Ultrasound targeting lipid nanoparticle and application thereof

Abstract

The invention belongs to the field of molecular biology, and particularly relates to lipid nanoparticles capable of achieving ultrasonic targeting and application of the lipid nanoparticles. The ultrasonic response type lipid nanoparticle provided by the invention comprises polyethylene glycol lipid, ionizable cationic lipid and steroid, the molar ratio of the polyethylene glycol lipid to the ionizable cationic lipid to the steroid is (5-20): (5-90): (5-80), and the lipid nanoparticles are loaded with nucleic acid. According to the lipid nanoparticles provided by the invention, the nucleic acid medicine can be subjected to targeted delivery to extrahepatic tissues under ultrasonic guidance, specific expression is realized, and the nucleic acid medicine is almost not expressed in the liver and the spleen.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to lipid nanoparticles capable of ultrasonic targeting and applications thereof. Background Art

[0002] Nucleic acid drugs, including mRNA, DNA, plasmids, sgRNA, circular RNA, siRNA, microRNA, etc., have attracted widespread attention in recent years due to their great potential in the treatment of cancer, genetic diseases and viral diseases. However, the application of these drugs faces the challenge of unstable chemical properties, especially in vitro and in vivo, they are easily degraded into single nucleotides by nucleases, resulting in loss of efficacy. Therefore, in order to ensure the effectiveness of nucleic acid drugs, it is necessary to rely on special carrier systems. Compared with viral vectors, non-viral vectors have become the research focus in the field of nucleic acid drug delivery due to their lower immunogenicity and higher safety. Non-viral vectors usually encapsulate nucleic acid drugs in the carrier through the electrostatic interaction between cationic materials and phosphate ions of nucleic acid drugs, thereby protecting the drugs from degradation and changing their cellular entry pathways. Among all non-viral vectors, lipid nanoparticles (LNPs) are one of the most commonly used carriers.

[0003] The combination of mRNA and lipid nanoparticles has proven to be a transformative technology, with the most typical example being the rapid development and application of the new coronavirus mRNA vaccine. However, in addition to vaccines, the application of mRNA drugs in protein replacement therapy and CRISPR-Cas gene editing still faces major challenges. Unlike mRNA vaccines, mRNA therapy usually requires higher protein expression levels, even up to 1000 times, which brings safety issues such as immunogenicity and toxicity. In addition, how to achieve targeted specific delivery in vivo without off-target expression remains a difficult problem. Intravenously injected lipid nanoparticles interact with serum proteins to form a protein corona, which allows lipid nanoparticles to be guided to organs such as the liver, spleen, and lungs. Although this endogenous targeting has certain benefits for specific organs, it also leads to significant off-target expression of mRNA in these organs. This problem is particularly prominent in mRNA therapy for cells that are not easily delivered through endogenous targeting. Although specificity can be enhanced by adding targeting ligands, off-target expression in the liver and spleen is still difficult to avoid.

[0004] To limit the formation of protein corona and reduce immunogenicity, polyethylene glycol (PEG)-modified nanoparticles have become a well-established technology. PEG lipids are essential for the formation of lipid nanoparticles and prevent their aggregation. However, PEG lipids may also hinder the cellular uptake of nanoparticles, which in turn affects the expression of mRNA. To address this issue, PEG lipids with C14 alkyl chains are often used to deliver mRNA. Such PEG lipids can enhance the stability of LNPs in vitro, and in vivo, their rapid dissociation leads to the formation of a protein corona, which promotes the expression of mRNA in major organs but also triggers immune responses. This effect is beneficial for mRNA vaccines, but for non-vaccine applications, it may cause off-target expression and immunogenicity issues. Although the use of PEG lipids with longer hydrophobic chains (such as C18 chains) can reduce the formation of protein corona, a high proportion of these lipids leads to a significant decrease in the transfection efficiency of lipid nanoparticles. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes to combine lipid nanoparticles prepared with PEG lipids with long hydrophobic chains with ultrasound and microbubbles that respond to ultrasound, and utilizes the sonoporation effect generated by the microbubbles under ultrasonic irradiation to solve the problem of low expression efficiency of nucleic acid drugs such as mRNA caused by long hydrophobic chain PEG lipids, thereby improving the delivery efficiency of nucleic acid drugs and reducing side effects caused by immune response and off-target expression.

[0006] The purpose of the present invention is to provide an ultrasound-responsive lipid nanoparticle.

[0007] Another object of the present invention is to provide a nucleic acid complex containing the lipid nanoparticles.

[0008] Another object of the present invention is to provide an ultrasound-targeted drug based on the above-mentioned complex.

[0009] According to a specific embodiment of the present invention, the ultrasonic responsive lipid nanoparticles are prepared by raw materials including: 1) Polyethylene glycol lipids; 2) ionizable cationic lipids; 3) Steroids; The molar percentage of polyethylene glycol lipid is 5%-20%, the molar percentage of ionizable cationic lipid is 5%-90%, and the molar percentage of steroid is 5%-80%; The lipid nanoparticles are loaded with nucleic acids, and the nucleic acids are selected from one or more of mRNA, DNA, plasmid, sgRNA, circular RNA, siRNA, and microRNA.

[0010] According to the lipid nanoparticles of a specific embodiment of the present invention, the polyethylene glycol lipid contains two saturated carbon chains of no less than 18 carbons.

[0011] According to the lipid nanoparticles of a specific embodiment of the present invention, the polyethylene glycol lipid is selected from one or more of DSPE-PEG2000, DSPE-PEG5000, and DSG-PEG2000.

[0012] According to the lipid nanoparticles of a specific embodiment of the present invention, the ionizable cationic lipid is a type of lipid that can carry a positive charge under acidic conditions (pH=5-7), and its structure includes an ionizable polar head and a hydrophobic chain.

[0013] According to the lipid nanoparticles of a specific embodiment of the present invention, the ionizable cationic lipid includes Dlin-MC3-DMA, SM102, ALC0315, cKK-E12 or C12-200.

[0014] Steroids include sitosterol, stigmasterol, lanosterol, ergosterol, fucoxanthol or cholesterol.

[0015] The present invention also provides application of the lipid nanoparticles in preparing ultrasound-targeted drugs.

[0016] The ultrasound-targeted drug according to a specific embodiment of the present invention comprises the above-mentioned lipid nanoparticles and ultrasound microbubbles, and also comprises a pharmaceutically acceptable carrier.

[0017] According to the ultrasound targeted drug of a specific embodiment of the present invention, the gas component in the ultrasound microbubbles is fluorocarbon gas or fluorine-sulfur gas; preferably, the gas component in the ultrasound microbubbles is perfluoropropane, perfluorobutane or sulfur hexafluoride.

[0018] According to the ultrasound targeted drug of a specific embodiment of the present invention, the lipid component in the ultrasound microbubbles is phospholipid or a phospholipid derivative.

[0019] The method of using the above-mentioned drug includes administering an effective amount of the above-mentioned drug to a subject. The administration method can be intravenous injection or local tissue injection, and combined with ultrasound, applied to the patient in need of treatment.

[0020] Pharmaceutically effective amount refers to a reasonable benefit / risk ratio that can be obtained by applying drug treatment, and a quantity sufficient to treat the disease. The level of effective dose can be determined depending on some factors, including the patient's disease type, severity, activity of the drug, sensitivity to the drug, application time, route of administration, excretion rate, treatment cycle, drugs used simultaneously, and other factors known in the medical field. The drug of the present invention can be used as an independent therapeutic agent, or combined with other therapeutic agents. In addition, the composition of the present invention can be added with typical therapeutic agents continuously or simultaneously, and the composition can be applied once or multiple times. It is important to consider all the above factors and apply the minimum dose that can produce the maximum effect without side effects, and the dosage can be determined by the physician according to the patient's condition, age, etc.

[0021] Beneficial effects of the present invention: The nucleic acid drug loaded by the lipid nanoparticles of the present invention will not exert its effect in major organs such as the liver and spleen after injection. However, when ultrasound is applied to the target area (heart, brain, blood vessels, muscles, etc.), the expression of the nucleic acid drug can be specifically activated in the area, thereby effectively solving the problems of serious off-target expression in the liver and spleen and difficulty in targeted delivery to extrahepatic tissues that are common in the current lipid nanoparticle delivery of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Shows the effects of different polyethylene glycol lipids on off-target expression of mRNA in major organs; a. The structures of the different polyethylene glycol lipids used; b. Lipid nanoparticles loaded with luciferase mRNA prepared using different polyethylene glycol lipids were injected intravenously to detect bioluminescent imaging of luciferase expression in various organs of mice; c. Quantitative results of luciferase expression in the liver of lipid nanoparticles loaded with luciferase mRNA prepared with different polyethylene glycol lipids; d. Quantitative results of luciferase expression in the spleen of lipid nanoparticles loaded with luciferase mRNA prepared with different polyethylene glycol lipids.

[0024] Figure 2 Effects of different ratios of polyethylene glycol lipids on off-target expression of mRNA in major organs; a. Bioluminescent imaging of luciferase expression in various organs of mice after intravenous injection of lipid nanoparticles loaded with luciferase mRNA prepared by using polyethylene glycol lipids with different doping ratios; b. Quantitative results of luciferase expression in the liver; c. Quantitative results of luciferase expression in spleen.

[0025] Figure 3 Display the expression levels of mRNA in various organs at different time points; a. Four different lipid nanoparticles loaded with luciferase mRNA were injected intravenously to detect bioluminescent imaging of luciferase expression in various organs of mice; b. Quantitative results of luciferase expression in the liver; c. Quantitative results of luciferase expression in spleen.

[0026] Figure 4 Show the effects of different ionizable lipids on the expression levels of mRNA in various organs; a Bioluminescence imaging to detect luciferase expression in various organs; b Quantitative results of luciferase expression in liver; c Quantitative results of luciferase expression in spleen; d Quantitative results of luciferase expression in the lung; e Quantitative results of luciferase expression in the heart; f Quantitative results of luciferase expression in kidney.

[0027] Figure 5 Four different lipid nanoparticles were mixed with microbubbles and dripped through the tail vein of mice. Ultrasound was applied to the right leg of the mice at the same time. Bioluminescence imaging was used for continuous monitoring for 7 days after ultrasound. The second administration was performed 30 days after the first administration. a. Representative bioluminescence imaging images; b. Changes in bioluminescent signals in the right leg of mice after the first administration; c. Changes in bioluminescent signals on the right side of the mouse after the second administration.

[0028] Figure 6 It shows that the fixed lipid nanoparticles contain 10% DSPE-PEG2000, and the proportions of other lipid components are changed. The obtained lipid nanoparticles loaded with luciferase mRNA are mixed with microbubbles and dripped through the tail vein of mice. At the same time, ultrasound is applied to the right leg of the mouse. One day later, the bioluminescence signal of the right leg is quantitatively analyzed using bioluminescence imaging.

[0029] Figure 7 It shows that the ionizable lipid MC3 was replaced with other ionizable lipids to prepare lipid nanoparticles, and the obtained lipid nanoparticles loaded with luciferase mRNA were mixed with microbubbles and dripped into the tail vein of mice. At the same time, ultrasound was applied to the right leg of the mouse. One day later, the bioluminescence signal of the right leg was quantitatively analyzed using bioluminescence imaging.

[0030] Figure 8 Shows cryo-electron microscopy images and particle size statistics of MC3-LNP and MC3-SLNP.

[0031] Fig. 9 showed that mRNA-loaded MC3-LNP or MC3-SLNP mixed with microbubbles were injected directly into mouse skeletal muscle, and the injection site was then sonicated; a. Quantitative results of luciferase expression levels in mouse skeletal muscle three days after administration; b. Bioluminescence imaging results of the main organs of mice at different time points after injection; c. Quantitative results of bioluminescence intensity in the liver; d. Quantification of bioluminescence in spleen.

[0032] Fig.10 It shows that MC3-LNP or MC3-SLNP loaded with plasmid DNA was mixed with microbubbles and then dripped into the tail vein of mice, while ultrasound was applied to the right leg of the mice, and then the expression of plasmid was continuously monitored; a. Representative bioluminescence images; b. Quantitative results of bioluminescent signals in the right leg of mice; c. Changes in mouse body weight after injection of different doses of MC3-LNP or MC3-SLNP loaded with plasmid DNA. A body weight of 0 represents the death of the mouse.

[0033] Fig.11 It was shown that MC3-SLNP loaded with Cre mRNA was mixed with microbubbles and then dripped through the tail vein of mice, combined with ultrasound irradiation to achieve targeted delivery of mRNA to multiple tissues; a. How Cre mRNA works in Ai9 mice; b. Skeletal muscle section results; c. Brain slice results; d. Fluorescence image of carotid artery; e. Heart section results.

[0034] Fig.12 Showing the effect of gene editing of the VCAM1 gene in endothelial cells. DETAILED DESCRIPTION

[0035] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0036] The present invention provides ultrasound-responsive lipid nanoparticles, the raw materials for preparing the particles include: 1) Polyethylene glycol lipids; 2) ionizable cationic lipids; 3) Steroids; The molar percentage of polyethylene glycol lipid is 5%-20%, the molar percentage of ionizable cationic lipid is 5%-90%, and the molar percentage of steroid is 5%-80%; The lipid nanoparticles are loaded with nucleic acids, and the nucleic acids are selected from one or more of mRNA, DNA, plasmid, sgRNA, circular RNA, siRNA, and microRNA.

[0037] Preferably, the structure of the polyethylene glycol lipid contains a hydrophobic saturated carbon chain of 18 carbon atoms and a hydrophilic carbon chain, wherein the number of the hydrophobic carbon chains is not less than 2, and the number of the hydrophilic carbon chains is 1.

[0038] Preferably, the polyethylene glycol lipid has one of the following two structures:

[0039] Wherein, m is an integer greater than 16, n represents the average value of the polymer, the value of n is an integer greater than 20, and X is selected from methyl, hydrogen, polypeptide, antibody, or X is selected from any other chemical structure.

[0040] Preferably, the polyethylene glycol lipid is DSPE-PEG (average molecular weight 2000-5000) or DSG-PEG (average molecular weight 2000-5000), and more preferably, the polyethylene glycol lipid is selected from DSPE-PEG2000, DSPE-PEG5000 or DSG-PEG2000.

[0041] The preferred lipid structure is as follows:

[0042] Preferably, the molar percentage of polyethylene glycol lipid in the three preparation raw materials of lipid nanoparticles is 5%-20%, more preferably, the proportion of polyethylene glycol lipid is 8-15%, or, the proportion of polyethylene glycol lipid is any value within the above range, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or other values, which are not described here one by one.

[0043] Ionizable cationic lipids are a class of lipids that can carry positive charges under acidic conditions (pH=5-7). Their structure includes an ionizable polar head and a hydrophobic chain, wherein the polar head can be a primary amine, a secondary amine, a tertiary amine, or a quaternary ammonium salt, as well as a phosphorus, guanidine, arsenic, imidazole, and pyridinium group, all of which can carry positive charges under acidic conditions. The main function of ionizable cationic lipids is to utilize the positive charge carried by ionization under acidic conditions to interact with negatively charged nucleic acid drugs, thereby achieving effective loading of nucleic acid drugs.

[0044] Preferably, the ionizable cationic lipid comprises Dlin-MC3-DMA, SM102, ALC0315, cKK-E12 or C12-200. The structure thereof is as follows.

[0045]

[0046] In addition to the above structures, the ionizable cationic lipids also include other compounds with ionizable polar head and hydrophobic chain structures.

[0047] The molar percentage of ionizable cationic lipids in the three raw materials for preparing lipid nanoparticles is 5%-90%. Preferably, the proportion of ionizable cationic lipids is 10%-80%, or 15%-70%, or 20%-60%, or 30%-50%, or 40%-50%. Alternatively, the proportion of ionizable cationic lipids is any value within the above range, for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or other values, which are not described one by one here.

[0048] Preferably, the steroid includes sitosterol, stigmasterol, lanosterol, ergosterol, fuccasterol or cholesterol, etc. More preferably, the steroid is selected from cholesterol.

[0049] Preferably, the molar percentage of steroids in the three raw materials for preparing lipid nanoparticles is 5-80%, preferably, the proportion of steroids is 15%-70%, or 20%-60%, or 30%-50%, or 40%-50%, or, the proportion of steroids is any value in the above range, for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or other values, which are not described here one by one.

[0050] The lipid nanoparticles are prepared by ethanol injection method or other conventional methods.

[0051] For example, this embodiment provides a method for preparing the above-mentioned lipid nanoparticles, comprising the following steps: According to the proportion, polyethylene glycol lipid, ionizable cationic lipid and steroid are dissolved in ethanol to obtain a lipid mixed solution; dissolving the nucleic acid in a buffered salt solution to obtain a nucleic acid solution; The lipid mixed solution and the nucleic acid solution are mixed and incubated at room temperature to obtain lipid nanoparticles loaded with nucleic acids.

[0052] Preferably, freshly prepared lipid nanoparticles are dialyzed against PBS.

[0053] Preferably, the nucleic acid is dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.1 μg / μL.

[0054] Preferably, the total mass ratio of nucleic acid to lipid is 1-20 μg:0.1-2 μmol. Wherein, lipid refers to polyethylene glycol lipid, ionizable cationic lipid and steroid. More preferably, the total mass ratio of nucleic acid to lipid is 15 μg:1 μmol, that is, 15g of nucleic acid for every 1 mol of lipid.

[0055] In addition to the above three components, other components such as phospholipids, small molecule drugs, peptides, etc. can also be incorporated into lipid nanoparticles.

[0056] After the lipid nanoparticles are loaded with nucleic acid, they are used in combination with microbubbles and ultrasound.

[0057] The microbubble structure is a bubble structure formed by lipid-encapsulated gas, the gas component in the microbubble structure is fluorocarbon gas or fluorine-sulfur gas, preferably perfluoropropane, perfluorobutane, sulfur hexafluoride; the lipid component in the microbubble structure is phospholipid or phospholipid derivative; the microbubble is preferably a phospholipid ultrasound contrast agent approved for clinical enhanced imaging.

[0058] Lipid nanoparticles need to be injected simultaneously with microbubbles, and the injection methods include intravenous injection, intramuscular injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, and intradermal injection. Injection can be performed by drip or bolus injection.

[0059] After or during the injection, ultrasound needs to be applied to the target tissue, and the ultrasound action frequency is below 10 MHz and the peak negative pressure is below 2 MPa. The preferred frequency range is 0.2-4 MHz, and the preferred peak negative pressure is 0.1-0.5 MPa.

[0060] The microbubble dose was 5 × 10 6 The dosage of lipid nanoparticles was 0.1 μg of mRNA per gram of body weight. After the two were mixed, an appropriate amount of physiological saline was added to make the total volume 250 μL.

[0061] By applying ultrasound to the target tissue, the ultrasound can interact with the microbubbles in the area, produce stable cavitation, and change the permeability of the cells, thereby allowing lipid nanoparticles loaded with nucleic acids to enter the cells and achieve the expression of nucleic acid drugs. In areas where ultrasound is not applied, the nucleic acid drugs have little or no effect.

[0062] Targeted tissues include heart, blood vessels, brain, skeletal muscle, tumors, eyes, bladder, pancreas, kidneys and other extrahepatic tissues and organs.

[0063] The delivered nucleic acids include mRNA, DNA, plasmids, sgRNA, circular RNA, siRNA, microRNA, etc., which can target endogenous targets through siRNA, microRNA, sgRNA, etc., or achieve the expression of exogenous proteins with therapeutic functions through mRNA, DNA, plasmids, circular RNA, etc.

[0064] Example 1 MC3, DSPC (distearoylphosphatidylcholine), and cholesterol were first mixed at a molar ratio of 50:10:38.5 and then combined with eight PEG lipids (DMPE-PEG2000, DSG-PEG2000, DPPE-PEG2000, DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG5000, and DOPE-PEG2000), respectively, with a molar percentage of PEG lipid of 10%.

[0065] The mRNA expressing the luciferase reporter gene was loaded separately, and the preparation method was as follows: The above PEG lipids were dissolved in ethanol at a concentration of 20 mM to prepare a stock solution. All lipids were then mixed in proportion, and mRNA was dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.1 μg / μL. The lipid mixture solution and the mRNA solution were then quickly mixed at a volume ratio of 3:1, and the total mass ratio of mRNA to lipid was 15 μg / 1 μmol. The mixture was incubated at room temperature for 10 minutes. Subsequently, the freshly prepared lipid nanoparticles were dialyzed in PBS for 1 hour to obtain lipid nanoparticles loaded with mRNA.

[0066] The obtained lipid nanoparticles loaded with luciferase mRNA were injected intravenously into mice at a dose of 0.1 mg / kg mRNA. Three hours after the injection, the mice were injected with luciferase substrates, and the mice were killed two minutes later. The tumors and major organs were removed, and bioluminescence imaging was performed to quantify the luminescence intensity of each tissue and organ.

[0067] The results are as follows Figure 1 As shown in bd. It can be seen that the PEG lipids with C18 hydrophobic chains (DSG-PEG2000, DSPE-PEG2000, DSPE-PEG5000) significantly reduced the expression level of mRNA in the liver and spleen compared with other lipids. When the C18 hydrophobic chain is an unsaturated chain (DOPE-PEG2000), the expression level of mRNA in the liver and spleen is also high. The longer the length of the hydrophobic chain, the lower the expression level of mRNA in the liver and spleen (DMPE-PEG2000>DPPE-PEG2000>DSPE-PEG2000). The longer the length of the PEG chain, the lower the expression level of mRNA in the liver and spleen (DSPE-PEG1000>DSPE-PEG2000>DSPE-PEG5000).

[0068] It can be concluded that in order to reduce the expression level of mRNA in liver and spleen, two saturated long hydrophobic chains (at least 18 carbons) should be used, the molecular weight of the PEG chain should be greater than 1000, and the preferred PEG lipids are DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000.

[0069] Example 2 This example further explores the effect of the doping ratio of DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000 on the mRNA expression level in the liver and spleen, and uses DMG-PEG2000 as a control.

[0070] The molar ratio of MC3, DSPC and cholesterol was fixed at 50:10:38.5, and the molar percentage of PEG lipid was changed (1.5%, 5%, 7.5%, 10%). The experimental method was referred to Example 1.

[0071]

[0072] The results are as follows Figure 2 As shown in the figure, the doping ratio of DMG-PEG2000 has almost no effect on the mRNA expression levels in the liver and spleen. For DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000, the higher the doping ratio, the lower the mRNA expression level in the liver and spleen. In order to achieve a lower mRNA expression level in the liver and spleen, the doping ratio of PEG lipids should be higher than 5%.

[0073] Example 3 This example further studies the expression levels of mRNA in various organs at different time points.

[0074] The molar ratio of MC3, DSPC and cholesterol was fixed at 50:10:38.5, the molar percentage of PEG lipid in the sample group was 10% (DSG-PEG2000, DSPE-PEG2000, DSPE-PEG5000), and the lipid in the control group was 1.5% DMG-PEG2000. The experimental method was referred to Example 1.

[0075] The results are as follows Figure 3 As shown, it can be seen that at different time points after injection, the lipid nanoparticles in the control group (1.5% DMG-PEG2000, DMG-PEG2000: MC3: DSPC: cholesterol = 1.5:50:10:38.5) had a high intensity of mRNA expression level in the liver and spleen within two days after injection. However, the mRNA expression levels of DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000 with a 10% doping ratio were very low in the liver and spleen at each time point.

[0076] Example 4 This example investigates the effects of different ionizable lipids on the expression levels of mRNA in various organs.

[0077] In addition to MC3, three representative ionizable lipids, SM102, cKK-E12, and C12-200, were selected as ionizable lipids. The molar ratio of ionizable lipids, DSPC, and cholesterol was fixed at 50:10:38.5, 10% DSPE-PEG2000 was selected as PEG lipid, and 1.5% DMG-PEG2000 was used as control. The experimental method was referred to Example 1.

[0078]

[0079] The results are as follows Figure 4 As shown, it can be seen that 3 hours after injection, compared with 1.5% DMG-PEG2000, 10% DSPE-PEG2000 can significantly reduce the mRNA expression in the heart, liver, spleen, lung, and kidney. This shows that the selection of polyethylene glycol lipids to shield nucleic acid expression in organs is a general strategy that is not affected by the type of ionizable lipids.

[0080] Example 5 Investigating the mRNA expression of lipid nanoparticles in the ultrasound irradiation area The four different lipid nanoparticles prepared in Example 3 were mixed with microbubbles (provided by Beijing Feiruida Medical Technology Co., Ltd., mainly composed of phospholipid-encapsulated perfluoropropane gas) (the dosage of microbubbles was 5 × 10 6 The dose of lipid nanoparticles was 0.1 μg of mRNA per gram of body weight. After the two were mixed, an appropriate amount of normal saline was added to make the total volume 250 μL) and dripped through the tail vein of mice. At the same time, ultrasound was applied to the right leg of the mice (ultrasound frequency was 1 MHz, sound pressure was 0.4 MPa), and bioluminescence imaging was used for continuous monitoring for 7 days after ultrasound.

[0081] Thirty days after the first administration, when the bioluminescence signal disappeared, a second injection of four different lipid nanoparticles and microbubbles was performed, and ultrasound was applied to the right leg of the mouse. After ultrasound, bioluminescence imaging was used for continuous monitoring for 7 days.

[0082] The results are as follows Figure 5 As shown, the control group lipid nanoparticles (1.5% DMG-PEG2000) not only had a high mRNA expression level at the ultrasound site after injection, but also had a high intensity of mRNA expression level in all parts of the body within the first two days. However, the lipid nanoparticles with a 10% doping ratio of DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000 showed mRNA expression almost only in the right leg where ultrasound was applied, while the mRNA expression levels in all parts of the body were very low. This result shows that the lipid nanoparticles with 10% DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000 not only showed a significant inhibition of off-target effects, but also achieved specific expression of mRNA at the ultrasound site.

[0083] It can be seen that the lipid nanoparticles formed by combining 10% PEG lipids containing saturated C18 hydrophobic chains (DSG-PEG2000, DSPE-PEG2000, DSPE-PEG5000) with four components: MC3, DSPC, and cholesterol can achieve specific expression of mRNA in the ultrasound site and inhibit off-target in various organs.

[0084] Example 6 In this example, the components of lipid nanoparticles were further optimized (PEG lipids were fixed to select 10% DSPE-PEG2000), lipid nanoparticles of different components were mixed with microbubbles and dripped through the tail vein of mice, and ultrasound was applied to the right leg of the mice. After 1 day, the luminescent signal of the right leg was quantified by bioluminescence imaging.

[0085]

[0086] All lipids were mixed in ethanol according to the ratios in the table above, with a total concentration of 20 mM. mRNA was dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.1 μg / μL. Then, the lipid mixture solution and the mRNA solution were quickly mixed at a volume ratio of 3:1, and the total mass ratio of RNA to lipid was 15 μg / 1 μmol. The mixture was incubated at room temperature for 10 minutes. Subsequently, the freshly prepared lipid nanoparticles were dialyzed in PBS for 1 hour to obtain lipid nanoparticles loaded with mRNA.

[0087] like Figure 6 As shown in the figure, compared with the four-component lipid nanoparticles, the mRNA expression level delivered by the two-component lipid nanoparticles (10% DSPE-PEG2000 and 90% MC3) was significantly reduced. In the three-component lipid nanoparticles without cholesterol, changing the ratio of DSPC did not significantly improve the expression of mRNA. In the three-component lipid nanoparticles without DSPC, when the cholesterol ratio was about 45%, the expression level of mRNA was significantly increased. Thus, when 10% DSPE-PEG2000, 45% MC3 and 45% cholesterol were matched, the expression level of mRNA was the highest.

[0088] Example 7 Investigating the effects of different ionizable lipids on mRNA expression at the ultrasound site In addition to MC3, four other ionizable lipids were selected in this example: SM102, ALC0315, cKK-E12, and C12-200.

[0089]

[0090] All lipids were mixed in ethanol in proportion to a total concentration of 20 mM. mRNA was dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.1 μg / μL. Then, the lipid mixture solution and the mRNA solution were quickly mixed at a volume ratio of 3:1, and the total mass ratio of RNA to lipid was 15 μg / 1 μmol. The mixture was incubated at room temperature for 10 minutes. Subsequently, the freshly prepared lipid nanoparticles were dialyzed in PBS for 1 hour to obtain lipid nanoparticles loaded with mRNA.

[0091] The lipid nanoparticles of different components were mixed with microbubbles (the dosage of microbubbles was 5 × 10 6 The dose of lipid nanoparticles was 0.1 μg of mRNA per gram of body weight. After the two were mixed, an appropriate amount of saline was added to the total volume of 250 μL) and the mouse tail vein was dripped. At the same time, ultrasound was applied to the right leg of the mouse. After 1 day, the luminescent signal of the right leg was quantified by bioluminescence imaging.

[0092] like Figure 7 As shown, except for MC3, lipid nanoparticles obtained from other ionizable lipids can achieve specific expression of mRNA at the ultrasonic treatment site, and compared with the LNP group, the overall mRNA expression level of the SLNP group without DSPC is higher.

[0093] Example 8 Investigating the particle size of MC3-LNP and MC3-SLNP The particle size and morphology characteristics of MC3-LNP and MC3-SLNP obtained in Example 7 were observed using cryo-electron microscopy.

[0094] The results are as follows Figure 8 As shown, the average particle size of lipid nanoparticles of MC3-LNP is 14.23 nm, which is smaller than the average particle size of 69.08 nm of MC3-LNP.

[0095] Example 9 This example investigates the feasibility of local injection.

[0096] After MC3-LNP or MC3-SLNP was mixed with microbubbles (the dose of microbubbles was 7×10 7 The microbubbles and lipid nanoparticles with a dose of 0.5 µg of mRNA were mixed and supplemented with an appropriate amount of saline to a total volume of 25 μL) and injected directly into the skeletal muscle of mice, and then ultrasound was applied to the injection site.

[0097] Four groups were set up for MC3-SLNP injection: (1) injection of MC3-SLNP only; (2) injection of MC3-SLNP and microbubbles simultaneously, but without ultrasound; (3) injection of MC3-SLNP only, followed by ultrasound after injection; (4) injection of MC3-SLNP and microbubbles simultaneously, followed by ultrasound after injection.

[0098] Two groups were set up for injection of MC3-LNP: injection of MC3-SLNP only; injection of MC3-SLNP and microbubbles at the same time, and ultrasound was applied after injection.

[0099] like Fig. 9 As shown in the figure, MC3-SLNP combined with microbubbles and ultrasound can achieve efficient expression of mRNA in mouse skeletal muscle. The expression level of mRNA is relatively low without ultrasound or microbubbles. Although MC3-LNP can also achieve efficient expression of mRNA in skeletal muscle, it will also cause a lot of off-target expression in the liver and spleen.

[0100] Example 10 Investigating the delivery of other nucleic acid drugs by MC3-SLNP MC3-SLNP and MC3-LNP loaded with plasmid DNA capable of expressing luciferase were prepared respectively.

[0101] All lipids were mixed in ethanol in proportion to a total concentration of 20 mM. Plasmid DNA expressing luciferase (containing a luciferase reporter gene driven by a CMV promoter) was dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.1 μg / μL. Then, the lipid mixture solution and the nucleic acid solution were quickly mixed at a volume ratio of 3:1, and the total mass ratio of nucleic acid to lipid was 15 μg / 1 μmol. The mixture was incubated at room temperature for 10 minutes. Subsequently, the freshly prepared lipid nanoparticles were dialyzed in PBS for 1 hour to obtain lipid nanoparticles MC3-SLNP and MC3-LNP loaded with plasmid DNA.

[0102] They were mixed with microbubbles and then intravenously dripped, while ultrasound was applied to the right leg of the mice.

[0103] like Fig.10 As shown in ab, MC3-SLNP can achieve the expression of plasmid DNA more efficiently without obvious off-target. In addition, MC3-SLNP has higher safety than MC3-LNP. When injected with a dose of 2.5 mg / kg DNA, MC3-LNP caused all mice to die after two days, while all mice injected with MC3-SLNP died ( Fig.10 c). This indicates the high safety and effectiveness of the three-component formulation of SLNP of the present invention.

[0104] Example 11 Investigating the expression of SLNP combinations in different organ tissues Ai9 mice were used to evaluate the effect of SLNP delivery. By delivering mRNA to express Cre recombinase in Ai9 mouse cells, the expression of the red fluorescent protein tdTomato can be achieved, so that the expression of mRNA can be indicated by the fluorescent protein.

[0105] To prepare MC3-SLNP loaded with Cre mRNA, DSPE-PEG2000, MC3, and cholesterol were mixed in ethanol at a molar ratio of 10:45:45 to a total concentration of 20 mM. The mRNA expressing Cre recombinase was dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.1 μg / μL. Then, the lipid mixed solution and the mRNA solution were quickly mixed at a volume ratio of 3:1, and the total mass ratio of RNA to lipid was 15 μg / 1 μmol. The mixture was incubated at room temperature for 10 minutes. Subsequently, the freshly prepared lipid nanoparticles were dialyzed in PBS for 1 hour to obtain lipid nanoparticles loaded with mRNA.

[0106] Prepare MC3-SLNP loaded with Cre mRNA and mix with microbubbles (microbubbles dose is 5 × 10 per gram body weight). 6 The dosage of lipid nanoparticles was 0.5 μg mRNA per gram body weight. The two were mixed and supplemented with an appropriate amount of saline to a total volume of 250 μL) for intravenous drip, while ultrasound was applied. The process was as follows Fig.11 As shown in a.

[0107] When the mouse legs were irradiated with ultrasound (ultrasound frequency of 1 MHz, sound pressure of 0.4 MPa), red fluorescent protein was specifically expressed in skeletal muscle ( Fig.11 b).

[0108] When ultrasound was used to irradiate the right side of the mouse brain (ultrasound frequency of 1 MHz, sound pressure of 0.2 MPa), red fluorescent protein specific expression could be observed in the right side of the brain ( Fig.11 c).

[0109] When the left carotid artery of mice was irradiated with ultrasound (ultrasound frequency of 3.2 MHz, sound pressure of 0.2 MPa), red fluorescent protein was specifically expressed in the left carotid artery, while no expression was found in the right carotid artery without ultrasound irradiation ( Fig.11 d).

[0110] When the mouse heart was irradiated with ultrasound (ultrasound frequency of 3.2 MHz, sound pressure of 0.3 MPa), red fluorescent protein specific expression could be observed in the heart ( Fig.11 e).

[0111] Example 12 Investigation of the application of SLNP combination in gene editing In order to evaluate the application of SLNP combination in treatment, this example used SLNP to simultaneously load mRNA expressing Cas9 nuclease and small guide RNA (gRNA) targeting VCAM1 gene, and evaluated its gene editing effect on VCAM1 gene in vascular endothelial cells.

[0112] To prepare SLNPs for gene editing, DSPE-PEG2000, MC3, and cholesterol were mixed in ethanol at a molar ratio of 10:45:45 to a total concentration of 20 mM. mRNA expressing Cas9 nuclease and gRNA targeting VCAM1 gene were dissolved in 10 mM citrate buffer (pH 4.0) at a mass ratio of 2:1 to a total concentration of 0.1 μg / μL. Then, the lipid mixed solution and RNA solution were quickly mixed at a volume ratio of 3:1, and the total mass ratio of RNA to lipid was 15 μg / 1 μmol. The mixture was incubated at room temperature for 10 minutes. Subsequently, the freshly prepared lipid nanoparticles were dialyzed in PBS for 1 hour to obtain lipid nanoparticles for gene editing.

[0113] The obtained lipid nanoparticles for VCAM1 gene editing were mixed with microbubbles (the dose of microbubbles was 5 × 10 6 The mice were injected with 100 μg of RNA per gram of body weight (100 μg of RNA per gram of body weight) and the two were mixed and supplemented with an appropriate amount of saline to a total volume of 250 μL) for intravenous drip through the tail vein. Ultrasound was applied to the left carotid artery of the mice, and the sound pressure of the ultrasound was selected to be 0.2 MPa, 0.3 MPa and 0.4 MPa respectively. One week after the experiment, the mice were killed, the carotid arteries of the mice were removed, the vascular endothelial cells of the mice were isolated, and the gene editing effect of the VCAM1 gene in the endothelial cells was analyzed by second-generation sequencing.

[0114] like Fig.12 As shown, compared with the right carotid artery (RCA) without ultrasound, the left carotid artery achieved efficient editing of the VCAM1 gene in vascular endothelial cells under three different acoustic pressures, with the highest editing efficiency reaching more than 20, while almost no editing was detected in RCA.

[0115] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An ultrasound-responsive lipid nanoparticle, characterized in that: The raw materials for preparing the lipid nanoparticles include: 1) Polyethylene glycol lipids; 2) ionizable cationic lipids; 3) Steroids; The molar percentage of polyethylene glycol lipid is 5%-20%, the molar percentage of ionizable cationic lipid is 5%-90%, and the molar percentage of steroid is 5%-80%; The lipid nanoparticles are loaded with nucleic acids, and the nucleic acids are selected from one or more of mRNA, DNA, plasmid, sgRNA, circular RNA, siRNA, and microRNA.

2. The lipid nanoparticle according to claim 1, characterized in that The polyethylene glycol lipid contains two saturated carbon chains with no less than 18 carbons.

3. The lipid nanoparticle according to claim 1, characterized in that The polyethylene glycol lipid is selected from one or more of DSPE-PEG2000, DSPE-PEG5000 and DSG-PEG2000.

4. The lipid nanoparticle according to claim 1, characterized in that The ionizable cationic lipid is a lipid with a positive charge under acidic conditions of pH 5-7, and its structure includes an ionizable polar head and a hydrophobic chain.

5. The lipid nanoparticle according to claim 1, characterized in that The ionizable cationic lipid is selected from Dlin-MC3-DMA, SM102, ALC0315, cKK-E12 or C12-200.

6. The lipid nanoparticle according to claim 1, characterized in that The steroid includes sitosterol, stigmasterol, lanosterol, ergosterol, fuccasterol or cholesterol.

7. Use of the lipid nanoparticles according to claim 1 in the preparation of ultrasound-targeted drugs.

8. An ultrasound-targeted drug, characterized in that: The method comprises the lipid nanoparticles according to claim 1 and ultrasound microbubbles.

9. The ultrasound targeted drug according to claim 8, characterized in that: The gas component in the ultrasonic microbubbles is fluorocarbon gas or fluorine-sulfur gas.

10. The ultrasound targeted drug according to claim 8, characterized in that: The gas component in the ultrasonic microbubble is perfluoropropane, perfluorobutane or sulfur hexafluoride.

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