Ultrasound-responsive lipid nanoparticle carrying gene and cell membrane, preparation method therefor, and use thereof

Abstract

An ultrasound-responsive lipid nanoparticle carrying a gene and a cell membrane, a preparation method therefor, and the use thereof. The lipid nanoparticle can carry both a small-molecular nucleic acid and a cell membrane and can serve as an in-vivo gene transfection vector. The surface of said lipid nanoparticle is linked to the small-molecular nucleic acid on the basis of the charge attraction principle, and a cell membrane component is mixed in a surface lipid layer component thereof; in addition, PFP coated in the nanoparticle can undergo a liquid-gas phase change under ultrasonic irradiation, and, explodes under continuous ultrasonic irradiation, the generated explosion energy forms reversible sonopores in the surface of the cell membrane, and under the pushing by an explosion driving force, a drug released after the explosion directly enters the interior of a cell via the sonopores, thereby achieving efficient intracellular nucleic acid release and transfection.

Description

Gene- and cell membrane-carrying ultrasound-responsive lipid nanoparticles, preparation method, and application Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, in particular to a gene-carrying and cell membrane ultrasound-responsive lipid nanoparticle, a preparation method, and applications of the gene-carrying and cell membrane ultrasound-responsive lipid nanoparticle. Background Art

[0002] Small nucleic acid drugs are generally composed of less than 30 bases or base pairs, and mainly target mRNA to regulate protein expression, including siRNA, aptamers, microRNA (miRNA), etc. They have the advantages of short preclinical research and development cycle, high specificity, and high efficiency, and can achieve precise treatment of diseases at the genetic level. However, small molecule nucleic acid drugs are unstable in the body circulation and easily degraded. If precise transfection in vivo is to be achieved, it is necessary to design and construct a targeting vector. Currently, commonly used vectors mainly include two categories: viral vectors and non-viral vectors. Among them, viral vectors have high transfection efficiency but poor application safety, and do not have targeting, and clinical transformation is limited. Non-viral vectors include cationic complexes such as cationic liposomes and polyethyleneimine. They mainly use the charge adsorption principle to adsorb nucleic acids on the surface of the vector to form a stable carrier. However, these types of nanocarriers generally lack the specific targeting function of specific regulatory cells in the disease area, and after binding and engulfing the cells, they cannot avoid lysosomal engulfment and digestion, resulting in low final transfection efficiency. If precise disease treatment based on small nucleic acid drugs is to be achieved, the nanocarriers must not only have the ability to break through biological barriers in the body and reach the diseased area, but also have the ability to release nucleic acids safely and efficiently into cells.

[0003] Ultrasound-targeted microbubble destruction (UTMD) is a new and rapidly developing method for mediated gene transfection in recent years. When microbubbles or ultrasonic phase-change nanoparticles are used as carriers, the gas cores formed inside the carriers can explode and release energy outward under the regional pressure changes generated by the acoustic field, instantly forming reversible holes on the cell membrane surface, forming a "sonoporation effect". The nucleic acids carried inside or on the surface can directly enter the cell through the sonoporation under the driving force generated by the explosion. At the same time, this method uses focused ultrasound irradiation. Due to the directionality of ultrasound, directional irradiation of specific tissue areas can be achieved, and a targeted acoustic field can be initially formed in the disease area. Combined with the specific targeting ability of microbubbles, precise intracellular gene transfection can be achieved, which has higher safety than electroporation.

[0004] Currently, microbubbles and ultrasonic phase-change nanoparticles still primarily rely on charge adsorption to deliver small nucleic acid drugs. Cationic gene carriers readily adsorb proteins in the blood, leaving room for improvement in their blood compatibility. They are also easily captured and cleared by immune cells, resulting in a smaller number of target cells being reached. Biomimetic nanotechnology has been another breakthrough in nano-drug delivery technology in recent years. Biomimetic nanoparticles, a technique that uses cell membranes to encapsulate gene- or drug-loaded nanoparticles, can alter the nano-biointerface properties through surface cell membrane coatings, mimicking the biological behavior of endogenous cells mediated by cell membranes to achieve various diagnostic and therapeutic purposes. Furthermore, endogenous cell membranes can cover the nanoparticle surface phagocytic and immune system recognition sites, significantly extending the nanoparticle's blood circulation time.

[0005] However, gene delivery using biomimetic nanotechnology combined with UTMD has yet to be reported. The primary reason is that nucleic acids, as water-soluble materials, cannot dissolve in the liquid fluorocarbon encapsulated within nanoparticles or the gas encapsulated within microbubbles. Therefore, cationic lipid nanoparticles must be prepared to utilize charge adsorption to adsorb microRNA onto the nanoparticle surface. However, biomimetic nanoparticle preparation involves coating the nanoparticle surface with a cell membrane, a process that requires a negative surface potential on the nanoparticle core. When using cationic nanoparticles as the core to prepare biomimetic nanoparticles, charge adsorption cross-links the nanoparticles and cell membranes during mixing, forming complex complexes. This makes it impossible to produce biomimetic nanoparticles with a stable core-shell structure and uniform shell thickness. Therefore, new strategies are needed to improve the preparation and functionality of small nucleic acid drug carriers.

[0006] Summary of the Invention

[0007] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a method for preparing gene- and cell membrane-carrying ultrasound-responsive lipid nanoparticles, which can simultaneously carry small molecule nucleic acids and cell membranes and can be used as in vivo gene transfection vectors.

[0008] The technical solution of the present invention is: this kind of gene- and cell membrane-carrying ultrasound-responsive lipid nanoparticles has a surface connected to small-molecule nucleic acids through the principle of charge adsorption, the surface lipid layer components are mixed with cell membrane components, the nanoparticles are wrapped with PFP, and a liquid-gas phase transition occurs under ultrasonic irradiation. Under continuous ultrasonic irradiation, the explosion energy generates a reversible sound hole on the cell membrane surface. The drug released after the explosion is driven by the explosion driving force and directly enters the cell through the sound hole, thereby achieving efficient release and transfection of nucleic acids in the cell.

[0009] Also provided is a method for preparing gene- and cell membrane-carrying ultrasound-responsive lipid nanoparticles, which comprises the following steps:

[0010] (1) Preparation of cationic ultrasonic phase change nanoparticles: Distearoylphosphatidylcholine DSPC, distearoylphosphatidylethanolamine DSPE, and 1,2-dioleoyl-3-trimethylammonium-propane DOTAP were mixed and dissolved in dichloromethane, and placed in a rotary evaporator to obtain a uniform translucent film, and ultrapure water was added. The film was placed in an ultrasonic cleaning machine to completely elute the film to form a homogeneous translucent suspension. The suspension was transferred to a centrifuge tube, pre-cooled on ice, and ultrasonic phase change material was added. The translucent suspension obtained after ultrasonic oscillation became an opaque emulsion to obtain a nanoparticle primary emulsion. After multiple low-temperature high-speed centrifugation, excess impurities were removed to obtain purified cationic ultrasonic phase change nanoparticles.

[0011] (2) Preparation of ultrasonic phase-change nanoparticles loaded with small nucleic acid drugs: The cationic ultrasonic phase-change nanoparticles prepared in step (1) are mixed with small molecule nucleic acids and incubated on ice. The nucleic acids are attached to the surface of the lipid nanoparticles by charge adsorption, and charge reversal is simultaneously formed on the surface of the lipid nanoparticles to prepare ultrasonic phase-change nanoparticles loaded with small nucleic acid drugs;

[0012] (3) Preparation of biomimetic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs: The cell membrane is mixed with the ultrasonic phase change nanoparticles loaded with small nucleic acid drugs obtained in step (2), and the cell membrane components are added to the lipid shell components of the nanoparticles by using the lipid membrane fluidity through the sonication method. The mixture is placed in an ice bath, and PFP is added thereto again. After ultrasonic oscillation, biomimetic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs are obtained. After multiple low-temperature high-speed centrifugation, excess impurities are removed to obtain purified biomimetic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs.

[0013] The present invention uses cationic liposomes as the core, reverses the surface charge of nanoparticles through charge adsorption, utilizes the fluidity of the cell membrane lipid layer, breaks and reorganizes the nanoparticles through ultrasonic vibration, and integrates the cell membrane on the surface of the gene-carrying nanoparticles, ultimately preparing gene-carrying bionic ultrasonic phase-change nanoparticles, which can carry small molecule nucleic acids and cell membranes at the same time and can be used as in vivo gene transfection vectors.

[0014] Also provided is the use of the gene-carrying and cell membrane ultrasound-responsive lipid nanoparticles in the targeted treatment of acute myocardial infarction, acute rejection after heart transplantation, breast cancer lung metastasis, and colon cancer liver metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG1 shows a transmission electron microscope structure photograph of the miR-125b-loaded biomimetic ultrasonic phase-change nanoparticles obtained in Example 3 of the present invention and the particle size potential detection results.

[0016] FIG2 shows the miR-125b and cell membrane carrying efficiency detection results obtained by flow cytometry detection of miR-125b-loaded biomimetic ultrasonic phase-change nanoparticles obtained in Example 3 of the present invention.

[0017] FIG3 shows the test results of the phase change ability of the miR-125b-loaded biomimetic ultrasonic phase-change nanoparticles obtained in Example 3 of the present invention at different LIFU irradiation times.

[0018] FIG4 shows the detection of the targeting ability of the miR-125b-loaded biomimetic ultrasonic phase-change nanoparticles obtained in Example 3 of the present invention to the area surrounding the infarcted myocardium in vivo.

[0019] FIG5 is a flow chart of the method for preparing gene-carrying and cell membrane ultrasound-responsive lipid nanoparticles according to the present invention. DETAILED DESCRIPTION

[0020] This gene-carrying and cell membrane ultrasound-responsive lipid nanoparticle has a surface that connects small-molecule nucleic acids through the principle of charge adsorption, and cell membrane components are mixed into the surface lipid layer components. PFP is wrapped inside the nanoparticle, and a liquid-gas phase transition occurs under ultrasonic irradiation. It explodes under continuous ultrasonic irradiation, and the explosion energy generated forms a reversible sound hole on the cell membrane surface. The drug released after the explosion enters the cell directly through the sound hole under the driving force of the explosion, thereby realizing the efficient release and transfection of nucleic acids in the cell.

[0021] The present invention uses cationic liposomes as the core, reverses the surface charge of nanoparticles through charge adsorption, utilizes the fluidity of the cell membrane lipid layer, breaks and reorganizes the nanoparticles through ultrasonic vibration, and integrates the cell membrane on the surface of the gene-carrying nanoparticles, ultimately preparing gene-carrying bionic ultrasonic phase-change nanoparticles, which can carry small molecule nucleic acids and cell membranes at the same time and can be used as in vivo gene transfection vectors.

[0022] Preferably, the lipid nanoparticle components include three lipid components: distearoylphosphatidylcholine DSPC, distearoylphosphatidylethanolamine DSPE, and 1,2-dioleoyl-3-trimethylammonium-propane DOTAP, among which 1,2-dioleoyl-3-trimethylammonium-propane DOTAP carries cationic components; the cell membranes selected are erythrocyte membranes, macrophage membranes, stem cell membranes, neutrophil membranes, and tumor cell membranes; the small nucleic acid drugs selected are siRNA, antisense oligonucleotides ASO, aptamers Aptamer, microRNA miRNA, and small activating RNA saRNA.

[0023] As shown in FIG5 , a method for preparing gene- and cell membrane-carrying ultrasound-responsive lipid nanoparticles is also provided, which comprises the following steps:

[0024] (1) Preparation of cationic ultrasonic phase change nanoparticles: Distearoylphosphatidylcholine DSPC, distearoylphosphatidylethanolamine DSPE, and 1,2-dioleoyl-3-trimethylammonium-propane DOTAP were mixed and dissolved in dichloromethane, and placed in a rotary evaporator to obtain a uniform translucent film, and ultrapure water was added. The film was placed in an ultrasonic cleaning machine to completely elute the film to form a homogeneous translucent suspension. The suspension was transferred to a centrifuge tube, pre-cooled on ice, and ultrasonic phase change material was added. The translucent suspension obtained after ultrasonic oscillation became an opaque emulsion to obtain a nanoparticle primary emulsion. After multiple low-temperature high-speed centrifugation, excess impurities were removed to obtain purified cationic ultrasonic phase change nanoparticles.

[0025] (2) Preparation of ultrasonic phase-change nanoparticles loaded with small nucleic acid drugs: The cationic ultrasonic phase-change nanoparticles prepared in step (1) are mixed with small molecule nucleic acids and incubated on ice. The nucleic acids are attached to the surface of the lipid nanoparticles by charge adsorption, and charge reversal is simultaneously formed on the surface of the lipid nanoparticles to prepare ultrasonic phase-change nanoparticles loaded with small nucleic acid drugs;

[0026] (3) Preparation of biomimetic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs: The cell membrane is mixed with the ultrasonic phase change nanoparticles loaded with small nucleic acid drugs obtained in step (2), and the cell membrane components are added to the lipid shell components of the nanoparticles by using the lipid membrane fluidity through the sonication method. The mixture is placed in an ice bath, and PFP is added thereto again. After ultrasonic oscillation, biomimetic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs are obtained. After multiple low-temperature high-speed centrifugation, excess impurities are removed to obtain purified biomimetic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs.

[0027] Preferably, in step (1), the lipid components distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) have a mass ratio of 10:4:2.

[0028] Preferably, in step (1), the ultrasonic oscillation is set to 90W using an acoustic cell disruptor, for a total of 5 minutes, with a duty cycle of 50%; the rotary evaporator is set to a temperature of 60°C and a rotation time of 1 hour; and repeated centrifugation purification is performed using a low-temperature high-speed centrifuge, with each centrifugation condition set to a speed of 12,000 rpm, a centrifugation time of 15 minutes, and a total of 3 centrifugations.

[0029] Preferably, in step (1), the phase change material added is liquid perfluoropentane PFP, which has a chemical formula of C5F12 and a molecular weight of 288.036 g / mol and is a perfluorinated analogue of pentane. When the ambient temperature reaches 29°C, a liquid-gas phase change occurs.

[0030] Preferably, in step (2), the cationic ultrasonic phase change nanoparticles and the small molecule nucleic acid are co-incubated at a charge ratio of 3:2.

[0031] Preferably, in step (2), the small nucleic acid drug is at least one of siRNA or microRNA; the cationic ultrasonic phase change nanoparticles and the small nucleic acid drug are co-incubated on ice for 30-40 minutes; and the particle size of the cationic ultrasonic phase change nanoparticles is 300-400 nm.

[0032] Preferably, in the step (3), the small nucleic acid drug-loaded ultrasonic phase change nanoparticles are mixed with the cell membrane in a phospholipid to membrane protein mass ratio of 1:1; the amount of PFP added before sonication is 200 μl, and the oil phase is added to increase the success rate of biomimetic nanoparticle preparation; the ultrasonic oscillation is set to 90W using a sonic cell disruptor, the time is 5 minutes, and the duty cycle is 50%; the excess impurities are removed after multiple low-temperature high-speed centrifugation, and the centrifugation conditions are set to 12000 rpm for 15 minutes each time, and a total of 3 centrifugations.

[0033] Also provided is the use of the gene-carrying and cell membrane ultrasound-responsive lipid nanoparticles in the targeted treatment of acute myocardial infarction, acute rejection after heart transplantation, breast cancer lung metastasis, and colon cancer liver metastasis.

[0034] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0035] Example 1 Preparation of mesenchymal stem cell biomimetic nanoparticles

[0036] The preparation of cationic nanoparticles (CNPs) was performed in the dark. A high-precision electronic balance was used to weigh the lipid components, DSPC, DSPE, and DOTAP, to a mass ratio of 10:4:2. Cationic lipid phase-change nanoparticles were synthesized using a thin-film hydration and ultrasonic oscillation method. Dichloromethane was added to a round-bottom flask using a 1ml micropipette. The flask was sealed with a rubber stopper to prevent evaporation of the dichloromethane. An ultrasonic cleaner was used to facilitate complete dissolution of the lipid components. A rotary evaporator was preheated to 50°C until a uniform, translucent film formed at the bottom of the flask. After completion, the flask was removed and placed in an ultrasonic cleaner for oscillation to completely elute the film, resulting in a homogeneous, translucent suspension. The suspension was then cooled on ice and 1ml of the ultrasonic phase-change material, PFP, was added. Set the ultrasonic cell disruptor to 90W for 5 minutes (5 seconds on, 5 seconds off). Place the probe below the liquid level, but away from the bottom of the bottle. Move the probe up and down to ensure uniform vibration. After the sonication ends, the translucent suspension becomes an opaque emulsion, which is the primary nanoparticle emulsion. Divide the nanoparticle emulsion into two 15ml centrifuge tubes and centrifuge them at 12,000 rpm for 15 minutes in a low-temperature high-speed centrifuge. Reconstitute the suspension in ultrapure water after centrifugation. Repeat this process three times to remove excess impurities.

[0037] The morphology of the nanoparticles was observed using an ordinary optical microscope, and the particle size and potential of the nanoparticles were measured using a Malvern laser particle size analyzer.

[0038] The nanoparticle sample appears as a milky white suspension. Preliminary observation of the CNPs in the liquid using an optical microscope revealed spherical shapes with uniform particle size and distribution. A Malvern laser particle size analyzer was used to measure the particle size and potential distribution of the nanoparticles, revealing an average particle size of 300-400 nm and a potential of 30-40 mV.

[0039] Example 2 Preparation of microRNA-loaded cationic nanoparticles

[0040] The cationic nanoparticles obtained in Experimental Example 1 were mixed with microRNA at a charge ratio of 3:2 and incubated on ice for 2 hours to obtain miR-CNPs by electrostatic adsorption. The mixture was centrifuged three times at 12,000 × g to remove unbound miRNA.

[0041] The particle size and potential of the nanoparticles were measured using a Malvern laser particle size analyzer. MicroRNA was labeled with FITC, and the miR-CNPs were diluted 10-fold with ultrapure water. The FITC fluorescence intensity on the surface of the miR-CNPs was then measured using a flow cytometer.

[0042] The average particle size of miR-CNPs is approximately 400 nm, and the average potential is approximately -20 mV. Compared to cationic nanoparticles, the nanoparticle size increases slightly after loading with miRNA, and the surface potential shifts from positive to negative. Flow cytometry analysis showed a microRNA loading efficiency of 92.6% on the nanoparticle surface.

[0043] Example 3 Preparation of Mesenchymal Stem Cell Biomimetic Nanoparticles

[0044] MSCs and macrophage membranes were extracted using the Thermo Fisher Scientific membrane protein extraction kit. Cells were scraped from the bottom of the culture dish using a plastic cell scraper to prepare a cell suspension. The suspension was centrifuged in a refrigerated centrifuge at 300 × g, 4°C, and 5 minutes. The supernatant was removed and the cells were resuspended in cell wash buffer. The centrifugation was repeated under the same conditions, and the supernatant was removed. The washed cell pellet was resuspended in permeabilization buffer and incubated on ice for 10 minutes. The permeabilized cells were centrifuged in a refrigerated high-speed centrifuge at 16,000 × g and 4°C for 15 minutes. The supernatant was removed to obtain the cell membranes. The cell membrane pellet was resuspended in ultrapure water and stored in a -80°C freezer until further use.

[0045] By using the sonication method to utilize the fluidity of the lipid membrane, cell membrane components were added to the nanoparticle lipid shell components to prepare miRNA-loaded biomimetic nanoparticles. 10 mg of the microRNA-loaded cationic nanoparticles prepared in Experimental Example 2 were mixed with the MSC cell membrane and macrophage membrane obtained in the previous step at a phospholipid to membrane protein mass ratio of 1:0.5:0.5 to prepare microRNA-loaded MSCs and macrophage mixed cell membrane biomimetic nanoparticles (miR-MRCNPs). The mixture was placed in an ice bath, and 200 μl of PFP was added to it again. The mixture was sonicated for 3 minutes (60w, on: 5 seconds; off: 5 seconds). The precipitate was collected by centrifugation at 15000 rpm for 15 minutes to obtain the biomimetic nanoparticles. The morphology, particle size distribution, and zeta potential of the biomimetic nanoparticles were measured.

[0046] The results of the Malvern laser particle size analyzer showed that compared with miR-CNPs, the average particle size and absolute value of the potential of the bionic nanoparticles increased, with an average particle size of 500-600nm and an average potential of about -40mV. The results of transmission electron microscopy observations showed that the internal structures of the three nanoparticles were typical core-shell structures.

[0047] Observation and evaluation of the ultrasonic phase transition ability of miR-MRCNPs

[0048] Bionic nanoparticles were added to the agarose gel holes, and a LIFU probe was applied with coupling agent and placed on the side wall of the gel hole. The irradiation parameters were set to 2W / cm2, focal length 1.5cm, duty cycle 50%, and pulse wave mode. The results showed that the acoustic intensity of the nanoparticles gradually increased with the extension of irradiation time, reaching the highest level after 4 minutes of irradiation.

[0049] Verification of cell membrane fusion and detection of cell membrane nanoparticle co-localization

[0050] DiO and DiR lipid-labeled probes were used to label the cell membranes of MSCs and macrophages, respectively, and the morphology and surface fluorescence labeling of the biomimetic nanoparticles were observed under a fluorescence microscope. The mixed cell membrane biomimetic nanoparticles were incubated with mouse endothelial cells for 2 hours, and the cell nuclei were counterstained with DAPI. Fluorescence colocalization was observed under a laser confocal microscope. The results showed that the biomimetic nanoparticles surrounding the cell nucleus could simultaneously detect green fluorescence from the cell membrane and red fluorescence from the nanoparticle lipid components, demonstrating good fusion efficiency between the cell membrane and the nanoparticles. The expression of specific proteins was detected by Coomassie Brilliant Blue, and the expression of three functional cell membrane proteins, CD49, CD47, and CD29, on the surfaces of MSCs and RAW 264.7 cells was detected by Western blotting. The results showed that CD29, CD49, and CD47 proteins were all expressed on the cell membranes of the biomimetic nanoparticles.

[0051] Detection of miR-MRCNPs' phagocytic escape ability in macrophages in vitro

[0052] DiO lipophilic dye was used to label CNPs, miR-CNPs, miR-MCNPs, and miR-MRCNPs.

[0053] Macrophages were seeded with 200 μl of nanoparticles in a 24-well plate, and 200 μl of nanoparticles were added to each well for incubation for 1 hour. Macrophages had a higher uptake efficiency of CNPs than other nanoparticles. Biomimetic nanoparticles modified with macrophage membranes had a higher ability to escape macrophages than pure MSC-inspired nanoparticles. These results confirm that modifying the biological interface can enhance the ability of nanoparticles to escape the phagocytic system.

[0054] Detection of adhesion ability of miR-MRCNPs to inflammatory endothelial cells and cardiomyocytes in vitro

[0055] Endothelial and cardiomyocytes were seeded in 24-well plates. 50 ng / mL of the inflammatory factor TNF-α was added to each well and incubated at a constant temperature for 24 hours to activate the endothelial and cardiomyocytes. Then, 100 μg of four nanoparticles, namely CNPs, miR-CNPs, miR-MCNPs, and miR-MRCNPs, were added and incubated for 1 hour. A large number of CNPs adhered to the surfaces of the inflammatory-activated cells, while the adhesion rate of the biomimetic nanoparticles was relatively low. No significant difference was observed between the miR-MRCNP and miR-MCNP groups.

[0056] Detection of the transfection ability and anti-apoptosis ability of myocardial and endothelial cells of miR-MRCNPs combined with UTMD in vitro

[0057] Endothelial cells and cardiomyocytes cultured with TNF-α were seeded in 24-well plates. The LIFU irradiation conditions were set at 1.03 MHz, 50% duty cycle, 2 W / cm2, and 10 minutes. Fluorescence microscopy was used to observe the intracellular miRNA fluorescence distribution 24 hours after transfection. The results showed that the green fluorescence signal of microRNA overlapped with the red fluorescence range representing the cytoskeleton, proving that miR-MRCNPs combined with UTMD were successfully transfected into cardiomyocytes and endothelial cells.

[0058] Example 4 In vivo efficacy and safety evaluation of miR-MRCNPs biomimetic ultrasonic phase-change nanoparticles

[0059] Male C57BL / 6 mice, 6-8 weeks old, were treated with an acute myocardial infarction model established by left anterior descending artery (LAD) ligation. 0.2 ml of microRNA-inspired phase-change nanoparticles were injected intravenously and irradiated in the precordial area using a low-frequency ultrasound probe coated with coupling agent. Irradiation parameters were 2 W / cm², a focal length of 1.5 cm, a 50% duty cycle, and pulsed wave mode for 10 minutes. Treatment was administered to mice 1, 3, 5, and 7 days after myocardial infarction, according to group. Treatment significantly reduced myocardial infarction size, significantly improved cardiac function, and Masson staining revealed a significant decrease in fibrosis. Immunofluorescence staining revealed an increase in endothelial density in the perimyocardial area.

[0060] The results of every other week body weight test of mice showed that the weight of mice in each group increased steadily, and there was no significant difference in the organ-to-body weight ratio of the four major organs: liver, kidney, spleen and lung. The main organs of the mice were sectioned and pathologically examined, and the results showed that the main organs in the bodies of mice in each group had uniform texture, and no edema or substantial lesions were observed.

[0061] Mice were randomly divided into four groups (n=30) according to the type of nanoparticle injection received via the tail vein: ① CNPs group; ② miR-CNPs group; ③ miR-MCPNs group; and ④ miR-MRCNPs group. Blood was collected from the medial canthal vein of each group 5 minutes and 1, 2, 4, 6, 8, 12, 24, and 48 hours after nanoparticle injection. The supernatant was collected after centrifugation at 12,500 rpm for 15 minutes. Absorbance at 635 / 680 nm was measured, demonstrating a significant increase in the nanoparticles' in vivo circulation time.

[0062] DiO lipid fluorescent probes were used to label the phospholipid components of four types of nanoparticles: CNPs, miR-CNPs, miR-MCPNs, and miR-MRCNPs. Three days after infarction, 200 μl of nanoparticles were injected into each mouse through the tail vein. One, eight, and 24 hours after injection, the mice in each group were anesthetized and perfused cardiacally. The lungs, liver, spleen, and kidneys were removed, and the distribution of the nanoparticles was detected using an in vivo fluorescence imaging system. The results showed that the fluorescence of the nanoparticles in the miR-CNPs, miR-MCNPs, and miR-MRCNPs groups was strong in the lungs at the beginning after injection, and then gradually increased in the liver and kidneys. By the 24th hour, the fluorescence signal in the miR-CNPs group was basically undetectable in all organs. In the biomimetic nanoparticle group, miR-MCNPs and miR-MRCNPs still had strong fluorescence signals in the liver 24 hours later, but the miR-MRCNPs group had stronger fluorescence signals than the miR-MCNPs group, indicating that the nanoparticles lasted longer in the body and had better stability than the non-bionic nanoparticles.

[0063] Each group of mice was injected with nanoparticles, and the mice were killed 10, 30 and 60 minutes after injection. The right atrial appendage of the mice was cut off, and normal saline was injected into the myocardium through the apex of the mouse heart. The mice were circulatory perfused to flush out the free nanoparticles in the myocardial microcirculation. The hearts were collected and placed on a small animal living imager to detect the fluorescence distribution and intensity in the heart; DiO and DiR dyes were used to label the surface cell membrane of miR-MRCNPs and the nanoparticle core, respectively, and the co-localization of the two fluorescences in the myocardium was detected using a small animal living imager. It can be seen that fluorescence aggregation was visible in the hearts of mice in the miR-MCNPs group and miR-MRCNPs group 10 minutes after injection, and the fluorescence intensity gradually increased over time.

[0064] After the miR-MRCNPs labeled with DiO and DiR in the previous step were injected into the mouse AMI model through the tail vein, the mice were killed and the heart tissue was perfused and embedded with OCT embedding medium. The obtained myocardial tissue was cut into 8-μm thick slices using a freezing microtome, and the cell nuclei were labeled with DAPI nuclear dye. Under a fluorescence microscope, the green and red fluorescence representing the biomimetic nanoparticles in the myocardium of the peri-infarct area were distributed in a short line-like manner, and the two fluorescence ranges overlapped.

[0065] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of protection of the technical solution of the present invention.

Claims

1. Gene-carrying and cell membrane ultrasound-responsive lipid nanoparticles, characterized in that: Its surface is connected to small molecule nucleic acids through the principle of charge adsorption, cell membrane components are mixed into the surface lipid layer components, PFP is wrapped inside the nanoparticles, liquid-gas phase transition occurs under ultrasonic irradiation, and explosion occurs under continuous ultrasonic irradiation. The explosion energy generated forms reversible sonopores on the cell membrane surface. The drugs released after the explosion enter the cell directly through the sonopores under the driving force of the explosion, realizing efficient release and transfection of nucleic acids in the cells.

2. The gene- and cell membrane ultrasound-responsive lipid nanoparticles according to claim 1, characterized in that: The lipid nanoparticle components include three lipid components: distearoylphosphatidylcholine DSPC, distearoylphosphatidylethanolamine DSPE, and 1,2-dioleoyl-3-trimethylammonium-propane DOTAP, among which 1,2-dioleoyl-3-trimethylammonium-propane DOTAP carries cationic components; the cell membranes selected are erythrocyte membranes, macrophage membranes, stem cell membranes, neutrophil membranes, and tumor cell membranes; the small nucleic acid drugs selected are siRNA, antisense oligonucleotides ASO, aptamers Aptamer, micro RNA miRNA, and small activating RNA saRNA.

3. The method for preparing gene- and cell membrane ultrasound-responsive lipid nanoparticles according to claim 1, characterized in that: It includes the following steps: (1) Preparation of cationic ultrasonic phase change nanoparticles: Distearoylphosphatidylcholine DSPC, distearoylphosphatidylethanolamine DSPE, and 1,2-dioleoyl-3-trimethylammonium-propane DOTAP were mixed and dissolved in dichloromethane, and placed in a rotary evaporator to obtain a uniform translucent film. Ultrapure water was added and placed in an ultrasonic cleaning machine to completely elute the film to form a homogeneous translucent suspension. The suspension was transferred to a centrifuge tube, precooled on ice, and ultrasonic phase change material was added. The translucent suspension obtained after the sonication becomes an opaque emulsion to obtain a primary emulsion of nanoparticles, and after multiple low-temperature high-speed centrifugation, excess impurities are removed to obtain purified cationic ultrasonic phase-change nanoparticles; (2) Preparation of ultrasonic phase change nanoparticles loaded with small nucleic acid drugs: the cationic ultrasonic phase change nanoparticles prepared in step (1) are mixed with small molecule nucleic acids and incubated on ice, the nucleic acids are connected to the surface of lipid nanoparticles by charge adsorption, and charge reversal is formed on the surface of lipid nanoparticles, thereby preparing ultrasonic phase change nanoparticles loaded with small nucleic acid drugs; (3) Preparation of bionic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs: The cell membrane is mixed with the ultrasonic phase change nanoparticles loaded with small nucleic acid drugs obtained in step (2), and the cell membrane components are added to the lipid shell components of the nanoparticles by using the sonication method to utilize the lipid membrane fluidity. The mixture is placed in an ice bath, and PFP is added thereto again. After ultrasonic oscillation, bionic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs are obtained. After multiple low-temperature high-speed centrifugation, excess impurities are removed to obtain purified bionic ultrasonic phase change nanoparticles loaded with small nucleic acid drugs.

4. The method for preparing gene- and cell membrane ultrasound-responsive lipid nanoparticles according to claim 3, characterized in that: In the step (1), the lipid components distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) have a mass ratio of 10:4:

2.

5. The method for preparing gene-carrying and cell membrane ultrasound-responsive lipid nanoparticles according to claim 4, characterized in that: In the step (1), the ultrasonic oscillation is set to 90W using an acoustic cell disruptor, the time is 5 minutes, and the duty cycle is 50%; the rotary evaporator is set to a temperature of 60°C and a rotation time of 1 hour; and a low-temperature high-speed centrifuge is used for repeated centrifugation purification, each centrifugation condition is set to a speed of 12000 rpm, the centrifugation time is 15 minutes, and a total of 3 centrifugations are performed.

6. The method for preparing gene- and cell membrane ultrasound-responsive lipid nanoparticles according to claim 5, characterized in that: In the step (1), the phase change material added is liquid perfluoropentane PFP, which has a chemical formula of C5F12 and a molecular weight of 288.036 g / mol and is a perfluorinated analogue of pentane. When the ambient temperature reaches 29°C, a liquid-gas phase change occurs.

7. The method for preparing gene- and cell membrane ultrasound-responsive lipid nanoparticles according to claim 6, characterized in that: In the step (2), the cationic ultrasonic phase change nanoparticles and the small molecule nucleic acid are co-incubated at a charge ratio of 3:

2.

8. The method for preparing gene- and cell membrane ultrasound-responsive lipid nanoparticles according to claim 7, characterized in that: In the step (2), the small nucleic acid drug is at least one of siRNA or microRNA; the cationic ultrasonic phase change nanoparticles and the small nucleic acid drug are co-incubated on ice for 30-40 minutes; and the particle size of the cationic ultrasonic phase change nanoparticles is 300-400 nm.

9. The method for preparing gene- and cell membrane ultrasound-responsive lipid nanoparticles according to claim 8, characterized in that: In the step (3), the small nucleic acid drug-loaded ultrasonic phase change nanoparticles are mixed with the cell membrane in a ratio of phospholipid to membrane protein of 1:1; 200 μl of PFP is added before sonication, and an oil phase is added to increase the success rate of biomimetic nanoparticle preparation; the ultrasonic oscillation is set to 90W using a sonic cell disruptor, the time is 5 minutes, and the duty cycle is 50%; excess impurities are removed after multiple low-temperature high-speed centrifugation, and the centrifugation conditions are set to a speed of 12000 rpm and a centrifugation time of 15 minutes each time, and a total of 3 centrifugations.

10. The gene-carrying and cell membrane ultrasound-responsive lipid nanoparticles according to claim 1 are used in the targeted treatment of acute myocardial infarction, acute rejection after heart transplantation, lung metastasis of breast cancer and liver metastasis of colon cancer.

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