Negative entropy input-ultrasonic mediation type composite liposome, negative entropy input-ultrasonic mediation type drug-loading composite liposome, and preparation methods and applications of negative entropy input-ultrasonic mediation type drug-loading composite liposome and negative entropy input-ultrasonic mediation type drug-loading composite liposome

Abstract

The invention provides a negentropy input-ultrasonic mediation type composite liposome and a negentropy input-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 negentropy input-ultrasound mediated composite liposome provided by the invention comprises a first liposome unit and a second liposome unit which are combined through charge attraction and hydrogen-bond interaction, the first liposome unit comprises a first shell layer and plasma gas wrapped in the first shell layer; the second liposome unit comprises a second shell layer and a transition phase wrapped in the second shell layer. The negentropy input-ultrasonic mediated drug-loaded composite liposome provided by the invention, as a carrier, can more effectively deliver a brain tumor treatment drug to brain tissues, and meanwhile, can perform in-situ repair on neurons based on plasma gas, so that the ordered health of a neuron system is recovered; namely, the negentropy effect is realized through the input of the plasma gas.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to negative entropy input-ultrasound mediated composite liposomes and negative entropy input-ultrasound mediated drug-loaded composite liposomes, as well as preparation methods and applications. Background Art

[0002] With the increasing incidence of brain tumors, brain infections caused by bacteria or viruses, and neurological diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD), the demand for technology that can accurately deliver drugs to the brain is also growing. Currently, 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 people to worry about the side effects of nerve cell damage during the operation. Despite this, 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. Summary of the invention

[0003] The purpose of the present invention is to provide negative entropy input-ultrasound mediated composite liposomes and negative entropy input-ultrasound mediated drug-loaded composite liposomes and preparation methods and applications. The negative entropy input-ultrasound mediated drug-loaded composite liposomes provided by the present invention are used as carriers, which are beneficial for brain tumor therapeutic drugs to have high blood-brain barrier penetration efficiency and can circulate in the blood for a long time. They can be co-administered with ultrasound and microbubbles, and can more effectively deliver brain tumor therapeutic drugs to brain tissue; at the same time, they can repair neurons in situ based on plasma gas, thereby restoring the "orderly" health of the neuronal system, that is, realizing the "negative entropy" effect through the input of plasma gas.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a negative entropy input-ultrasound-mediated 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 plasma gas encapsulated in the first shell layer; the second liposome unit comprises a second shell layer and a transition phase encapsulated in the second shell layer.

[0006] Preferably, the plasma gas is one or more of air, O2 and N2 activated by plasma discharge; the transition phase includes one or more of air, O2, CO2, fluorine-containing gas and water.

[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 negative entropy input-ultrasound-mediated composite liposome is 0.2 to 2:1.

[0008] Preferably, the raw materials for preparing the first shell layer and the second shell layer independently include phospholipid materials, emulsifiers, stabilizers and antioxidants.

[0009] 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; the molar ratio of the phospholipid material, emulsifier, stabilizer and antioxidant is 1-50:0.1-20:1-20:0.1-20.

[0010] The present invention provides a method for preparing the negative entropy input-ultrasound-mediated composite liposome described in the above technical solution, including method one or method two;

[0011] The method 1 comprises 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; mixing a second liposome unit with the first pre-treated dispersion, and performing a homogenization treatment, thereby obtaining the negative entropy input-ultrasound-mediated composite liposome;

[0012] The second method comprises the following steps: pre-treating a dispersion containing a second liposome unit so that the zeta potential value of the second liposome unit becomes negative, thereby obtaining a second pre-treated dispersion; mixing the first liposome unit with the second pre-treated dispersion, and performing a homogenization treatment to obtain the negative entropy input-ultrasound-mediated composite liposome.

[0013] The present invention provides a negative entropy input-ultrasound-mediated drug-loaded composite liposome, comprising a negative entropy input-ultrasound-mediated composite liposome and a brain tumor therapeutic drug encapsulated in a transition phase in the negative entropy input-ultrasound-mediated composite liposome; the negative entropy input-ultrasound-mediated composite liposome is the negative entropy input-ultrasound-mediated composite liposome described in the above technical scheme or the negative entropy input-ultrasound-mediated composite liposome prepared by the preparation method described in the above technical scheme.

[0014] Preferably, the brain tumor therapeutic drug includes one or more of selenoprotein P, vincristine, levodopa, valrubicin, belotecan and doxorubicin.

[0015] The present invention provides a method for preparing the negative entropy input-ultrasound-mediated drug-loaded composite liposomes described in the above technical solution, including method three or method four;

[0016] The method three comprises 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; mixing the drug-loaded second liposome unit with the first pre-treated dispersion, and performing a homogenization treatment, thereby obtaining the negative entropy input-ultrasound-mediated drug-loaded composite liposome;

[0017] The method 4 comprises the following steps: pre-treating a dispersion containing a second drug-loaded liposome unit so that the zeta potential value of the second drug-loaded liposome unit is negative, thereby obtaining a second pre-treated dispersion; mixing the first liposome unit with the second pre-treated dispersion, and performing a homogenization treatment to obtain the negative entropy input-ultrasound-mediated drug-loaded composite liposome;

[0018] The drug-loaded 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.

[0019] The present invention provides the use of the negative entropy input-ultrasound-mediated composite liposomes described in the above technical scheme, the negative entropy input-ultrasound-mediated composite liposomes prepared by the preparation method described in the above technical scheme, the negative entropy input-ultrasound-mediated drug-loaded composite liposomes described in the above technical scheme, or the negative entropy input-ultrasound-mediated drug-loaded composite liposomes prepared by the preparation method described in the above technical scheme in the preparation of negative entropy input-ultrasound-mediated brain drug delivery preparations that penetrate the blood-brain barrier.

[0020] The present invention provides a negative entropy input-ultrasound mediated 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 plasma gas encapsulated in the first shell layer; the second liposome unit comprises a second shell layer and a transition phase encapsulated in the second shell layer. The negative entropy input-ultrasound mediated composite liposome provided by the present invention is filled with plasma gas in the first liposome unit, and the plasma gas can improve the permeability of the cell membrane, enter the brain neuron tissue through the blood-brain barrier, stimulate the growth and proliferation of neurons, promote the process of neuronal material exchange, and realize in situ repair of neurons. Specifically, the plasma gas easily generates O - OH -Active free radicals such as oxidizing agents can eliminate inflammation in neuronal tissues, thereby restoring the "orderly" health of the neuronal system, that is, the "negative entropy" effect is achieved through the input of plasma gas; at the same time, due to the high activity of plasma gas, it has a significant oxidizing effect on the first shell layer, which can easily destroy its stable structure. The negative entropy input-ultrasound-mediated drug-loaded composite liposome is used as a carrier. Compared with filling ordinary gas that has not been activated by plasma discharge, the drug release rate is significantly improved, and the gas components are released after the first shell structure is destroyed, which is beneficial for the active ions to enter the neuronal cells for anti-inflammatory effects.

[0021] At the same time, the present invention provides a negative entropy input-ultrasound-mediated drug-loaded composite liposome, that is, the negative entropy input-ultrasound-mediated composite liposome is used as a carrier to load brain tumor therapeutic drugs, wherein the first liposome unit is a directional pressure boosting end, and the brain tumor therapeutic drugs are encapsulated in the transition phase in the second liposome unit to achieve drug loading (i.e., the drug-loaded second liposome unit), which is the 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 to cause it to rupture, and because ultrasound has an obvious "cavitation" effect, the first liposome unit mainly contains plasma 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 plasma 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, and the drug-loaded second liposome unit is directional and accelerated to pass through the blood-brain barrier into the neuronal tissue to release the brain tumor therapeutic drugs for treatment. Moreover, 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 negative entropy input-ultrasound-mediated drug-loaded composite liposomes provided by the present invention are micron-level drugs. Compared with the above-mentioned traditional anticancer drugs, the blood circulation time of micron-level drugs can be extended to 48 to 72 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the structure of the negative entropy input-ultrasound-mediated drug-loaded composite liposome in an embodiment of the present invention, in which 1 is the first shell layer, 2 is the plasma gas, 3 is the brain tumor therapeutic drug, 4 is the second shell layer, and 5 is the transition phase;

[0023] Figure 2Flow chart of preparing the drug-loaded 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 drug-loaded second liposome unit. DETAILED DESCRIPTION

[0024] The present invention provides a negative entropy input-ultrasound-mediated 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 plasma gas encapsulated in the first shell layer; the second liposome unit comprises a second shell layer and a transition phase encapsulated in the second shell layer.

[0025] Brain neuron activity, especially the generation of lesions, such as neurodegenerative diseases such as PD and AD, can be regarded as the development of neuron activity from an ordered low-entropy state to a disordered high-entropy direction. In order to ensure the activity of neurons, the brain will carry out a series of "self-rescue" activities, including clearing β-amyloid protein and tau protein, avoiding the accumulation of fibrotic lesions, and transferring other parts of energy to support the activity of the lesion part of the neurons. The above purposes are to reduce the entropy value of the neuron system and ensure the "orderly" health. However, this will cause the energy input of other tissues to decrease, the function will gradually be lost, and it will seriously affect human health. In view of this, if "negative entropy" can be input into the brain neuron system to maintain the low entropy state of the system, it will have a certain degree of inhibitory effect on the occurrence and development of neurodegenerative diseases, similar to moving the chemical equilibrium in the opposite direction, thereby inhibiting the production of β-amyloid protein, tau protein, etc. There is no relevant research in the existing technology.

[0026] The negative entropy input-ultrasound-mediated composite liposome provided by the present invention has excellent drug loading efficiency and ultrasound-induced drug release effect as a carrier, and can effectively penetrate the blood-brain barrier during ultrasound stimulation; in particular, based on the environmental sensitivity of the negative entropy input-ultrasound-mediated composite liposome, using it as a carrier can ensure that while having better blood-brain barrier penetration efficiency, it can directly and accurately deliver drugs to neurons, avoid the physical damage of cells and blood vessels to cells by focused ultrasound, and have an excellent killing effect on brain tumor cells; at the same time, the negative entropy input-ultrasound-mediated composite liposome provided by the present invention can repair neurons in situ based on plasma gas, thereby restoring the "orderly" health of the neuronal system, that is, realizing the "negative entropy" effect through the input of plasma gas.

[0027] The negative entropy input-ultrasound-mediated composite liposome of the present invention 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, wherein the first liposome unit is a directional pressure boosting end, and the brain tumor therapeutic drug is encapsulated in the transition phase in the second liposome unit to achieve drug loading (i.e., the drug-loaded second liposome unit), which is the brain tumor therapeutic drug delivery end. In the present invention, the particle sizes of the first liposome unit and the second liposome unit are independently preferably 1 to 500 μm, more preferably 1 to 50 μm; the particle size of the first liposome unit can be specifically 25 μm, 35 μm, 40 μm, 45 μm, 75 μm or 85 μm; the particle size of the second liposome unit can be specifically 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 negative entropy input-ultrasound-mediated composite liposome is preferably 0.2 to 2:1, more preferably 0.5 to 1:1, 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.

[0028] In the present invention, the first liposome unit includes a first shell layer and a plasma gas wrapped in the first shell layer; the first liposome unit is specifically a large unilamellar vesicle (LUVs), or called an O / W type microbubble. In the present invention, the plasma gas is one or more of air, O2 and N2 gases activated by plasma discharge, more preferably air, O2 or N2 gases activated by plasma discharge, specifically air plasma, O2 plasma or N2 plasma; the plasma 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%. The present invention fills the first liposome unit with plasma gas, because the gas activated by plasma discharge easily produces O - OH -Active free radicals such as phospholipids are oxidative, and have anti-inflammatory effects on neuronal inflammation, etc.; at the same time, the density of the gas after plasma discharge activation has not changed. Compared with the gas filled with ordinary gas without plasma discharge activation, the release rate of brain tumor therapeutic drugs has been significantly improved. This is mainly due to the high activity of plasma gas, which has a significant oxidative effect on the shell composed of phospholipids, and is easy to destroy its stable structure, thereby achieving the release of gas components, which helps active ions enter the neuronal cells for anti-inflammatory effects. Specifically, the high-energy electrons and ions in the plasma gas can produce a strong electric field effect, which may cause the charge distribution of the cell membrane to change, thereby affecting the permeability of the cell membrane; and the plasma gas may induce the formation of pores on the cell membrane, so that the permeability of the cell membrane to substances increases. These pores can make ions and molecules pass through the cell membrane more easily, thereby affecting the exchange of substances inside and outside the cell. Therefore, the present invention fills the plasma gas in the first liposome unit, which is conducive to improving the permeability of the cell membrane, entering the brain neuronal tissue through the blood-brain barrier, and releasing plasma gas, which can stimulate the growth and proliferation of neurons, promote the process of neuronal substance exchange, and realize the in situ repair of neurons, thereby restoring the "orderliness" health of the neuronal system.

[0029] In the present invention, the thickness of the first shell layer is preferably 0.05-250 μm, more preferably 50-250 nm, specifically 55 nm, 80 nm, 85 nm or 90 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 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; 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 stabilizers, which can promote the accumulation of lipid chains and the formation of bilayers, reduce the fluidity of 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.

[0030] In the present invention, the method for preparing the first liposome unit preferably comprises the following steps:

[0031] The phospholipid material, the emulsifier, the stabilizer and the antioxidant are mixed and homogenized to obtain a homogenized liquid;

[0032] Plasma gas is introduced into the homogenized liquid in the form of microbubbles to obtain the first liposome unit.

[0033] 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 -1 The 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.

[0034] After obtaining the homogenized liquid, the present invention passes plasma gas into the homogenized liquid in the form of microbubbles to obtain the first liposome unit. The present invention preferably uses a plasma gas generator to pass plasma gas into the homogenized liquid in the form of microbubbles. In the present invention, the plasma gas generator is preferably a micro-nano bubble generator, and the bubble particle size is preferably controlled to be 0.01 to 100 μm, specifically 1 μm, 10 μm or 100 μm. The flow rate of the plasma gas of the present invention is preferably 1 to 500 mL / min, more preferably 10 to 250 mL / min, specifically 25 mL / min, 50 mL / min, 75 mL / min, 100 mL / min or 200 mL / min; the passage time is preferably 15 to 30 min, specifically 15 min, 20 min or 30 min; the plasma gas passage process 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 to -20°C, more preferably -25°C, and the time is preferably 12 to 24 hours, more preferably 20 to 24 hours.

[0035] In the present invention, the gas content of the first liposome unit prepared by the above method is Preferably, it is 0.05 to 0.30, preferably 0.08 to 0.20, and specifically, it can be 0.09, 0.102, 0.155, 0.161, 0.205 or 0.25; the average bubble diameter (d 32 ) is preferably 0.01 to 100 μm, preferably 0.1 to 50 μm, and specifically can be 1.25 μm, 1.45 μm, 2.0 μm, 5 μm, 8.5 μm or 35 μm; the average bubble rise speed (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 brain tumor treatment drug delivery efficiency.

[0036] In the present invention, the second liposome unit includes a second shell layer and a transition phase encapsulated in the second shell layer. 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, 65 nm, 80 nm or 85 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.

[0037] In the present invention, the second liposome unit is preferably prepared by different methods according to the type of transition phase, which will be described in detail later in combination with the drug loading condition.

[0038] The present invention provides a method for preparing the negative entropy input-ultrasound-mediated composite liposome described in the above technical solution, including method one or method two;

[0039] The method 1 comprises 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; mixing a second liposome unit with the first pre-treated dispersion, and performing a homogenization treatment, thereby obtaining the negative entropy input-ultrasound-mediated composite liposome;

[0040] The second method comprises the following steps: pre-treating a dispersion containing a second liposome unit so that the zeta potential value of the second liposome unit becomes negative, thereby obtaining a second pre-treated dispersion; mixing the first liposome unit with the second pre-treated dispersion, and performing a homogenization treatment to obtain the negative entropy input-ultrasound-mediated composite liposome.

[0041] 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 negative entropy input-ultrasound-mediated composite liposome. 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 negative entropy input-ultrasound-mediated composite liposome. A specific explanation is given below.

[0042] 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.

[0043] After obtaining the pretreated dispersion, the present invention mixes the first liposome unit prepared by the above technical solution with the pretreated dispersion, performs a homogenization treatment, and obtains the negative entropy input-ultrasound-mediated 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 pretreated dispersion is preferably 19 to 35wt%, specifically 19.5wt%, 24.2wt%, 24.5wt%, 32.4wt%, 33.0wt% or 33.2wt%. 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.

[0044] The present invention provides a negative entropy input-ultrasound mediated drug-loaded composite liposome, including the negative entropy input-ultrasound mediated composite liposome and a brain tumor therapeutic drug encapsulated in the transition phase of the negative entropy input-ultrasound mediated composite liposome; the negative entropy input-ultrasound mediated composite liposome is the negative entropy input-ultrasound mediated composite liposome described in the above technical solution or the negative entropy input-ultrasound mediated composite liposome prepared by the preparation method described in the above technical solution. Specifically, Figure 1 As shown, the negative entropy input-ultrasound-mediated drug-loaded composite liposome of the present invention includes a first liposome unit and a drug-loaded second liposome unit, and the first liposome unit and the second liposome unit are combined by charge attraction and hydrogen bonding; the first liposome unit includes a first shell layer and a plasma gas encapsulated in the first shell layer; the drug-loaded 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 drug-loaded second liposome unit of the present invention 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 drug-loaded liposome unit is preferably 0.05-10wt%, specifically 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3.5wt% or 5.2wt%.

[0045] In the present invention, the drug-loaded 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.

[0046] In the present invention, when the transition phase is a gas (such as air, O2, CO2, fluorine-containing gas), the method for preparing the drug-loaded second liposome unit preferably comprises the following steps:

[0047] The phospholipid material, the emulsifier, the stabilizer and the antioxidant are mixed and homogenized to obtain a homogenized liquid;

[0048] The brain tumor therapeutic drug is sprayed into the flowing transition phase by high-pressure atomization to obtain atomized core droplets;

[0049] The mist-like inner core droplets are passed into the homogenized liquid to obtain the drug-loaded second liposome unit.

[0050] 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.

[0051] 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.

[0052] After obtaining the mist-like core liquid droplets and the homogenized liquid, the present invention passes the mist-like core liquid droplets into the homogenized liquid to obtain the drug-loaded second liposome unit. The present invention preferably uses a gas generator to pass the mist-like 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-like core liquid is preferably 15 to 30 minutes, specifically 15 minutes, 20 minutes or 30 minutes; the process of passing the mist-like 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 drug-loaded 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 drug-loaded 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 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 or 4.5 cm / s.

[0053] In the present invention, when the transition phase is a liquid (such as water), the method for preparing the drug-loaded second liposome unit preferably comprises the following steps:

[0054] The phospholipid material, the emulsifier, the stabilizer and the antioxidant are mixed and homogenized to obtain a homogenized liquid;

[0055] mixing the brain tumor therapeutic drug with the transition phase and homogenizing the mixture to obtain an emulsion;

[0056] The homogenized liquid is mixed with the emulsion to perform a microemulsification reaction to obtain the drug-loaded second liposome unit.

[0057] 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.

[0058] 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.

[0059] 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 drug-loaded second liposome unit. In the present invention, the volume ratio of the emulsion to the homogenized liquid is preferably 1:8-12, 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 30-60 minutes, specifically 30 minutes, 45 minutes or 60 minutes. In the present invention, the drug-loaded second liposome unit liquid is obtained after the microemulsification reaction, which can be directly used to prepare the negative entropy input-ultrasound-mediated drug-loaded composite liposomes without post-treatment.

[0060] The present invention provides a method for preparing the negative entropy input-ultrasound-mediated drug-loaded composite liposomes described in the above technical solution, including method three or method four;

[0061] The method three comprises 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; mixing the drug-loaded second liposome unit with the first pre-treated dispersion, and performing a homogenization treatment, thereby obtaining the negative entropy input-ultrasound-mediated drug-loaded composite liposome;

[0062] The method 4 comprises the following steps: pre-treating a dispersion containing a second drug-loaded liposome unit so that the zeta potential value of the second drug-loaded liposome unit is negative, thereby obtaining a second pre-treated dispersion; mixing the first liposome unit with the second pre-treated dispersion, and performing a homogenization treatment to obtain the negative entropy input-ultrasound-mediated drug-loaded composite liposome;

[0063] The drug-loaded 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.

[0064] In the present invention, the relevant operation steps and conditions in Method 3 and Method 4 refer to Method 2 and will not be repeated here.

[0065] In an embodiment of the present invention, the particle size of the negative entropy input-ultrasound-mediated drug-loaded composite liposome is preferably 50-500 μm, specifically 55 μm, 105 μm, 185 μm, 190 μm, 205 μm or 430 μm; the loading efficiency of brain tumor therapeutic drugs in the negative entropy input-ultrasound-mediated drug-loaded composite liposome is preferably 91.5-97.5%, specifically 92%, 95.3%, 95.4%, 96.8%, 96.9%, 97.1%; the release rate is preferably 60-80%, specifically 61.2%, 72.3%, 73.9%, 75.2%, 75.4%.

[0066] The present invention provides the use of the negative entropy input-ultrasound-mediated composite liposomes described in the above technical scheme, the negative entropy input-ultrasound-mediated composite liposomes prepared by the preparation method described in the above technical scheme, the negative entropy input-ultrasound-mediated drug-loaded composite liposomes described in the above technical scheme, or the negative entropy input-ultrasound-mediated drug-loaded composite liposomes prepared by the preparation method described in the above technical scheme in the preparation of negative entropy input-ultrasound-mediated brain drug delivery preparations that penetrate the blood-brain barrier.

[0067] 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.

[0068] Example 1

[0069] Prepare the first liposome unit, the steps are as follows:

[0070] 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;

[0071] Air plasma (gas activated by plasma discharge of air) was introduced into the homogenized liquid by using a micro-nano bubble generator (the bubble diameter was set to 1 μm), the gas flow rate was set to 100 mL / min, the introduction time was set to 30 min, and after the introduction, the liquid was freeze-dried at -25 ° C for 24 h to obtain a first liposome unit (including a shell layer and bubbles formed by air plasma wrapped in the shell layer); the average particle size of the first liposome unit was 35 μm, the shell thickness was 80 nm, and the gas content was is 0.161, and the average bubble diameter d 32 is 5 μm, and the average rising velocity of the bubble is ν 32 3.5cm / s.

[0072] Example 2

[0073] Prepare the first liposome unit, the steps are as follows:

[0074] 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. After the homogenization treatment was completed, the obtained homogenized liquid was sealed for standby use;

[0075] Air plasma (gas activated by plasma discharge of air) was introduced into the homogenized liquid by using a micro-nano bubble generator (the bubble particle size was set to 1 μm), the gas flow rate was set to 100 mL / min, the introduction time was 30 min, and 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 25 μm, and the gas content was is 0.205, the average bubble diameter d 32is 1.25 μm, the shell thickness is 55 nm, and the average bubble rising speed ν 32 2.5cm / s.

[0076] Example 3

[0077] Prepare the first liposome unit, the steps are as follows:

[0078] 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;

[0079] A micro-nano bubble generator (the bubble particle size is set to 1 μm) is used to pass N2 plasma (N2 gas activated by plasma discharge) into the homogenized liquid, the gas flow rate is set to 200 mL / min, the passing time is 15 min, and after the passing is completed, it is freeze-dried at -25 ° C for 24 h to obtain the first liposome unit; the average particle size of the first liposome unit is 45 μm, the shell thickness is 80 nm, and the gas content is is 0.155, the average bubble diameter d 32 is 2.0 μm, and the average rising velocity of the bubble is ν 32 It is 3.7cm / s.

[0080] Example 4

[0081] Prepare the first liposome unit, the steps are as follows:

[0082] 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. After the homogenization treatment was completed, the obtained homogenized liquid was sealed for standby use;

[0083] A micro-nano bubble generator (the bubble particle size was set to 1 μm) was used to pass N2 plasma (gas after N2 was activated by plasma discharge) into the homogenized liquid, the gas flow rate was set to 75 mL / min, the passing time was 30 min, and after the passing was completed, it 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 shell thickness was 90 nm, and the gas content was is 0.205, the average bubble diameter d 32 is 1.45 μm, and the average rising velocity of the bubble is ν 32 It is 2.75cm / s.

[0084] Example 5

[0085] Prepare the first liposome unit, the steps are as follows:

[0086] 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;

[0087] A micro-nano bubble generator (the bubble particle size is set to 100 μm) is used to pass N2 plasma (gas after N2 is activated by plasma discharge) into the homogenized liquid, the gas flow rate is set to 50 mL / min, the passing time is 15 min, and after the passing is completed, it is freeze-dried at -25 ° C for 24 h to obtain the first liposome unit; the average particle size of the first liposome unit is 75 μm, the shell thickness is 80 nm, and the gas content is is 0.102, the average bubble diameter d 32 is 8.5μm, and the average rising velocity of the bubble is ν 32 3.5cm / s.

[0088] Example 6

[0089] Prepare the first liposome unit, the steps are as follows:

[0090] 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;

[0091] A micro-nano bubble generator (the bubble particle size was set to 10 μm) was used to pass N2 plasma (gas after N2 was activated by plasma discharge) into the homogenized liquid, the gas flow rate was set to 25 mL / min, the passing time was 30 min, and after the passing was completed, it 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 85 μm, the shell thickness was 85 nm, and the gas content was is 0.090, 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.

[0092] Example 7

[0093] The steps for preparing the drug-loaded second liposome unit are as follows:

[0094] 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 atomized droplets is 0.15μm;

[0095] 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;

[0096] The mist-like core droplets were introduced into the homogenized liquid by 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 drug-loaded 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 drug-loaded second liposome unit was 35 μm, the 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 drug-loaded liposome unit is 2.5wt%.

[0097] Example 8

[0098] The steps for preparing the drug-loaded second liposome unit are as follows:

[0099] 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 atomized droplets is 10μm;

[0100] 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;

[0101] 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 the drug-loaded second liposome unit. The average particle size of the drug-loaded second liposome unit was 90 μm, the 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 4.5cm / s; the content of the brain tumor therapeutic drug in the second drug-loaded liposome unit is 1wt%.

[0102] Example 9

[0103] The steps for preparing the drug-loaded second liposome unit are as follows:

[0104] 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, 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 atomized droplets is 10μm;

[0105] 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;

[0106] 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 the second drug-loaded liposome unit. The average particle size of the second drug-loaded liposome unit was 95 μm, the 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 drug-loaded liposome unit is 1.5wt%.

[0107] Example 10

[0108] The steps for preparing the drug-loaded second liposome unit are as follows:

[0109] 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, 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 atomized droplets is 10μm;

[0110] 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;

[0111] 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 the second drug-loaded liposome unit. The average particle size of the second drug-loaded liposome unit was 25 μm, the 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 drug-loaded liposome unit is 2wt%.

[0112] Embodiment 11

[0113] The steps for preparing the drug-loaded second liposome unit are as follows:

[0114] 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;

[0115] 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;

[0116] 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 the second drug-loaded liposome unit; the average particle size of the second drug-loaded liposome unit is 350 μm, the shell thickness is 80 nm, and the content of the brain tumor therapeutic drug in the second drug-loaded liposome unit is 3.5 wt%.

[0117] Example 12

[0118] The steps for preparing the drug-loaded second liposome unit are as follows:

[0119] 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;

[0120] 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;

[0121] 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 drug-loaded second liposome unit; the average particle size of the drug-loaded second liposome unit is 100 μm, the shell thickness is 65 nm, and the content of the brain tumor therapeutic drug in the drug-loaded second liposome unit is 5.2 wt%.

[0122] Embodiment 13

[0123] The steps for preparing the composite liposomes are as follows:

[0124] The drug-loaded second liposome unit prepared in Example 7 was mixed with a dispersion medium (specifically pure water) to obtain a dispersion containing the drug-loaded second liposome unit (the concentration of the drug-loaded second liposome unit was 15wt%); the dispersion containing the drug-loaded second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 8m / s, and continuous circulation was performed at room temperature for 120min, and the zeta potential value was measured to be -28mV, to obtain a pretreated dispersion;

[0125] The first liposome unit prepared in Example 1 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 24.5 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 55 μm, the loading efficiency of the brain tumor treatment drug was 95.3%, and the release rate was 72.3%.

[0126] Embodiment 14

[0127] The steps for preparing the composite liposomes are as follows:

[0128] The drug-loaded second liposome unit prepared in Example 8 was mixed with a dispersion medium (specifically ethylene glycol) to obtain a dispersion containing the drug-loaded second liposome unit (the concentration of the drug-loaded second liposome unit was 15 wt %); the dispersion containing the drug-loaded 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;

[0129] The first liposome unit prepared in Example 5 was mixed with the pretreated dispersion to obtain a mixture with a concentration of the first liposome unit of 19.5 wt %. 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 430 μm, the loading efficiency of the brain tumor therapeutic drug was 92%, and the release rate was 61.2%.

[0130] Embodiment 15

[0131] The steps for preparing the composite liposomes are as follows:

[0132] The second drug-loaded liposome unit-containing liquid prepared in Example 12 was mixed with a dispersion medium (specifically pure water) to obtain a dispersion containing the second drug-loaded liposome unit (the concentration of the second drug-loaded liposome unit was 10 wt %); the dispersion containing the second drug-loaded 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;

[0133] The first liposome unit prepared in Example 3 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 33.2 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 205 μm, the loading efficiency of the brain tumor treatment drug was 97.1%, and the release rate was 75.4%.

[0134] Example 16

[0135] The steps for preparing the composite liposomes are as follows:

[0136] The drug-loaded second liposome unit prepared in Example 9 was mixed with a dispersion medium (specifically pure water) to obtain a dispersion containing the drug-loaded second liposome unit (the concentration of the drug-loaded second liposome unit was 20wt%); the dispersion containing the drug-loaded second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 8m / s, and continuous circulation was performed at room temperature for 90min, and the zeta potential value was measured to be -28mV, to obtain a pretreated dispersion;

[0137] The first liposome unit prepared in Example 2 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 32.4 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 75.4%.

[0138] Embodiment 17

[0139] The steps for preparing the composite liposomes are as follows:

[0140] The drug-loaded second liposome unit prepared in Example 10 was mixed with a dispersion medium (specifically pure water) to obtain a dispersion containing the drug-loaded second liposome unit (the concentration of the drug-loaded second liposome unit was 15wt%); the dispersion containing the drug-loaded second liposome unit was injected into a tubular continuous reactor, a circulation pump was turned on, the flow rate was controlled at 8m / s, and continuous circulation was performed at room temperature for 90min, and the zeta potential value was measured to be -28mV, to obtain a pretreated dispersion;

[0141] The first liposome unit prepared in Example 4 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 33.0 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 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 loading efficiency of the brain tumor treatment drug was 96.8%, and the release rate was 75.2%.

[0142] Embodiment 18

[0143] The steps for preparing the composite liposomes are as follows:

[0144] The drug-loaded second liposome unit-containing liquid prepared in Example 11 was mixed with a dispersion medium (specifically ethylene glycol) to obtain a dispersion containing the drug-loaded second liposome unit (the concentration of the drug-loaded second liposome unit was 20 wt %); the dispersion containing the drug-loaded 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;

[0145] The first liposome unit prepared in Example 6 was mixed with the pretreated dispersion to obtain a mixture with a concentration of 24.2 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 185 μm, the brain tumor therapeutic drug loading efficiency was 95.4%, and the release rate was 73.9%.

[0146] Comparative Example 1

[0147] The composite liposomes were prepared by referring to the method of Example 17, except that N2 plasma was replaced by N2 when preparing the first liposome unit.

[0148] Test Example 1

[0149] 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 compared with the composite liposome in Comparative Example 1. The specific steps are as follows:

[0150] 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. Then, the cells were 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, and the anti-inflammatory effect was analyzed using serum CRP levels as an indicator.

[0151] The cancer cell killing effect and anti-inflammatory effect of the composite liposomes prepared in Example 17 under different concentration conditions are shown in Table 1, and the cancer cell killing effect and anti-inflammatory effect of the composite liposomes prepared in Comparative Example 1 are shown in Table 2. It can be seen that 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 ultrasonic conditions, the cancer cell killing effect and anti-inflammatory effect are significantly enhanced when the composite liposomes provided by the present invention are administered under ultrasonic 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 and anti-inflammatory effects on brain tumor cells.

[0152] Table 1 Cancer cell killing effect and anti-inflammatory effect of the composite liposomes prepared in Example 17

[0153]

[0154] Table 2 Cancer cell killing effect and anti-inflammatory effect of the composite liposomes prepared in Comparative Example 1

[0155]

[0156] 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 negative entropy input-ultrasound-mediated 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 plasma gas encapsulated in the first shell layer; the second liposome unit comprises a second shell layer and a transition phase encapsulated in the second shell layer.

2. The negative entropy input-ultrasound-mediated drug-loaded composite liposome according to claim 1, characterized in that: The plasma gas is one or more of air, O2 and N2 that are activated by plasma discharge; the transition phase includes one or more of air, O2, CO2, fluorine-containing gas and water.

3. The negative entropy input-ultrasound-mediated complex liposome according to claim 1 or 2, 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 negative entropy input-ultrasound-mediated composite liposome is 0.2 to 2:

1.

4. The negative entropy input-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.

5. The negative entropy input-ultrasound-mediated drug-loaded composite liposome according to claim 4, 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; the molar ratio of the phospholipid material, emulsifier, stabilizer and antioxidant is 1-50:0.1-20:1-20:0.1-20.

6. The method for preparing the negative entropy input-ultrasound mediated composite liposome according to any one of claims 1 to 5, comprising method 1 or method 2; The method 1 comprises 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 liquid, and homogenized to obtain the negative entropy input-ultrasound-mediated composite liposome; The second method comprises the following steps: pre-treating a dispersion containing a second liposome unit so that the zeta potential value of the second liposome unit becomes negative, thereby obtaining a second pre-treated dispersion; mixing the first liposome unit with the second pre-treated dispersion, and performing a homogenization treatment to obtain the negative entropy input-ultrasound-mediated composite liposome.

7. A negative entropy input-ultrasound-mediated drug-loaded composite liposome, comprising a negative entropy input-ultrasound-mediated composite liposome and a brain tumor therapeutic drug encapsulated in the transition phase of the negative entropy input-ultrasound-mediated composite liposome; the negative entropy input-ultrasound-mediated composite liposome is the negative entropy input-ultrasound-mediated composite liposome described in any one of claims 1 to 5 or the negative entropy input-ultrasound-mediated composite liposome prepared by the preparation method described in claim 6.

8. The negative entropy input-ultrasound-mediated drug-loaded composite liposome according to claim 7, characterized in that: The brain tumor treatment drugs include one or more of selenoprotein P, vincristine, levodopa, valrubicin, belotecan and doxorubicin.

9. The method for preparing the negative entropy input-ultrasound-mediated drug-loaded composite liposome according to claim 7 or 8, comprising method three or method four; The method three comprises 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 drug-loaded liposome unit is mixed with the first pre-treated dispersion liquid, and homogenized to obtain the negative entropy input-ultrasound-mediated drug-loaded composite liposome; The method 4 comprises the following steps: pre-treating a dispersion containing a second drug-loaded liposome unit so that the zeta potential value of the second drug-loaded liposome unit is negative, thereby obtaining a second pre-treated dispersion; mixing the first liposome unit with the second pre-treated dispersion, and performing a homogenization treatment to obtain the negative entropy input-ultrasound-mediated drug-loaded composite liposome; The drug-loaded 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.

10. Use of the negative entropy input-ultrasound-mediated composite liposomes as described in any one of claims 1 to 5, the negative entropy input-ultrasound-mediated composite liposomes prepared by the preparation method described in claim 6, the negative entropy input-ultrasound-mediated drug-loaded composite liposomes as described in claim 7 or 8, or the negative entropy input-ultrasound-mediated drug-loaded composite liposomes prepared by the preparation method described in claim 9 in the preparation of negative entropy input-ultrasound-mediated brain drug delivery preparations that penetrate the blood-brain barrier.

Images