Method for applying gigahertz bulk acoustic wave resonator to active drug loading of lipidosome

Abstract

The invention discloses a method for applying a gigahertz bulk acoustic wave resonator to active drug loading of lipidosome, and belongs to the technical field of drug loading. The present invention is a universal method for efficiently, quantitatively loading one or more drug molecules having different solubilities into liposomal vesicles. On the basis of a high-speed vortex-shaped acoustic streaming effect generated by a kilomegahertz bulk acoustic wave resonator, the permeability of the liposome with the flowable phospholipid bilayer membrane is changed, so that drugs with different solubilities can be loaded into the liposome at the same time; and the loading capacity of the medicine can be quantitatively controlled by regulating and controlling the working power and time of the GHz bulk acoustic wave resonator. The application range of the method is not limited to specific phospholipid molecules assembled by lipidosome types, and the method is effective for vesicle cavities with phospholipid bilayer structures. The loading method used in the invention can improve the preparation efficiency of the drug carrier, is not limited by the hydrophilic and hydrophobic properties of the drug, and can realize the preparation of the drug carrier with quantitative concentration.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical drug delivery, and relates to a general method for active drug delivery of liposomes based on gigahertz bulk acoustic wave resonator acoustic fluid control. Background Art

[0002] Bulk Acoustic Wave Resonator (BAW) is a piezoelectric resonator that converts electrical energy into sound waves to form resonance through the inverse piezoelectric effect of piezoelectric film. BAW resonators prepared by MEMS technology can greatly reduce the size of the device and can obtain extremely high resonance frequencies, which can generate gigahertz-level bulk acoustic waves. There are two main structures of BAW resonators, namely thin film bulk acoustic wave resonators and solid-mounted resonators. Their core structure is a piezoelectric resonance layer sandwiched by two layers of electrodes. The main difference is that the thin film bulk acoustic wave resonator blocks and reflects sound waves by etching a cavity on the back of the resonance layer, while the solid-mounted resonator reflects sound waves by depositing a Bragg reflection layer on the back of the resonance layer, thereby reducing energy loss and improving resonator performance.

[0003] The acoustic streaming effect refers to the nonlinear fluid phenomenon caused by the attenuation of sound waves inside a viscous fluid medium. When the GHz BAW resonator operates in the thickness shear mode, the longitudinally transmitted sound waves it excites generate volume forces in the liquid due to rapid attenuation, thereby driving the liquid to generate high-speed vortices and forming a three-dimensional acoustic streaming field with a specific flow velocity distribution. The distribution range of the acoustic streaming field is mainly affected by the device size of the resonator, which is usually in the sub-millimeter order. The flow velocity of the acoustic streaming field is determined by the device resonator frequency and the device power, which is usually in the m / s order. Based on the acoustic streaming effect generated by the device, cells, liposomes, vesicles, nanoparticles, etc. can be precisely manipulated.

[0004] Liposomes are small vesicles with a phospholipid bilayer structure formed by amphiphilic molecules through self-assembly methods, which can contain single or multiple cavities inside. Among them, the hydrophilic layer of the liposome membrane is composed of phosphate groups and other polar groups (such as choline, ethanolamine, and serine). These groups are polar and can interact with water molecules through hydrogen bonds and electrostatics, and are hydrophilic; the hydrophobic layer of the liposome membrane is composed of non-polar long-chain fatty acids, which do not interact with water molecules and are hydrophobic. The membrane structure of liposomes is highly similar to that of cell membranes, and the phospholipid molecules used to assemble liposomes can also be selected from types close to cell membrane components. Therefore, they are often used as drug carriers to deliver drug molecules to specific tissues or cells in the body. Drugs are loaded into liposomes, which can protect them from degradation and improve their pathways to target sites. Liposomes can also improve the pharmacokinetic properties of drugs, prolong their circulation time in the body and reduce toxicity; and by modifying different protein molecules on the surface of liposomes, targeted delivery of drug molecules can be achieved, reducing the side effects of drug treatment.

[0005] However, using liposomes as carriers, achieving controllable and quantitative loading of drug molecules is still a major challenge in the field of drug carrier preparation. At present, the methods commonly used to prepare drug lipid carriers include passive drug loading and active drug loading. In passive drug loading, phospholipids are usually first dissolved in an organic solvent, and the organic solvent is volatilized by negative pressure rotary evaporation to form a uniform phospholipid film, and then a water-based solvent dissolved with drug molecules is added to allow the drug molecules to be encapsulated in the vesicle cavity during the spontaneous hydration and swelling of the phospholipid membrane to form vesicles. The passive drug loading method is simple to operate, but since the presence of drug molecules will reduce the efficiency of liposome self-assembly, the efficiency of drug carrier preparation is low, and the encapsulation process and drug loading are uncontrollable. The active drug loading method refers to applying external physical or chemical stimulation, such as adjusting the pH value, adding additional ion solutions, etc. to form an ion concentration difference inside and outside the liposome, based on this concentration gradient, the hydrophilic drug molecules outside the liposome are actively diffused into the liposome, and the introduction of drug molecules into the liposome is promoted by applying physical signals such as acoustics and electricity. The active drug loading method has a high loading efficiency and the process is relatively controllable, but there is still a lack of a loading method that can be applied to drugs of different chemical properties. Generally, the solubility of the drug has a great influence on its loading rate in liposomes: water-soluble drugs are easier to load into liposomes, while fat-soluble drugs are more difficult to load. However, in current clinical practice, especially in cancer treatment and anti-infection treatment, multiple drugs are usually required to be administered in combination to enhance synergistic effects. These drugs have different or even completely opposite properties, which poses great difficulties for multi-drug therapy. Therefore, it is crucial to provide a universal and controllable liposome drug loading method that is not limited by the physical and chemical properties of drug molecules. Summary of the invention

[0006] In order to solve the problem of the difficulty of existing multi-drug co-encapsulation, the purpose of the present invention is to provide a method for using a gigahertz body acoustic wave resonator for active drug loading in liposomes, which is a general method for efficiently and quantitatively loading one or more drug molecules with different solubility into liposome vesicles, and the method uses an acoustic fluidic chip prepared based on a gigahertz body acoustic wave resonator to produce an acoustic flow effect, and acts on the liposomes, and the drug molecules are controllably loaded by changing the permeability of the liposomes. This method can achieve a high loading efficiency for both water-soluble and fat-soluble drug molecules, and the prepared drug lipid carrier has good biocompatibility. The present invention can further load a variety of drug molecules with different solubility into liposomes together, prepare a multifunctional drug carrier, and achieve the synergistic effect of a variety of drug molecules in the treatment of diseases based on drug delivery, thereby improving the therapeutic effect.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The method for using a gigahertz bulk acoustic wave resonator for active drug loading in liposomes disclosed in the present invention comprises the following steps:

[0009] Step 1, dissolving drug molecules in a solvent sterilized by ultraviolet light to obtain a drug solution to be loaded; preparing a liposome suspension; and then mixing the drug solution to be loaded with the liposome suspension to obtain a mixed solution;

[0010] Step 2: placing the mixed solution on the surface of a gigahertz bulk acoustic wave resonator; the resonator changes the permeability of the liposome to achieve effective drug loading, thereby obtaining micron-sized liposome drugs encapsulating the target drug molecules.

[0011] The method for preparing the micron-sized liposome drug obtained in step 2 into a nano-sized drug is as follows: the micron-sized liposome is passed through a filter membrane with a diameter of 100 nm by an extrusion method to be converted into a drug lipid carrier with a uniform size and a phospholipid bilayer structure with a diameter of 100 nm.

[0012] The preparation method of the liposome suspension is as follows: 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPC is dissolved in an organic solvent, and liposomes with a diameter of micrometer order are prepared by a molecular self-assembly method.

[0013] The drug molecules include at least one of water-soluble drug molecules and fat-soluble drug molecules.

[0014] Water-soluble drug molecules are dissolved in ultrapure water after UV sterilization; fat-soluble drugs are dissolved in water-based solvents added with organic co-solvents after UV sterilization.

[0015] The drug molecules include at least one of water-soluble drug molecules and fat-soluble drug molecules.

[0016] Water-soluble drug molecules are dissolved in ultrapure water after UV sterilization; fat-soluble drugs are dissolved in water-based solvents with added cosolvents after UV sterilization.

[0017] The resonance frequency of the BAW resonator used should be in the gigahertz range. Preferably, a BAW resonator with a resonance frequency of 2.5 GHz should be used.

[0018] If only water-soluble drug molecules need to be encapsulated, the drug molecules are directly dissolved in a water-based solvent in step (1). Preferably, the water-soluble drug molecules are selected from one or more of doxorubicin, aspirin, and quinacrine.

[0019] If only fat-soluble drug molecules need to be encapsulated, part of the organic cosolvent needs to be added to the water-based solvent in step (1) to improve the solubility of the fat-soluble drug molecules. Preferably, the organic cosolvent is dimethyl sulfoxide (DMSO), and the fat-soluble drug molecule is niclosamide. More preferably, the ratio of the organic cosolvent to the water-based solvent is: ultrapure water: DMSO = (100-1000): 1.

[0020] If it is necessary to encapsulate both water-soluble and fat-soluble drug molecules at the same time, follow the above two steps in sequence.

[0021] Beneficial effects:

[0022] The present invention discloses a method for using a gigahertz bulk acoustic wave resonator for active drug loading in liposomes, which is a general method for controllably loading one or more hydrophilic and hydrophobic drug molecules into liposomes. The method is based on the high-speed vortex acoustic streaming effect generated by the gigahertz bulk acoustic wave resonator, thereby changing the permeability of liposomes with a fluid phospholipid bilayer membrane, so that drugs of different solubility can be loaded into the liposomes at the same time, and the loading amount of the drug can be quantitatively controlled by regulating the working power and time of the GHz bulk acoustic wave resonator. The present invention has low selectivity for the type of liposomes, is not limited to the specific phospholipid molecules assembled therein, and is effective for vesicle cavities with a phospholipid bilayer structure. The loading method used in the present invention can improve the preparation efficiency of drug carriers, is not limited by the hydrophilicity of the drug, and can achieve the preparation of drug carriers with quantitative concentrations. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The schematic diagram of the structure of the acoustofluidic chip prepared based on the GHz BAW resonator and the cavity fixed on its surface, wherein 1 is the substrate, 2 is the acoustic fluid action cavity, and 3 is the BAW resonator;

[0024] Figure 2This is a comparison diagram of the particle size distribution of the lipid carrier (DOX-SUVs) loaded with doxorubicin drug molecules prepared by applying acoustic flow stimulation in Example 1 and its blank control group (empty liposome SUVs without acoustic flow stimulation);

[0025] Figure 3 The absorbance change of the lipid carrier (DOX-SUVs) loaded with doxorubicin drug molecules at 490 nm (characteristic wavelength of doxorubicin) under the action of acoustic flow of different powers (0, 100, 200, 300, 500 mW) in Example 1;

[0026] Figure 4 The absorbance change at 340 nm (characteristic wavelength of chloranthamine) of the lipid carrier (BAY2353-SUVs) loaded with chloranthamine drug molecules under the action of acoustic streaming of different powers (0, 100, 200, 300, 500 mW) in Example 2. DETAILED DESCRIPTION

[0027] In order to better illustrate the purpose and advantages of the present invention, the invention is further described below with reference to the accompanying drawings and examples.

[0028] The steps of the determination method in the following example are as follows:

[0029] 1. Calculation of DOX and BAY2353 drug loading rate:

[0030] The UV absorption values ​​of the prepared drug lipid carriers were measured at 490 nm and 340 nm by UV-visible spectrophotometry, and the concentrations of DOX and BAY2353 were determined based on the standard curve. The loading rate was calculated as follows:

[0031]

[0032] 2. Dialysis method to filter out free drug molecules outside the drug lipid carrier:

[0033] The prepared drug lipid carrier solution is placed in a dialysis bag with a molecular weight cutoff of 1000, and the dialysis bag is placed in a large-capacity beaker filled with ultrapure water, and is continuously stirred on a magnetic stirrer for 12 to 24 hours. During this period, the ultrapure water in the beaker is replaced every 2 hours, and the absorbance value of the replaced waste liquid is measured. When the absorbance of the waste liquid is consistent with the measured absorbance value of the ultrapure water, it is determined that all free drug molecules outside the drug lipid carrier have been filtered out.

[0034] 3. Construct the acoustic streaming cavity of GHz BAW resonator:

[0035] A 1mm thick annular cavity is made using polydimethylsiloxane (PDMS), and the center of the PDMS annular cavity is aligned with the center of the bulk acoustic wave resonator, and sealed and fixed to the top of the resonator to form an acoustic flow action cavity. The loaded drug molecule solution and liposome suspension are injected into the cavity, and an electrical signal of a predetermined power is applied to the resonator to perform acoustic flow stimulation.

[0036] Example 1: Preparation of lipid carriers loaded with a hydrophilic drug molecule DOX (DOX-SUVs) by acoustic flow stimulation:

[0037] (1) Preparation of DOX solution: DOX was slowly dissolved in ultrapure water after UV sterilization in a fume hood, mixed and stirred at room temperature to prepare a DOX solution with a concentration of 80 μM, and placed in a 4°C refrigerator for later use;

[0038] (2) Preparation of micron-sized DOPC liposomes: Weigh 50 mg of DOPC powder in a fume hood and place it in a brown glass bottle. Slowly inject 5 mL of chloroform into the brown glass bottle and slowly shake the brown glass bottle at the same time until all the DOPC powder is dissolved to prepare a 10 mg / mL DOPC chloroform solution. Use a microinjector to transfer 100 μL of DOPC solution into a new, unused small glass bottle, tilt the bottle and slowly rotate the glass bottle, while passing nitrogen into the bottle to evaporate the chloroform and form a uniform DOPC lipid film on the inner wall of the glass bottle. Place the glass bottle in a vacuum box and evacuate for 12 hours to completely evaporate the chloroform. Then, take 1 mL of ultrapure water that has been sterilized by ultraviolet light in the fume hood and place it in the glass bottle with the DOPC lipid film, shake it evenly on a shaker, and then let it stand at room temperature for 2 hours to allow the phospholipids to fully swell to obtain a DOPC liposome suspension;

[0039] (3) Preparation of nanoscale drug lipid carrier DOX-SUVs: The DOPC liposome suspension prepared in step (2) and the DOX solution prepared in step (1) were mixed at a volume ratio of 1:1 and placed in an acoustic flow chamber. Figure 1 As shown. Turn on the electrical signal to make the gigahertz bulk acoustic wave resonator work and generate acoustic flow to promote the enrichment of DOX into DOPC liposomes. The output powers of 100mW, 200mW, 300mW, 400mW and 500mW were applied for 10 minutes respectively to obtain a liposome suspension encapsulating DOX. The liposome suspension was then placed in an extruder, and a filter membrane with a pore size of 100nm was used in the extruder. The extruder was pushed repeatedly 11 times so that the liposomes were reorganized into DOX-SUVs with a diameter of 100nm. The particle size distribution was compared with that of empty SUVs. Figure 2The obtained DOX-SUVs solution was placed in a dialysis bag and dialyzed with ultrapure water for 12 hours to precipitate the residual drug molecules in the extracellular fluid of the vesicles, and finally a lipid carrier solution loaded with DOX was obtained. The test results of drug loading under different powers are shown in Figure 3 shown.

[0040] (4) Test results: The DOX encapsulation rates in the experimental groups using powers of 100 mW, 200 mW, 300 mW, 400 mW and 500 mW were calculated to be 27.84%, 36.18%, 42.76%, 49.25% and 53.51%, respectively.

[0041] Example 2: Preparation of lipid carriers loaded with a hydrophobic drug molecule BAY2353 (BAY2353-SUVs) by acoustic streaming effect:

[0042] (1) Preparation of BAY2353 water-based solution: In a fume hood, slowly dissolve BAY2353 in UV-sterilized DMSO, mix and stir at room temperature to prepare a 10 mM BAY2353 solution. Mix the resulting solution with ultrapure water sterilized by UV at a volume ratio of 1:500, and shake thoroughly on a shaker until the solution is completely clear to obtain a BAY2353 water-based solution for later use;

[0043] (2) Preparation of micron-sized DOPC liposomes: Weigh 50 mg of DOPC powder in a fume hood and place it in a brown glass bottle. Slowly inject 5 mL of chloroform into the brown glass bottle and slowly shake the brown glass bottle at the same time until all the DOPC powder is dissolved to prepare a 10 mg / mL DOPC chloroform solution. Use a microinjector to transfer 100 μL of DOPC solution into a new, unused small glass bottle, tilt the bottle and slowly rotate the glass bottle, while passing nitrogen into the bottle to evaporate the chloroform and form a uniform DOPC lipid film on the inner wall of the glass bottle. Place the glass bottle in a vacuum box and evacuate for 12 hours to completely evaporate the chloroform. Then, take 1 mL of ultrapure water that has been sterilized by ultraviolet light in the fume hood and place it in the glass bottle with the DOPC lipid film, and quickly shake it on a shaker, then let it stand at room temperature for 2 hours to allow the phospholipids to fully swell to obtain a DOPC liposome suspension;

[0044] (3) Preparation of nanoscale drug lipid carrier BAY2353-SUVs: The DOPC liposome suspension prepared in step (2) and the BAY2353 solution prepared in step (1) were mixed at a volume ratio of 1:1 and placed in an acoustic flow chamber. An electrical signal was turned on to operate the gigahertz bulk acoustic wave resonator and generate acoustic flow to promote the enrichment of BAY2353 in the DOPC liposomes. The output powers of 100 mW, 200 mW, 300 mW, 400 mW and 500 mW were applied for 10 min respectively to obtain a liposome suspension encapsulating BAY2353. The suspension was then placed in an extruder, and a filter membrane with a pore size of 100 nm was used in the extruder. The extruder was repeatedly pushed 11 times so that the liposomes were reorganized into BAY2353-SUVs with a diameter of 100 nm. The BAY2353-SUVs solution was placed in a dialysis bag and dialyzed with ultrapure water for 12 hours to precipitate the drug molecules remaining in the extracellular fluid of the vesicles, and finally a lipid carrier solution loaded with BAY2353 was obtained. The test results of drug loading at different powers are shown in Figure 2. Figure 4 shown.

[0045] (4) Test results: The loading rates of BAY2353 in the experimental groups using output powers of 100 mW, 200 mW, 300 mW, 400 mW and 500 mW were calculated to be 18.43%, 21.78%, 25.62%, 31.49% and 36.95%.

[0046] Example 3: Preparation of lipid carriers loaded with two different hydrophilic and hydrophobic drug molecules, DOX and BAY2353 (DOX-BAY2353-SUVs) by acoustic flow stimulation:

[0047] (1) Preparation of DOX-BAY2353 solution: In a fume hood, slowly dissolve DOX in ultrapure water after UV sterilization, mix and stir at room temperature to prepare a DOX solution with a concentration of 80 μM. Slowly dissolve BAY2353 in DMSO after UV sterilization, mix and stir at room temperature to prepare a BAY2353 solution with a concentration of 10 mM. Mix the resulting solution with the DOX solution at a volume ratio of 1:500, and shake thoroughly on a shaker until the solution is completely clear to obtain a DOX-BAY2353 solution for use;

[0048] (2) Preparation of micron-sized DOPC liposomes: Weigh 50 mg of DOPC powder in a fume hood and place it in a brown glass bottle. Slowly inject 5 mL of chloroform into the brown glass bottle and slowly shake the brown glass bottle until all the DOPC powder is dissolved to prepare a 10 mg / mL DOPC chloroform solution. Slowly introduce argon gas into the bottle and quickly tighten the bottle cap. Use a sealing film to seal the gap in the bottle cap and place it in a -20°C refrigerator. Use a microinjector to transfer 100 μL of DOPC solution into a new, unused small glass bottle, tilt the bottle and slowly rotate the glass bottle while introducing nitrogen gas into the bottle to evaporate the chloroform and form a uniform DOPC lipid film on the inner wall of the glass bottle. Place the glass bottle in a vacuum box and evacuate it for 12 hours to completely evaporate the chloroform. Then, 1 mL of ultrapure water sterilized by ultraviolet light was taken in a fume hood and placed in a glass bottle with DOPC lipid film, and then shaken on a shaker, and then left to stand at room temperature for 2 hours to allow the phospholipids to swell fully, so as to obtain a DOPC liposome suspension;

[0049] (3) Preparation of nanoscale drug lipid carrier DOX-BAY2353-SUVs: The DOPC liposome suspension prepared in step (2) and the DOX-BAY2353 solution prepared in step (1) were mixed at a volume ratio of 1:1 and placed in an acoustic flow chamber. An electrical signal was turned on to operate the gigahertz bulk acoustic wave resonator and generate acoustic flow to promote the enrichment of DOX and BAY2353 in the DOPC liposomes. After the liposome suspension encapsulated with DOX and BAY2353 was obtained at an output power of 500 mW for 10 minutes, the liposome suspension was placed in an extruder, and a filter membrane with a pore size of 100 nm was used in the extruder. The extruder was repeatedly pushed 11 times so that the liposomes were reorganized into DOX-BAY2353-SUVs with a diameter of 100 nm. The DOX-BAY2353-SUVs solution was placed in a dialysis bag and dialyzed with ultrapure water for 12 h to precipitate the drug molecules remaining in the extraliposome fluid, and finally a lipid carrier solution loaded with DOX and BAY2353 was obtained.

[0050] (4) Test results: The calculated encapsulation rate of DOX under this method is 47.63%; the encapsulation rate of BAY2353 is 31.35%.

[0051] Example 4: Drug molecules (DOX and BAY2353) with different hydrophilicity were loaded sequentially to form lipid carriers (DOX-BAY2353-SUVs) by acoustic flow stimulation:

[0052] (1) Preparation of DOX solution: DOX was slowly dissolved in ultrapure water after UV sterilization in a fume hood, mixed and stirred at room temperature to prepare a DOX solution with a concentration of 80 μM, and placed in a 4°C refrigerator for later use;

[0053] (2) Preparation of micron-sized DOPC liposomes: Weigh 50 mg of DOPC powder in a fume hood and place it in a brown glass bottle. Slowly inject 5 mL of chloroform into the brown glass bottle and slowly shake the brown glass bottle at the same time until all the DOPC powder is dissolved to prepare a 10 mg / mL DOPC chloroform solution. Use a microinjector to transfer 100 μL of DOPC solution into a new, unused small glass bottle, tilt the bottle and slowly rotate the glass bottle, while passing nitrogen into the bottle to evaporate the chloroform and form a uniform DOPC lipid film on the inner wall of the glass bottle. Place the glass bottle in a vacuum box and evacuate for 12 hours to completely evaporate the chloroform. Then, take 1 mL of ultrapure water that has been sterilized by ultraviolet light in the fume hood and place it in the glass bottle with the DOPC lipid film, shake it evenly on a shaker, and then let it stand at room temperature for 2 hours to allow the phospholipids to fully swell to obtain a DOPC liposome suspension;

[0054] (3) Preparation of nanoscale drug lipid carrier DOX-BAY2353-SUVs: The DOPC liposome suspension prepared in step (2) and the DOX solution prepared in step (1) were mixed at a volume ratio of 1:1 and placed in an acoustic flow chamber. An electrical signal was turned on to make the gigahertz body acoustic wave resonator work and generate acoustic flow to promote the enrichment of DOX into the DOPC liposomes. The power was applied at 500 mW for 10 minutes to obtain a liposome suspension encapsulating DOX. Then, BAY2353 was slowly dissolved in DMSO after UV sterilization in a fume hood, mixed and stirred at room temperature to prepare a BAY2353 solution with a concentration of 10 mM. The obtained solution was mixed with the liposome suspension encapsulating DOX at a volume ratio of 1:500 and placed on a shaker to be fully shaken. Subsequently, an electrical signal was turned on to make the gigahertz body acoustic wave resonator work and generate acoustic flow to promote the enrichment of BAY2353 into the liposome encapsulating DOX. The liposome suspension encapsulating DOX and BAY2353 was obtained by applying an output power of 500 mW for 10 minutes. The obtained liposome suspension was then placed in an extruder, and a filter membrane with a pore size of 100 nm was used in the extruder. The extruder was repeatedly pushed 11 times so that the liposomes were reorganized into DOX-BAY2353-SUVs with a diameter of 100 nm. The DOX-BAY2353-SUVs solution was placed in a dialysis bag and dialyzed with ultrapure water for 12 hours to precipitate the drug molecules remaining in the extracellular fluid of the vesicles, and finally a lipid carrier solution loaded with DOX and BAY2353 was obtained.

[0055] (4) Test results: The calculated encapsulation rate of DOX under this method is 40.88%; the encapsulation rate of BAY2353 is 37.26%.

[0056] Example 5: Analysis of the effect of acoustic flow stimulation on liposome loading rate

[0057] By comparing the drug loading rates of Examples 1 to 4, it can be seen that the application of acoustic flow stimulation can promote the loading of water-soluble and fat-soluble drugs into liposomes, and the corresponding drug loading rate can be increased by 4 times. From Examples 1 and 2, it can be seen that the liposome drug loading rate is positively correlated with the action power of the GHz BAW resonator, and quantitative drug loading can be achieved by adjusting the power.

[0058] In order to better illustrate the purpose and advantages of the present invention, the advantages of the present invention are further described below in conjunction with comparative examples.

[0059] Comparative Example 1: Preparation of lipid carriers (DOX-SUVs) loaded with a hydrophilic drug molecule DOX by hydration method without using the acoustic flow effect generated by the device

[0060] (1) Preparation of DOX solution: DOX was slowly dissolved in ultrapure water after UV sterilization in a fume hood, mixed and stirred at room temperature to prepare a DOX solution with a concentration of 40 μM, and placed in a 4°C refrigerator for later use;

[0061] (2) Preparation of micron-sized DOPC liposomes: Weigh 50 mg of DOPC powder in a fume hood and place it in a brown glass bottle. Slowly inject 5 mL of chloroform into the brown glass bottle and slowly shake the brown glass bottle until all the DOPC powder is dissolved to prepare a 10 mg / mL DOPC chloroform solution. Use a microinjector to transfer 100 μL of DOPC solution into a new, unused small glass bottle, tilt the bottle and slowly rotate the glass bottle, while passing nitrogen into the bottle to evaporate the chloroform and form a uniform DOPC lipid film on the inner wall of the glass bottle. Place the glass bottle in a vacuum box and evacuate for 12 hours to completely evaporate the chloroform. Then use the hydration method to prepare micron-sized liposomes encapsulating DOX: In a fume hood, take 1 mL of the doxorubicin hydrochloride solution prepared in step (1) into the glass bottle that forms the DOPC lipid film, and slowly shake it on a shaker, then let it stand at room temperature for 2 hours to allow the phospholipids to fully swell to obtain a liposome suspension containing DOX;

[0062] (3) Preparation of nanoscale drug lipid carrier DOX-SUVs: The liposome suspension prepared in step (3) was placed in an extruder, and a filter membrane with a pore size of 100 nm was used in the extruder. The extruder was repeatedly pushed 11 times so that the liposomes were reorganized into SUVs with a diameter of 100 nm. The DOX-SUVs solution was placed in a dialysis bag and dialyzed with ultrapure water for 12 hours to precipitate the drug molecules remaining in the extracellular fluid of the vesicles, and finally a lipid carrier solution loaded with DOX was obtained.

[0063] (4) Test results: The DOX encapsulation rate under this method was calculated to be 12.46%. Compared with Example 1, the encapsulation rate of Example 1 was significantly higher than that of the control example.

[0064] Comparative Example 2: Preparation of lipid carriers loaded with a hydrophobic drug molecule BAY2353 (BAY2353-SUVs) by hydration without using the acoustic streaming effect generated by the device

[0065] (1) Preparation of DOPC-BAY2353 solution: Weigh 50 mg of DOPC powder in a fume hood and place it in a brown glass bottle. Slowly inject 5 mL of chloroform into the brown glass bottle and slowly shake the brown glass bottle at the same time until all the DOPC powder is dissolved to prepare a 10 mg / mL DOPC solution. Afterwards, weigh a certain amount of BAY2353 and slowly dissolve it in the newly obtained DOPC solution so that the final concentration of BAY2353 is 20 μM, and finally prepare a DOPC-BAY2353 solution. Slowly pass argon gas into the bottle and quickly tighten the bottle cap. Use sealing film to seal the gap in the bottle cap and place it in a -20°C refrigerator for use;

[0066] (2) Preparation of nano-DOPC-BAY2353 liposomes: In a fume hood, use a microinjector to transfer 100 μL of the DOPC-BAY2353 solution prepared in step (1) into a new, unused small glass bottle, tilt the bottle and slowly rotate the bottle while introducing nitrogen into the bottle to evaporate the chloroform and form a uniform DOPC-BAY2353 lipid film on the inner wall of the glass bottle. Place the glass bottle in a vacuum box and evacuate for 12 hours to completely evaporate the chloroform. Then, take 1 mL of ultrapure water sterilized by ultraviolet light in the fume hood and place it in the glass bottle with the DOPC-BAY2353 lipid film formed, shake it on a shaker, and then let it stand at room temperature for 2 hours to allow the phospholipids to fully swell, so as to obtain a liposome suspension encapsulating BAY2353. Place the suspension in an extruder and perform 11 extrusions using a filter membrane with a pore size of 100 nm to obtain BAY2353-SUVs with a diameter of 100 nm. The BAY2353-SUVs solution was placed in a dialysis bag and dialyzed with ultrapure water for 12 h to precipitate the drug molecules remaining in the extracellular fluid of the vesicles, and finally a BAY2353-SUVs solution was prepared.

[0067] (3) Test results: The inclusion rate of BAY2353 under this method was calculated to be 9.58%. Compared with Example 2, the inclusion rate of Example 2 was significantly higher than that of the control example.

[0068] In summary, using the drug loading method provided by the present invention, the encapsulation rate for a single water-soluble drug can reach 53.51%, and the encapsulation rate for a single fat-soluble drug can reach 36.95%. In the combined encapsulation of water-soluble and fat-soluble drugs, the encapsulation rate of water-soluble drugs can reach 47.63%, and the encapsulation rate of fat-soluble drugs can reach 31.35%. In the control group, the encapsulation rate of water-soluble drugs was 12.46%, and the encapsulation rate of fat-soluble drugs was 9.58%, and it was difficult to achieve the purpose of encapsulating water-soluble and fat-soluble drugs at the same time. Therefore, the use of the drug loading method provided by the present invention can greatly improve the encapsulation rate of water-soluble and fat-soluble drugs in liposomes.

[0069] The specific description above further illustrates the purpose, technical solutions and beneficial effects of the invention in detail. It should be understood that the above is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for using a gigahertz bulk acoustic wave resonator for active drug loading in liposomes, characterized in that: The following steps are included: Step 1, dissolving drug molecules in a solvent sterilized by ultraviolet light to obtain a drug solution to be loaded; preparing a liposome suspension; and then mixing the drug solution to be loaded with the liposome suspension to obtain a mixed solution; Step 2, placing the mixed solution on the surface of a gigahertz bulk acoustic wave resonator; The resonator changes the permeability of liposomes to achieve effective drug loading, thereby obtaining micron-sized liposome drugs encapsulating target drug molecules.

2. The method according to claim 1, characterized in that: The method for preparing the micron-sized liposome drug obtained in step 2 into a nano-sized drug is to make the micron-sized liposome pass through a filter membrane with a diameter of 100 nm by extrusion, and convert it into a drug lipid carrier with a uniform size and a phospholipid bilayer structure with a diameter of 100 nm.

3. The method according to claim 1, characterized in that: The method for preparing a liposome suspension comprises dissolving 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPC in an organic solvent and preparing liposomes with a diameter of micrometer order by a molecular self-assembly method.

4. The method according to claim 1, characterized in that: The drug molecules include at least one of water-soluble drug molecules and fat-soluble drug molecules.

5. The method according to claim 4, characterized in that: Water-soluble drug molecules are dissolved in ultrapure water after UV sterilization; fat-soluble drugs are dissolved in water-based solvents added with organic co-solvents after UV sterilization.

6. The method according to claim 1, characterized in that: The drug molecules include at least one of water-soluble drug molecules and fat-soluble drug molecules.

7. The method according to claim 6, characterized in that: Water-soluble drug molecules are dissolved in ultrapure water after UV sterilization; fat-soluble drugs are dissolved in water-based solvents with added cosolvents after UV sterilization.

8. The method according to claim 1, characterized in that: The BAW resonator uses a BAW resonator having a resonance frequency of 2.5 GHz.

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