplement), wherein the first liposome unit is a directional pressure boosting end, and the second liposome unit is a brain tumor treatment drug delivery end.

[0026] In the present invention, the particle size of the first liposome unit and the second liposome unit is preferably 1 to 500 μm, more preferably 1 to 50 μm; the particle size of the first liposome unit can be 25 μm, 35 μm, 40 μm, 45 μm, 75 μm or 85 μm; the particle size of the second liposome unit can be 25 μm, 35 μm, 90 μm, 95 μm, 100 μm or 350 μm; the particle size ratio of the first liposome unit to the second liposome unit in the microenvironment response-ultrasound-mediated drug-loaded composite liposome is preferably 0.2 to 2:1, more preferably 0.5 to 1:1, and can be specifically 0.24:1, 0.26:1, 0.45:1, 0.8:1, 1:1, 1.3:1 or 1.6:1. The first liposome unit and the second liposome unit are described in detail below.

[0027] In the present invention, the first liposome unit includes a first shell layer and a filling gas encapsulated in the first shell layer; the first liposome unit is specifically a large unilamellar vesicle (LUVs), or O / W type microbubbles. In the present invention, the filling gas is one or more of air, O2, CO2 and fluorine-containing gas, more preferably air, O2, CO2 or fluorine-containing gas; the fluorine-containing gas preferably includes C3F8, C4F 10 and SF6, more preferably C3F8, C4F 10or SF6; the filling gas of the present invention is preferably at the ppm level, wherein the content of the gas component in the air is preferably ≥99.9999%, and the content of other impurity components is preferably ≤0.0001%. In the present invention, the thickness of the first shell layer is preferably 0.05-250 μm, more preferably 50-250 nm, specifically 50 nm, 65 nm, 80 nm or 85 nm. In the present invention, the raw materials for preparing the first shell layer preferably include phospholipid materials, emulsifiers, stabilizers and antioxidants, and the molar ratio of the phospholipid materials, emulsifiers, stabilizers and antioxidants is preferably 1-50:0.1-20:1-20:0.1-20, specifically 25:1:5:1, 5:1:1:1 or 1:0.1:10:1. In the present invention, the phospholipid material preferably includes glycerophospholipids and / or sphingomyelin, more preferably glycerophospholipids or sphingomyelin; the emulsifier preferably includes polysorbate and / or stearic acid monoglyceride, more preferably polysorbate or stearic acid monoglyceride; the antioxidant preferably includes vitamin E; the stabilizer preferably includes one or more of cholesterol, oligosaccharides, chitosan, alginate and whey protein, more preferably cholesterol, oligosaccharides, chitosan, alginate or whey protein. The present invention preferably uses the above-mentioned types of stabilizers, which can promote the accumulation of lipid chains and the formation of bilayers, reduce the fluidity of the bilayers, reduce the transmembrane transport of water-soluble drugs (i.e., brain tumor therapeutic drugs), and can reduce the interaction between the first liposome unit and proteins in the body, reduce the loss of phospholipids, thereby improving the stability of the first liposome unit.

[0028] In the present invention, the method for preparing the first liposome unit preferably comprises the following steps:

[0029] The phospholipid material, the emulsifier, the stabilizer and the antioxidant are mixed and homogenized to obtain a homogenized liquid;

[0030] The filling gas is introduced into the homogenized liquid in the form of microbubbles to obtain the first liposome unit.

[0031] The present invention mixes phospholipid materials, emulsifiers, stabilizers and antioxidants, and performs homogenization treatment to obtain a homogenized liquid. In the present invention, the shear rate of the homogenization treatment is preferably 10000 to 30000·s -1 , specifically 10000·s -1 , 15000·s -1 , 20000·s -1 or 30000 s -1The time is preferably 15 to 60 minutes, specifically 15 minutes, 30 minutes, 45 minutes or 60 minutes. The present invention preferably performs the homogenization process at room temperature. In an embodiment of the present invention, the homogenization process is specifically performed in a high shear homogenization pump.

[0032] After obtaining the homogenized liquid, the present invention introduces the filling gas into the homogenized liquid in the form of microbubbles to obtain the first liposome unit. The present invention preferably uses a gas generator to introduce the filling gas into the homogenized liquid in the form of microbubbles. In the present invention, the gas generator is preferably a micro-nano bubble generator, and the bubble particle size is preferably controlled to be 0.01-100 μm, specifically 1 μm, 10 μm or 100 μm. The flow rate of the filling gas in the present invention is preferably 10-100 mL / min, specifically 25 mL / min, 50 mL / min or 75 mL / min; the introduction time is preferably 15-30 min, specifically 15 min, 20 min or 30 min; the introduction process of the filling gas is preferably carried out at room temperature. In the present invention, freeze drying is preferably performed after the introduction to obtain the first liposome unit; the freeze drying temperature is preferably -30--20 ° C, more preferably -25 ° C, and the time is preferably 12-24 h, more preferably 20-24 h.

[0033] In the present invention, the gas content of the first liposome unit prepared by the above method is Preferably, it is 0.05 to 0.25, preferably 0.08 to 0.2, and specifically, it can be 0.092, 0.112, 0.125, 0.156, 0.175 or 0.182; the average bubble diameter (d 32 ) is preferably 0.01 to 100 μm, preferably 0.1 to 50 μm, specifically 1 μm, 1.25 μm, 1.5 μm, 5 μm, 8.5 μm or 35 μm; the average bubble rise velocity (ν 32 ) is preferably 2.0 to 7.0 cm / s, specifically 2.5 cm / s, 2.75 cm / s, 3.5 cm / s, 3.7 cm / s or 5.5 cm / s. The larger the value, the greater the amount of dissolved gas. 32 The smaller the bubble, the smaller the 32 The smaller the value, the longer the gas residence time. The above indicators reflect the amount of gas encapsulated in the first liposome unit. The larger the amount of gas encapsulated in the first liposome unit, the higher the subsequent drug delivery efficiency.

[0034] In the present invention, the second liposome unit includes a second shell layer, a transition phase encapsulated in the second shell layer, and a brain tumor therapeutic drug encapsulated in the transition phase; the second liposome unit is specifically an O / W / O type microbubble or an O / W / O type microemulsion. In the present invention, the brain tumor therapeutic drug is preferably a water-soluble drug, more preferably including one or more of selenoprotein P, vincristine, levodopa, valrubicin, belotecan and doxorubicin, more preferably selenoprotein P (SELENOP), vincristine, levodopa, valrubicin, belotecan or doxorubicin; the content of the brain tumor therapeutic drug in the second liposome unit is preferably 0.05-10wt%, specifically 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3.5wt% or 5.2wt%. In the present invention, the transition phase preferably includes one or more of air, O2, CO2, fluorine-containing gas and water, more preferably air, O2, CO2, fluorine-containing gas or water; the fluorine-containing gas preferably includes C3F8, C4F 10 and SF6, more preferably C3F8, C4F 10 Or SF6. In the present invention, the thickness of the second shell layer is preferably 0.05-250 μm, more preferably 50-250 nm, specifically 55 nm, 60 nm, 80 nm, 85 nm or 100 nm. In the present invention, the raw materials for preparing the second shell layer preferably include phospholipid materials, emulsifiers, stabilizers and antioxidants, and the molar ratio of the phospholipid materials, emulsifiers, stabilizers and antioxidants is preferably 1-50:0.1-20:1-20:0.1-20, specifically 25:1:5:1, 5:1:1:1 or 1:0.1:10:1. In the present invention, the phospholipid material preferably includes glycerophospholipids and / or sphingomyelin, more preferably glycerophospholipids or sphingomyelin; the emulsifier preferably includes polysorbate and / or stearic acid monoglyceride, more preferably polysorbate or stearic acid monoglyceride; the stabilizer preferably includes one or more of cholesterol, oligosaccharides, chitosan, alginate and whey protein, more preferably cholesterol, oligosaccharides, chitosan, alginate or whey protein; the antioxidant preferably includes one or more of vitamin E, hydroxydecylubiquinone, kinetin, coenzyme Q10 and α-lipoic acid, more preferably vitamin E, hydroxydecylubiquinone, kinetin, coenzyme Q10 or α-lipoic acid.

[0035] In the present invention, the second liposome unit (preparation flow chart as shown in Figure 2 It is preferred to adopt different preparation methods according to the type of transition phase, which will be described in detail below.

[0036] In the present invention, when the transition phase is a gas (such as air, O2, CO2, fluorine-containing gas), the method for preparing the second liposome unit preferably comprises the following steps:

[0037] The phospholipid material, the emulsifier, the stabilizer and the antioxidant are mixed and homogenized to obtain a homogenized liquid;

[0038] The brain tumor therapeutic drug is sprayed into the flowing transition phase by high-pressure atomization to obtain atomized core droplets;

[0039] The mist-like inner core droplets are passed into the homogenized liquid to obtain the second liposome unit.

[0040] The present invention mixes phospholipid materials, emulsifiers, stabilizers and antioxidants, and performs homogenization treatment to obtain a homogenized liquid. The preparation method of the homogenized liquid of the present invention preferably refers to the preparation method of the homogenized liquid of the above technical solution, which will not be repeated here.

[0041] The present invention uses a high-pressure atomization method to spray brain tumor therapeutic drugs into a flowing transition phase to obtain mist-like core droplets. In the present invention, the molar ratio of the transition phase to the brain tumor therapeutic drug is preferably 8 to 12:1, and can be specifically 10:1. In the present invention, the conditions of the high-pressure atomization method include: the atomization pressure is preferably 1 to 10 MPa, and can be specifically 1 MPa, 8 MPa or 10 MPa; the atomization time is preferably 0.5 to 60 s, and can be specifically 1 s, 20 s, 30 s or 60 s; the flow rate of the transition phase is preferably 0.1 to 5 m / s, and can be specifically 1 m / s or 3.5 m / s. In the present invention, the particle size of the mist-like core droplets is preferably 0.01 to 10 μm, and can be specifically 0.15 μm, 1 μm, 5 μm or 10 μm. In an embodiment of the present invention, the mist-like core droplets are specifically prepared in a T-type micromixer.

[0042] After obtaining the mist core liquid droplets and the homogenized liquid, the present invention passes the mist core liquid droplets into the homogenized liquid to obtain the second liposome unit. The present invention preferably uses a gas generator to pass the mist core liquid into the homogenized liquid; the gas generator is preferably a micro-nano bubble generator, and the bubble particle size is preferably controlled to be 0.01 to 10 μm, specifically 0.25 μm, 1 μm, 5 μm or 10 μm. In the present invention, the time for passing the mist core liquid is preferably 15 to 30 minutes, specifically 15 minutes, 20 minutes or 30 minutes; the process of passing the mist core liquid is preferably carried out at room temperature. In the present invention, after the passing is completed, freeze drying is preferably carried out to obtain the second liposome unit; the freeze drying temperature is preferably -30 to -20°C, more preferably -25°C; the time is preferably 12 to 24 hours, more preferably 20 to 24 hours. In the present invention, the gas content of the second liposome unit prepared by the above method is Preferably, it is 0.10 to 0.25, specifically, it can be 0.105, 0.125, 0.20 or 0.25; the average bubble diameter (d 32 ) is preferably 0.01 to 100 μm, more preferably 1 to 90 μm, specifically 20 μm, 40 μm, 80 μm or 85 μm; the average bubble rise velocity (ν 32 ) is preferably 3.0 to 7.0 cm / s, specifically 3.0 cm / s, 3.5 cm / s, 4.5 cm / s or 5.0 cm / s.

[0043] In the present invention, when the transition phase is a liquid (such as water), the method for preparing the second liposome unit preferably comprises the following steps:

[0044] The phospholipid material, the emulsifier, the stabilizer and the antioxidant are mixed and homogenized to obtain a homogenized liquid;

[0045] mixing the brain tumor therapeutic drug with the transition phase and homogenizing the mixture to obtain an emulsion;

[0046] The homogenized liquid is mixed with the emulsion to perform a microemulsification reaction to obtain the second liposome unit.

[0047] The present invention mixes phospholipid materials, emulsifiers, stabilizers and antioxidants, and performs homogenization treatment to obtain a homogenized liquid. The preparation method of the homogenized liquid of the present invention preferably refers to the preparation method of the homogenized liquid of the above technical solution, which will not be repeated here.

[0048] The present invention mixes the brain tumor therapeutic drug with the transition phase and performs homogenization treatment to obtain an emulsion. In the present invention, the molar ratio of the transition phase to the brain tumor therapeutic drug is preferably 7 to 11:2, specifically 10:2. In the present invention, the shear rate of the homogenization treatment is preferably 10,000 to 30,000·s -1 , specifically 15000·s -1 or 20000 s -1 ; The time is preferably 30 to 90 minutes, specifically 45 minutes or 60 minutes; The present invention preferably performs the homogenization process at room temperature. In an embodiment of the present invention, the homogenization process is specifically performed in a high shear homogenizer. The emulsion of the present invention is specifically an O / W emulsion.

[0049] After obtaining the homogenized liquid and the emulsion, the present invention mixes the homogenized liquid with the emulsion for a microemulsification reaction to obtain the second liposome unit. In the present invention, the volume ratio of the emulsion to the homogenized liquid is preferably 1:8 to 12, specifically 1:10. In the present invention, the microemulsification reaction is preferably carried out at room temperature, and the time of the microemulsification reaction is preferably 30 to 60 minutes, specifically 30 minutes, 45 minutes or 60 minutes. In the present invention, the second liposome unit-containing liquid is obtained after the microemulsification reaction, which can be directly used to prepare the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes without post-treatment, which will be described in detail later.

[0050] The present invention provides a method for preparing the microenvironment responsive-ultrasound mediated drug-loaded composite liposomes described in the above technical solution. Specifically, the microenvironment responsive-ultrasound mediated drug-loaded composite liposomes can be prepared by method one or method two, which is described in detail below.

[0051] Method 1: pre-treating a dispersion containing a first liposome unit so that the zeta potential value of the first liposome unit becomes negative, thereby obtaining a first pre-treated dispersion;

[0052] The second liposome unit is mixed with the first pre-treated dispersion and homogenized to obtain the microenvironment responsive-ultrasound-mediated drug-loaded composite liposome.

[0053] Method 2: pre-treating the dispersion containing the second liposome unit so that the zeta potential value of the second liposome unit is negative, thereby obtaining a pre-treated dispersion;

[0054] The first liposome unit is mixed with the pre-treated dispersion and homogenized to obtain the microenvironment responsive-ultrasound-mediated drug-loaded composite liposome.

[0055] The following is an explanation using method 2 as an example (the relevant operating steps and conditions in method 1 can refer to method 2). The present invention utilizes the principle that nanopowder particles are very likely to agglomerate during preparation to prepare the microenvironment response-ultrasound-mediated drug-loaded composite liposomes. Specifically, nanoparticles are prone to generate negative charges on their surfaces during the process of continuous collision and movement. After accumulating to a certain extent, the nanoparticles aggregate and adhere to each other due to electrostatic attraction; in this way, this principle can be used to produce directional movement inside the emulsion system after the second liposome unit is prepared and stabilized. After a certain period of time, negative charges will accumulate on the surface of the second liposome unit to form an aggregated state. At this time, the prepared first liposome unit is added to the second liposome unit and homogenized to obtain the microenvironment response-ultrasound-mediated drug-loaded composite liposomes. The following is a specific explanation.

[0056] The present invention pre-treats the dispersion containing the second liposome unit so that the zeta potential value of the second liposome unit is negative, thereby obtaining a pre-treated dispersion. In the present invention, the zeta potential value of the second liposome unit in the pre-treated dispersion is preferably -10 to -30 mV, specifically -28 mV, -20 mV or -10 mV. In the present invention, the pre-treatment preferably comprises: mixing the second liposome unit (or the liquid containing the second liposome unit) prepared by the above technical solution with a dispersion medium to obtain a dispersion containing the second liposome unit; injecting the dispersion containing the second liposome unit into a tubular continuous reactor, turning on a circulation pump, and performing continuous circulation at room temperature, and measuring the zeta potential value to meet the above requirements to complete the circulation and obtain a pre-treated dispersion. In the present invention, the dispersion medium preferably comprises water and / or ethylene glycol; the water is preferably pure water; the concentration of the second liposome unit in the dispersion containing the second liposome unit is preferably 0.1 to 30 wt%, more preferably 5 to 25 wt%, specifically 10 wt%, 15 wt% or 20 wt%. In the present invention, the flow rate of the continuous circulation is preferably 1-10 m / s, specifically 1 m / s, 5 m / s or 8 m / s; the time is preferably 60-120 min, specifically 60 min, 90 min or 120 min.

[0057] After obtaining the pre-treated dispersion, the present invention mixes the first liposome unit prepared by the above technical solution with the pre-treated dispersion, performs a homogenization treatment, and obtains the microenvironment response-ultrasound-mediated drug-loaded composite liposome. In the present invention, the concentration of the first liposome unit in the mixed material obtained after the first liposome unit is mixed with the pre-treated dispersion is preferably 20 to 35wt%, specifically 20.6wt%, 22.5wt%, 23.8wt%, 31.8wt%, 32.5wt% or 33.9wt%. In the present invention, the shear rate of the homogenization treatment is preferably 20,000 to 30,000·s -1 , specifically 20000·s -1 , 25000·s -1 or 30000 s -1 The homogenization treatment is preferably carried out at room temperature.

[0058] In an embodiment of the present invention, the particle size of the microenvironment response-ultrasound-mediated drug-loaded composite liposomes is preferably 50-500 μm, specifically 50 μm, 105 μm, 185 μm, 190 μm, 210 μm or 425 μm; the loading efficiency of brain tumor therapeutic drugs in the microenvironment response-ultrasound-mediated drug-loaded composite liposomes is preferably 91.5-97.5%, specifically 91.8%, 95.2%, 95.5%, 96.8%, 96.9% or 97.0%; the release rate is preferably 60-80%, specifically 60.5%, 72.5%, 74.2%, 75.2%, 75.3% or 75.4%.

[0059] The present invention provides the use of the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes described in the above technical scheme or the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes prepared by the preparation method described in the above technical scheme in the preparation of ultrasound-mediated blood-brain barrier penetrating brain drug delivery preparations.

[0060] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Example 1

[0062] Prepare the first liposome unit, the steps are as follows:

[0063] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at room temperature in a molar ratio of 25:1:5:1, and the resulting mixture was homogenized at a shear rate of 20000 s -1 The homogenization treatment was carried out for 30 minutes under the above conditions, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0064] A micro-nano bubble generator (the bubble particle size is set to 1 μm) is used to introduce a filling gas (specifically air) into the homogenized liquid at a flow rate of 50 mL / min for 30 min. After the introduction, the liquid is freeze-dried at -25 ° C for 24 h to obtain a first liposome unit (including a shell layer and bubbles formed by the filling gas in the shell layer); the average particle size of the first liposome unit is 35 μm, the average shell thickness is 80 nm, and the gas content is is 0.156, the average bubble diameter d 32 is 1 μm, and the average rising velocity of the bubble is ν32 3.5cm / s.

[0065] Example 2

[0066] Prepare the first liposome unit, the steps are as follows:

[0067] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 20000·s using a high shear homogenizer. -1 The homogenization treatment was carried out for 30 minutes under the above conditions, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0068] A micro-nano bubble generator (the bubble particle size was set to 1 μm) was used to introduce a filling gas (specifically air) into the homogenized liquid at a flow rate of 75 mL / min for 30 min. After the introduction, the liquid was freeze-dried at -25 °C for 24 h to obtain a first liposome unit. The first liposome unit had an average particle size of 25 μm, an average shell thickness of 50 nm, and a gas content of is 0.175, the average bubble diameter d 32 is 0.5μm, and the average rising velocity of the bubble is ν 32 2.5cm / s.

[0069] Example 3

[0070] Prepare the first liposome unit, the steps are as follows:

[0071] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 15000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 15 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0072] A micro-nano bubble generator (the bubble particle size was set to 1 μm) was used to introduce the filling gas (specifically CO2) into the homogenized liquid at a flow rate of 50 mL / min for 15 min. After the introduction, the liquid was freeze-dried at -25 °C for 24 h to obtain the first liposome unit. The average particle size of the first liposome unit was 45 μm, the average shell thickness was 65 nm, and the gas content was is 0.125, the average bubble diameter d 32 is 1.5 μm, and the average rising velocity of the bubble is ν32 It is 3.7cm / s.

[0073] Example 4

[0074] Prepare the first liposome unit, the steps are as follows:

[0075] The phospholipid material (specifically sphingomyelin), the emulsifier (specifically stearic acid monoglyceride), the stabilizer (specifically whey protein) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 20000·s using a high shear homogenizer. -1 The homogenization treatment was carried out for 30 minutes under the above conditions, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0076] A micro-nano bubble generator (the bubble particle size was set to 1 μm) was used to introduce the filling gas (specifically CO2) into the homogenized liquid at a flow rate of 75 mL / min for 30 min. After the introduction, the liquid was freeze-dried at -25 °C for 24 h to obtain the first liposome unit. The average particle size of the first liposome unit was 40 μm, the average shell thickness was 85 nm, and the gas content was is 0.182, the average bubble diameter d 32 is 1.25 μm, and the average rising velocity of the bubble is ν 32 It is 2.75cm / s.

[0077] Example 5

[0078] Prepare the first liposome unit, the steps are as follows:

[0079] The phospholipid material (specifically sphingomyelin), the emulsifier (specifically stearic acid monoglyceride), the stabilizer (specifically whey protein) and the antioxidant (specifically vitamin E) were mixed at room temperature in a molar ratio of 1:0.1:10:1, and the resulting mixture was homogenized at a shear rate of 10000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 15 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0080] A micro-nano bubble generator (the bubble particle size was set to 100 μm) was used to introduce the filling gas (specifically C3F8) into the homogenized liquid at a flow rate of 50 mL / min for 15 min. After the introduction, the liquid was freeze-dried at -25 °C for 24 h to obtain a first liposome unit. The first liposome unit had an average particle size of 75 μm, an average shell thickness of 80 nm, and a gas content of is 0.112, the average bubble diameter d 32is 8.5μm, and the average rising velocity of the bubble is ν 32 3.5cm / s.

[0081] Example 6

[0082] Prepare the first liposome unit, the steps are as follows:

[0083] The phospholipid material (specifically sphingomyelin), the emulsifier (specifically stearic acid monoglyceride), the stabilizer (specifically whey protein) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 15000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 60 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0084] A micro-nano bubble generator (the bubble particle size was set to 10 μm) was used to introduce the filling gas (specifically C3F8) into the homogenized liquid at a flow rate of 25 mL / min for 30 min. After the introduction, the liquid was freeze-dried at -25 °C for 24 h to obtain a first liposome unit. The first liposome unit had an average particle size of 85 μm, an average shell thickness of 85 nm, and a gas content of is 0.092, the average bubble diameter d 32 is 35 μm, and the average rising velocity of the bubble is ν 32 It is 5.5cm / s.

[0085] Example 7

[0086] The second liposome unit is prepared as follows:

[0087] The transition phase (specifically CO2) and the brain tumor therapeutic drug (specifically selenoprotein P) are mixed in a T-type micromixer at a molar ratio of 10:1 based on a high-pressure atomization method to prepare a mist core droplets, specifically, selenoprotein P is sprayed into flowing CO2 gas by a high-pressure atomization method to prepare the mist core droplets, wherein the atomization pressure is 8MPa, the atomization time is 30s, the CO2 flow rate is 3.5m / s, and the average particle size of the mist core droplets is 0.15μm;

[0088] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 10000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 15 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0089] The mist-like core droplets were introduced into the homogenized liquid using a micro-nano bubble generator (the bubble diameter was set to 0.25 μm) for 20 min. After the introduction, the liquid was freeze-dried at -25°C for 24 h to obtain a second liposome unit (including a shell layer, bubbles formed by the transition phase and encapsulated in the shell layer, and a brain tumor therapeutic drug encapsulated in the bubbles); the average particle size of the second liposome unit was 35 μm, the average shell thickness was 55 nm, and the gas content was is 0.25, the average bubble diameter d 32 is 20 μm, and the average rising velocity of the bubble is ν 32 is 3.0 cm / s; the content of the brain tumor therapeutic drug in the second liposome unit is 2.5wt%.

[0090] Example 8

[0091] The second liposome unit is prepared as follows:

[0092] The transition phase (specifically CO2) and the brain tumor therapeutic drug (specifically selenoprotein P) are mixed in a T-type micromixer at a molar ratio of 10:1 based on a high-pressure atomization method to prepare a mist core droplets, specifically, selenoprotein P is sprayed into flowing CO2 gas by a high-pressure atomization method to prepare the mist core droplets, wherein the atomization pressure is 1MPa, the atomization time is 1s, the CO2 flow rate is 1m / s, and the average particle size of the mist core droplets is 10μm;

[0093] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 20000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 45 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0094] The mist-like core droplets were introduced into the homogenized liquid using a micro-nano bubble generator (the bubble particle size was set to 10 μm) for 15 min. After the introduction, the liquid was freeze-dried at -25 °C for 12 h to obtain a second liposome unit. The average particle size of the second liposome unit was 90 μm, the average shell thickness was 85 nm, and the gas content was is 0.105, the average bubble diameter d 32 is 80μm, and the average rising velocity of the bubble is ν 32 is 4.5 cm / s; the content of the brain tumor therapeutic drug in the second liposome unit is 1wt%.

[0095] Example 9

[0096] The second liposome unit is prepared as follows:

[0097] The transition phase (specifically C3F8) and the brain tumor therapeutic drug (specifically selenoprotein P) are mixed in a T-type micromixer at a molar ratio of 10:1 based on a high-pressure atomization method to prepare a mist core droplets, specifically, the selenoprotein P is sprayed into flowing CO2 gas by a high-pressure atomization method to prepare the mist core droplets, wherein the atomization pressure is 1MPa, the atomization time is 1s, the CO2 flow rate is 1m / s, and the average particle size of the mist core droplets is 10μm;

[0098] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 20000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 45 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0099] The mist-like core droplets were introduced into the homogenized liquid using a micro-nano bubble generator (the bubble particle size was set to 10 μm) for 15 min. After the introduction, the liquid was freeze-dried at -25 °C for 12 h to obtain a second liposome unit. The average particle size of the second liposome unit was 95 μm, the average shell thickness was 80 nm, and the gas content was is 0.125, the average bubble diameter d 32 is 85μm, and the average rising velocity of the bubble is ν 32 is 3.5cm / s; the content of the brain tumor therapeutic drug in the second liposome unit is 1.5wt%.

[0100] Example 10

[0101] The second liposome unit is prepared as follows:

[0102] The transition phase (specifically C3F8) and the brain tumor therapeutic drug (specifically selenoprotein P) are mixed in a T-type micromixer at a molar ratio of 10:1 based on a high-pressure atomization method to prepare a mist core droplets, specifically, the selenoprotein P is sprayed into flowing CO2 gas by a high-pressure atomization method to prepare the mist core droplets, wherein the atomization pressure is 1MPa, the atomization time is 1s, the CO2 flow rate is 1m / s, and the average particle size of the mist core droplets is 10μm;

[0103] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 10000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 15 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0104] The mist-like core droplets were introduced into the homogenized liquid using a micro-nano bubble generator (the bubble particle size was set to 10 μm) for 15 min. After the introduction, the liquid was freeze-dried at -25 °C for 12 h to obtain a second liposome unit. The average particle size of the second liposome unit was 25 μm, the average shell thickness was 60 nm, and the gas content was is 0.105, the average bubble diameter d 32 is 40μm, and the average rising velocity of the bubble is ν 32 is 3.5cm / s; the content of the brain tumor therapeutic drug in the second liposome unit is 2wt%.

[0105] Embodiment 11

[0106] The second liposome unit is prepared as follows:

[0107] The transition phase (specifically water) and the brain tumor treatment drug (specifically vincristine) were placed in a high shear homogenizer at a molar ratio of 10:2 and the shear rate was 15000 s -1 The mixture was homogenized for 60 min under the above conditions to obtain an emulsion;

[0108] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 20000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 45 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0109] The emulsion and the homogenized liquid are placed in a microemulsion reactor at a volume ratio of 1:10, and a microemulsification reaction is carried out at room temperature for 30 minutes to obtain a liquid containing a second liposome unit; the average particle size of the second liposome unit is 350 μm, the shell thickness is 80 nm, and the content of the brain tumor therapeutic drug in the second liposome unit is 3.5 wt%.

[0110] Example 12

[0111] The second liposome unit is prepared as follows:

[0112] The transition phase (specifically water) and the brain tumor treatment drug (specifically vincristine) were placed in a high shear homogenizer at a molar ratio of 10:2 and the shear rate was 15000 s -1 The mixture was homogenized for 60 min under the above conditions to obtain an emulsion;

[0113] The phospholipid material (specifically glycerophospholipid), the emulsifier (specifically polysorbate), the stabilizer (specifically cholesterol) and the antioxidant (specifically vitamin E) were mixed at a molar ratio of 5:1:1:1 at room temperature, and the resulting mixture was homogenized at a shear rate of 30000·s using a high shear homogenizer. -1 The homogenization treatment was carried out under the following conditions for 60 minutes, and after the homogenization treatment was completed, the obtained homogenized liquid was sealed and set aside for use;

[0114] The emulsion and the homogenized liquid are placed in a microemulsion reactor at a volume ratio of 1:10, and a microemulsification reaction is carried out at room temperature for 60 minutes to obtain a liquid containing a second liposome unit; the average particle size of the second liposome unit is 100 μm, the shell thickness is 100 nm, and the content of the brain tumor therapeutic drug in the second liposome unit is 5.2 wt%.

[0115] Embodiment 13

[0116] The steps for preparing the composite liposomes are as follows:

[0117] The second liposome unit prepared in Example 7 was mixed with a dispersion medium (specifically pure water) to obtain a dispersion containing the second liposome unit (the concentration of the second liposome unit was 15 wt %); the dispersion containing the second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 8 m / s, and continuous circulation was performed at room temperature for 120 min, and the zeta potential value was measured to be -28 mV, to obtain a pretreated dispersion;

[0118] The first liposome unit prepared in Example 1 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 22.5 wt %. The mixture was placed in a high shear homogenizer at a shear rate of 30,000 s -1 The homogenization treatment was carried out for 60 minutes under the conditions to obtain composite liposomes; the average particle size of the composite liposomes was 50 μm, the loading efficiency of the brain tumor therapeutic drug was 95.5%, and the release rate was 72.5%.

[0119] Embodiment 14

[0120] The steps for preparing the composite liposomes are as follows:

[0121] The second liposome unit prepared in Example 8 was mixed with a dispersion medium (specifically ethylene glycol) to obtain a dispersion containing the second liposome unit (the concentration of the second liposome unit was 15 wt %); the dispersion containing the second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 1 m / s, and continuous circulation was performed at room temperature for 60 min, and the zeta potential value was measured to be -10 mV to obtain a pretreated dispersion;

[0122] The first liposome unit prepared in Example 5 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 20.6 wt % of the first liposome unit. The mixture was placed in a high shear homogenizer at a shear rate of 25000·s -1 The homogenization treatment was carried out for 15 minutes under the conditions to obtain composite liposomes; the average particle size of the composite liposomes was 425 μm, the brain tumor therapeutic drug loading efficiency was 91.8%, and the release rate was 60.5%.

[0123] Embodiment 15

[0124] The steps for preparing the composite liposomes are as follows:

[0125] The second liposome unit-containing liquid prepared in Example 12 was mixed with a dispersion medium (specifically pure water) to obtain a dispersion containing the second liposome unit (the concentration of the second liposome unit was 10 wt %); the dispersion containing the second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 5 m / s, and continuous circulation was performed at room temperature for 90 min, and the zeta potential value was measured to be -20 mV, to obtain a pretreated dispersion;

[0126] The first liposome unit prepared in Example 3 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 32.5 wt % of the first liposome unit. The mixture was placed in a high shear homogenizer at a shear rate of 20,000 s -1 The homogenization treatment was carried out for 45 minutes under the conditions to obtain composite liposomes; the average particle size of the composite liposomes was 210 μm, the brain tumor therapeutic drug loading efficiency was 96.8%, and the release rate was 75.2%.

[0127] Example 16

[0128] The steps for preparing the composite liposomes are as follows:

[0129] The second liposome unit prepared in Example 9 was mixed with a dispersion medium (specifically pure water) to obtain a dispersion containing the second liposome unit (the concentration of the second liposome unit was 20 wt %); the dispersion containing the second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 8 m / s, and continuous circulation was performed at room temperature for 90 min, and the zeta potential value was measured to be -28 mV, thereby obtaining a pretreated dispersion;

[0130] The first liposome unit prepared in Example 2 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 31.8 wt % of the first liposome unit. The mixture was placed in a high shear homogenizer at a shear rate of 20,000 s -1 The homogenization treatment was carried out under the conditions for 45 minutes to obtain composite liposomes; the average particle size of the composite liposomes was 190 μm, the brain tumor therapeutic drug loading efficiency was 96.9%, and the release rate was 75.3%.

[0131] Embodiment 17

[0132] The steps for preparing the composite liposomes are as follows:

[0133] The second liposome unit prepared in Example 10 was mixed with a dispersion medium (specifically pure water) to obtain a dispersion containing the second liposome unit (the concentration of the second liposome unit was 15 wt %); the dispersion containing the second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 8 m / s, and continuous circulation was performed at room temperature for 90 min, and the zeta potential value was measured to be -28 mV, to obtain a pretreated dispersion;

[0134] The first liposome unit prepared in Example 4 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 33.9 wt % of the first liposome unit. The mixture was placed in a high shear homogenizer at a shear rate of 30,000 s -1 The homogenization treatment was carried out for 60 minutes under the conditions to obtain composite liposomes; the average particle size of the composite liposomes was 105 μm, the brain tumor therapeutic drug loading efficiency was 97.0%, and the release rate was 75.4%.

[0135] Embodiment 18

[0136] The steps for preparing the composite liposomes are as follows:

[0137] The second liposome unit-containing liquid prepared in Example 11 was mixed with a dispersion medium (specifically ethylene glycol) to obtain a dispersion containing the second liposome unit (the concentration of the second liposome unit was 20 wt %); the dispersion containing the second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 8 m / s, and continuous circulation was performed at room temperature for 90 min, and the zeta potential value was measured to be -20 mV, to obtain a pretreated dispersion;

[0138] The first liposome unit prepared in Example 6 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 23.8 wt %. The mixture was placed in a high shear homogenizer at a shear rate of 20,000 s -1 The homogenization treatment was carried out under the conditions for 45 minutes to obtain composite liposomes; the average particle size of the composite liposomes was 185 μm, the brain tumor therapeutic drug loading efficiency was 95.2%, and the release rate was 74.2%.

[0139] Test Example 1

[0140] The anticancer effect of the composite liposome prepared in Example 17 of the present invention was verified based on brain tumor cells U87MG cells, and the specific steps are as follows:

[0141] U87MG cells were plated at 5 × 10 4 The cells were inoculated into a 96-well plate and cultured for 24 hours. Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics was used as a cell culture medium. In order to evaluate the efficacy, the composite liposomes were added to the cells at different concentrations (0, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL) and incubated for 4 hours. Ultrasonic conditions were applied during the incubation process, and no ultrasonic conditions were set for control. After the incubation, the cells were filtered and washed three times with phosphate buffered saline (0.01 M, pH = 7.4) to remove residual drugs. The cells were then cultured with the above-mentioned cell culture medium for 72 hours, and the proliferation of cancer cells under ultrasonic conditions and without ultrasonic conditions was compared. Specifically, the cancer cell killing effect was quantitatively analyzed by performing MTT analysis and measuring the absorbance at a wavelength of 570 nm.

[0142] The cancer cell killing effect of the composite liposomes under different concentration conditions is shown in Table 1. As can be seen from Table 1, the composite liposomes provided by the present invention are used to penetrate the blood-brain barrier to kill cancer cells. Compared with the administration without ultrasound conditions, the cancer cell killing effect is significantly enhanced when the composite liposomes are administered under ultrasound conditions. These results show that the composite liposomes provided by the present invention can be used to penetrate the blood-brain barrier to administer drugs and have excellent killing effects on brain tumor cells.

[0143] Table 1 Cancer cell killing effect of composite liposomes

[0144]

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A microenvironment-responsive-ultrasound-mediated drug-loaded composite liposome, comprising a first liposome unit and a second liposome unit, wherein the first liposome unit and the second liposome unit are combined by charge attraction and hydrogen bonding; the first liposome unit comprises a first shell layer and a filling gas encapsulated in the first shell layer, wherein the filling gas is one or more of air, O2, CO2 and fluorine-containing gas; the second liposome unit comprises a second shell layer, a transition phase encapsulated in the second shell layer and a brain tumor therapeutic drug encapsulated in the transition phase.

2. The microenvironment-responsive-ultrasound-mediated drug-loaded composite liposome according to claim 1, characterized in that: The particle sizes of the first liposome unit and the second liposome unit are independently 1 to 500 μm; the particle size ratio of the first liposome unit to the second liposome unit in the microenvironment response-ultrasound-mediated drug-loaded composite liposome is 0.2 to 2:

1.

3. The microenvironment-responsive-ultrasound-mediated drug-loaded composite liposome according to claim 1 or 2, characterized in that: The brain tumor treatment drugs include one or more of selenoprotein P, vincristine, levodopa, valrubicin, belotecan and doxorubicin.

4. The microenvironment-responsive-ultrasound-mediated drug-loaded composite liposome according to claim 1 or 2, characterized in that: The transition phase includes one or more of air, O2, CO2, fluorine-containing gas and water.

5. The microenvironment-responsive-ultrasound-mediated drug-loaded composite liposome according to claim 1 or 2, characterized in that: The raw materials for preparing the first shell layer and the second shell layer independently include phospholipid materials, emulsifiers, stabilizers and antioxidants.

6. The microenvironment-responsive-ultrasound-mediated drug-loaded composite liposome according to claim 5, characterized in that: The phospholipid material includes glycerophospholipids and / or sphingomyelin; the emulsifier includes polysorbate and / or stearic acid monoglyceride; the stabilizer includes one or more of cholesterol, oligosaccharides, chitosan, alginate and whey protein; the antioxidant includes one or more of vitamin E, hydroxydecylubiquinone, kinetin, coenzyme Q10 and α-lipoic acid.

7. The microenvironment-responsive-ultrasound-mediated drug-loaded complex liposome according to claim 5, characterized in that: The molar ratio of the phospholipid material, the emulsifier, the stabilizer and the antioxidant is 1-50:0.1-20:1-20:0.1-20.

8. The method for preparing the microenvironment responsive-ultrasound mediated drug-loaded composite liposome according to any one of claims 1 to 7, comprising the following steps: pre-treating a dispersion containing a first liposome unit so that the zeta potential value of the first liposome unit is negative, thereby obtaining a first pre-treated dispersion; The second liposome unit is mixed with the first pre-treated dispersion and homogenized to obtain the microenvironment responsive-ultrasound-mediated drug-loaded composite liposome.

9. The method for preparing the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposome according to any one of claims 1 to 7, comprising the following steps: pre-treating the dispersion containing the second liposome unit so that the zeta potential value of the second liposome unit is negative, thereby obtaining a second pre-treated dispersion; The first liposome unit is mixed with the second pre-treated dispersion liquid, and homogenized to obtain the microenvironment responsive-ultrasound-mediated drug-loaded composite liposome.

10. Use of the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes according to any one of claims 1 to 7 or the microenvironment-responsive-ultrasound-mediated drug-loaded composite liposomes prepared by the preparation method according to claim 8 or 9 in the preparation of ultrasound-mediated blood-brain barrier penetrating brain drug delivery preparations.

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