An ultrasound-targetable lipid nanoparticle and its application

Abstract

The present invention belongs to the field of molecular biology, and particularly relates to an ultrasound-targetable lipid nanoparticle and its application. The ultrasound-responsive lipid nanoparticle of the present invention comprises: polyethylene glycol lipid, ionizable cationic lipid, and steroid; the molar ratio of polyethylene glycol lipid, ionizable cationic lipid, and steroid is 5-20:5-90:5-80, and the lipid nanoparticle is loaded with nucleic acid. The lipid nanoparticle provided by the present invention can target and deliver nucleic acid drugs to extrahepatic tissues under ultrasound guidance and achieve specific expression, while hardly expressing in the liver and spleen.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and particularly relates to an ultrasound-targetable lipid nanoparticle and its application. Background Art

[0002] Nucleic acid drugs, including mRNA, DNA, plasmids, sgRNA, circular RNA, siRNA, microRNA, etc., have received extensive 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 chemical instability, especially being easily degraded into individual nucleotides by nucleases in vitro and in vivo, resulting in the loss of drug efficacy. Therefore, in order to ensure the effectiveness of nucleic acid drugs, a special carrier system must be relied on. 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 the phosphate ions of nucleic acid drugs, thereby protecting the drugs from degradation and changing their endocytosis 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 been proven to be a transformative technology, and the most typical example is the rapid development and application of the novel 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. Different from mRNA vaccines, mRNA therapy usually requires a much higher protein expression level, 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. Lipid nanoparticles injected intravenously interact with serum proteins to form a protein corona, which causes the lipid nanoparticles to be directed 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 by endogenous targeting. Although the 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 the protein corona and reduce immunogenicity, polyethylene glycol (PEG)-modified nanoparticles have become a well-established technology. PEG lipids are crucial for the formation of lipid nanoparticles and can prevent their aggregation. However, PEG lipids may also hinder the cellular uptake of nanoparticles, thereby affecting mRNA expression. 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. In vivo, their rapid dissociation leads to the formation of the protein corona, which promotes mRNA expression in major organs but also triggers an immune response. This effect is beneficial for mRNA vaccines, but for non-vaccine applications, it may cause off-target expression and immunogenicity problems. Although the use of PEG lipids with longer hydrophobic chains (such as C18 chains) can reduce the formation of the protein corona, a high proportion of these lipids will significantly reduce the transfection efficiency of lipid nanoparticles. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes to combine lipid nanoparticles prepared with PEG lipids having long hydrophobic chains with ultrasound and microbubbles responsive to ultrasound, and utilize the sonoporation effect generated by microbubbles under ultrasound irradiation to solve the problem of low expression efficiency of nucleic acid drugs such as mRNA caused by PEG lipids with long hydrophobic chains, and while improving the delivery efficiency of nucleic acid drugs, reduce the side effects brought about by immune reactions and off-target expression.

[0006] The object 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 above lipid nanoparticles.

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

[0009] The ultrasound-responsive lipid nanoparticle according to the specific embodiment of the present invention has the following preparation raw materials:

[0010] 1) Polyethylene glycol lipid;

[0011] 2) Ionizable cationic lipid;

[0012] 3) Steroid;

[0013] The molar percentage of the polyethylene glycol lipid is 5% - 20%, the molar ratio of the ionizable cationic lipid is 5% - 90%, and the molar percentage of the steroid is 5% - 80%;

[0014] The lipid nanoparticle is loaded with nucleic acid, and the nucleic acid is selected from one or more of mRNA, DNA, plasmid, sgRNA, circular RNA, siRNA, and microRNA.

[0015] According to the lipid nanoparticles of the specific embodiments of the present invention, the polyethylene glycol lipid contains two saturated carbon chains with no less than 18 carbons.

[0016] According to the lipid nanoparticles of the specific embodiments of the present invention, the polyethylene glycol lipid is selected from one or more of DSPE-PEG2000, DSPE-PEG5000, and DSG-PEG2000.

[0017] According to the lipid nanoparticles of the specific embodiments 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.

[0018] According to the lipid nanoparticles of the specific embodiments of the present invention, the ionizable cationic lipid includes Dlin-MC3-DMA, SM102, ALC0315, cKK-E12, or C12-200.

[0019] Steroids include sitosterol, stigmasterol, lanosterol, ergosterol, fucosterol, or cholesterol.

[0020] The present invention also provides the application of the above lipid nanoparticles in the preparation of ultrasound-targeted drugs.

[0021] According to the ultrasound-targeted drug of the specific embodiments of the present invention, it comprises the above lipid nanoparticles and ultrasound microbubbles, and also includes a pharmaceutically acceptable carrier.

[0022] According to the ultrasound-targeted drug of the specific embodiments of the present invention, the gas component in the ultrasound microbubbles is a fluorocarbon gas or a fluorosulfur gas; preferably, the gas component in the ultrasound microbubbles is perfluoropropane, perfluorobutane, or sulfur hexafluoride.

[0023] According to the ultrasound-targeted drug of the specific embodiments of the present invention, the lipid component in the ultrasound microbubbles is a phospholipid or a phospholipid derivative.

[0024] For the usage method of the above drug, it includes administering an effective amount of the above drug to a subject. The administration method can be intravenous injection or local tissue injection, and ultrasound is used in combination and applied to the patient in need of treatment.

[0025] A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio achievable by the application of a drug. The level of the effective dose can be determined depending on several factors, including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route, the excretion rate, the treatment period, the drugs used simultaneously, and other factors well-known in the medical field. The drugs of the present invention can be administered as an independent therapeutic agent or in combination with other therapeutic agents. Moreover, the compositions of the present invention can be added to typical therapeutic agents continuously or simultaneously, and the compositions can be administered once or multiple times. It is important to consider all the above factors and administer at the minimum dose that can produce the maximum effect without side effects, and such dose can be determined by a physician according to the patient's condition, age, etc.

[0026] Advantages of the present invention:

[0027] The nucleic acid drug loaded in the lipid nanoparticles of the present invention does not exert its effect in major organs such as the liver and spleen after injection, while 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 this area, thus effectively solving the problems of serious off-target expression in the liver and spleen and difficult targeted delivery to extrahepatic tissues that are commonly present when lipid nanoparticles deliver nucleic acid drugs at present. Description of the drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0029] Figure 1 Showing the effects of different polyethylene glycol lipids on the off-target expression of mRNA in major organs; a. Structures of different polyethylene glycol lipids used;

[0030] b. Bioluminescence imaging of luciferase expression detected in various organs of mice after intravenous injection of lipid nanoparticles loaded with luciferase mRNA prepared with different polyethylene glycol lipids;

[0031] c. Quantitative results of luciferase expression in the liver of lipid nanoparticles loaded with luciferase mRNA prepared with different polyethylene glycol lipids;

[0032] d. Quantitative results of luciferase expression in the spleen of lipid nanoparticles loaded with luciferase mRNA prepared with different polyethylene glycol lipids.

[0033] Figure 2The effects of polyethylene glycol lipids with different ratios on the off-target expression of mRNA in major organs;

[0034] a. Bioluminescent imaging of luciferase expression was detected in various organs of mice after intravenous injection of lipid nanoparticles loaded with luciferase mRNA prepared with polyethylene glycol lipids with different doping ratios;

[0035] b. Quantitative results of luciferase expression in the liver;

[0036] c. Quantitative results of luciferase expression in the spleen.

[0037] Figure 3 Showing the expression levels of mRNA in various organs at different time points;

[0038] a. Bioluminescent imaging of luciferase expression was detected in various organs of mice after intravenous injection of four different lipid nanoparticles loaded with luciferase mRNA;

[0039] b. Quantitative results of luciferase expression in the liver;

[0040] c. Quantitative results of luciferase expression in the spleen.

[0041] Figure 4 Showing the effects of different ionizable lipids on the expression levels of mRNA in various organs;

[0042] a. Bioluminescent imaging of luciferase expression detected in various organs;

[0043] b. Quantitative results of luciferase expression in the liver;

[0044] c. Quantitative results of luciferase expression in the spleen;

[0045] d. Quantitative results of luciferase expression in the lung;

[0046] e. Quantitative results of luciferase expression in the heart;

[0047] f. Quantitative results of luciferase expression in the kidney.

[0048] Figure 5 Showing that four different lipid nanoparticles were respectively mixed with microbubbles and infused through the tail vein of mice, and ultrasound was applied to the right leg of the mice at the same time. After ultrasound, bioluminescent imaging was used to continuously monitor for 7 days, and the second administration was carried out 30 days after the first administration;

[0049] a. Representative bioluminescent imaging pictures;

[0050] b. Bioluminescence signal change graph of the right leg of the mouse after the first administration;

[0051] c. Bioluminescence signal change graph of the right side of the mouse after the second administration.

[0052] Figure 6 It is shown that the fixed lipid nanoparticles contain 10% DSPE-PEG2000. By changing the proportion of other lipid components, the obtained lipid nanoparticles loaded with luciferase mRNA are respectively mixed with microbubbles and infused through the tail vein of mice. At the same time, ultrasound is applied to the right leg of the mice. One day later, the bioluminescence signal of the right leg is quantitatively analyzed by bioluminescence imaging.

[0053] Figure 7 It is shown that the ionizable lipid MC3 is respectively replaced with other ionizable lipids to prepare lipid nanoparticles. The obtained lipid nanoparticles loaded with luciferase mRNA are respectively mixed with microbubbles and infused through the tail vein of mice. At the same time, ultrasound is applied to the right leg of the mice. One day later, the bioluminescence signal of the right leg is quantitatively analyzed by bioluminescence imaging.

[0054] Figure 8 Show the cryo-electron microscopy images and particle size statistics results of MC3-LNP and MC3-SLNP.

[0055] Figure 9 Show that the MC3-LNP or MC3-SLNP loaded with mRNA is mixed with microbubbles and directly injected into the skeletal muscle of mice, and then ultrasound is applied to the injection site;

[0056] a. Quantitative results of luciferase expression levels in the skeletal muscle of mice three days after administration;

[0057] b. Bioluminescence imaging results in the major organs of mice at different time points after injection;

[0058] c. Quantitative results of bioluminescence intensity in the liver;

[0059] d. Quantitative results of bioluminescence in the spleen.

[0060] Figure 10 Show that the MC3-LNP or MC3-SLNP loaded with plasmid DNA is mixed with microbubbles and infused through the tail vein of mice. At the same time, ultrasound is applied to the right leg of the mice, and then the plasmid expression is continuously monitored;

[0061] a. Representative bioluminescence images;

[0062] b. Quantitative results of bioluminescence signals in the right leg of mice;

[0063] c. Changes in body weight of mice after injection of different doses of MC3-LNP or MC3-SLNP loaded with plasmid DNA. A body weight of 0 indicates mouse death.

[0064] Figure 11 It shows that after mixing MC3-SLNP loaded with Cre mRNA with microbubbles and then performing tail vein infusion in mice, combined with ultrasound irradiation, mRNA targeted delivery to multiple tissues is achieved.

[0065] a. Working principle of Cre mRNA in Ai9 mice.

[0066] b. Results of skeletal muscle sections.

[0067] c. Results of brain sections.

[0068] d. Fluorescent images of carotid arteries.

[0069] e. Results of heart sections.

[0070] Figure 12 It shows the gene editing effect of the VCAM1 gene in endothelial cells. Detailed implementation manners

[0071] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the scope protected by the present invention.

[0072] The embodiments of the present invention provide ultrasound-responsive lipid nanoparticles, and the raw materials for their preparation include:

[0073] 1) Polyethylene glycol lipid;

[0074] 2) Ionizable cationic lipid;

[0075] 3) Steroid;

[0076] The molar percentage of the polyethylene glycol lipid is 5% - 20%, the molar ratio of the ionizable cationic lipid is 5% - 90%, and the molar percentage of the steroid is 5% - 80%;

[0077] 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.

[0078] Preferably, the poly(ethylene glycol) lipid structure contains a hydrophobic saturated carbon chain and a hydrophilic carbon chain with 18 carbon atoms. Among them, the number of hydrophobic carbon chains is not less than 2, and the number of hydrophilic carbon chains is 1.

[0079] Preferably, the poly(ethylene glycol) lipid has one of the following two structures

[0080]

[0081] 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.

[0082] Preferably, the poly(ethylene glycol) lipid is DSPE-PEG (average molecular weight 2000 - 5000) or DSG-PEG (average molecular weight 2000 - 5000). More preferably, the poly(ethylene glycol) lipid is selected from DSPE-PEG2000, DSPE-PEG5000 or DSG-PEG2000.

[0083] The preferred lipid structure is as follows:

[0084]

[0085] Preferably, the molar percentage of the poly(ethylene glycol) lipid in the three raw materials for preparing the lipid nanoparticles is 5% - 20%. More preferably, the proportion of the poly(ethylene glycol) lipid is 8 - 15%, or the proportion of the poly(ethylene 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 will not be elaborated here one by one.

[0086] Ionizable cationic lipids are a class of lipids that can carry a positive charge under acidic conditions (pH = 5 - 7). Their structure includes an ionizable polar head and a hydrophobic chain. Among them, the polar head can be primary amine, secondary amine and tertiary amine, or quaternary ammonium salt, as well as phosphorus, guanidine, arsenic, imidazole and pyridinium groups. The above groups can carry a positive charge under acidic conditions. The main role of ionizable cationic lipids is to utilize the positive charge carried by their ionization under acidic conditions to interact with negatively charged nucleic acid drugs to achieve effective loading of nucleic acid drugs.

[0087] Preferably, the ionizable cationic lipid includes Dlin-MC3-DMA, SM102, ALC0315, cKK-E12 or C12-200. Their structures are as follows.

[0088]

[0089] In addition to the above structure, the ionizable cationic lipid also includes other compounds with an ionizable polar head and a hydrophobic chain structure.

[0090] The molar percentage of the ionizable cationic lipid in the three raw materials for preparing the lipid nanoparticles is 5% - 90%. Preferably, the proportion of the ionizable cationic lipid is 10% - 80%, or 15% - 70%, or 20% - 60%, or 30% - 50%, or 40% - 50%. Or, the proportion of the ionizable cationic lipid 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 will not be elaborated here one by one.

[0091] Preferably, the steroid includes sitosterol, stigmasterol, lanosterol, ergosterol, fucosterol, cholesterol, etc. More preferably, the steroid is selected from cholesterol.

[0092] Preferably, the molar percentage of the steroid in the three raw materials for preparing the lipid nanoparticles is 5 - 80%. Preferably, the proportion of the steroid is 15% - 70%, or 20% - 60%, or 30% - 50%, or 40% - 50%. Or, the proportion of the steroid 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 will not be elaborated here one by one.

[0093] The above lipid nanoparticles are prepared by the ethanol injection method or can be obtained by other conventional methods.

[0094] For example, this embodiment provides a method for preparing the above lipid nanoparticles, including the following steps:

[0095] Dissolve the polyethylene glycol lipid, ionizable cationic lipid, and steroid in ethanol according to the ratio to obtain a lipid mixed solution;

[0096] Dissolve the nucleic acid in a buffer salt solution to obtain a nucleic acid solution;

[0097] Mix the lipid mixed solution and the nucleic acid solution, and incubate at room temperature to obtain lipid nanoparticles loaded with nucleic acid.

[0098] Preferably, dialyze the freshly prepared lipid nanoparticles in PBS.

[0099] Preferably, the nucleic acid is dissolved in 10 mM citrate buffer (pH 4.0) at a concentration of 0.1 μg / μL.

[0100] Preferably, the total mass ratio of nucleic acid to lipid is 1 - 20 μg : 0.1 - 2 μmol. Among them, the 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, 15 g of nucleic acid corresponds to 1 mol of lipid.

[0101] In the lipid nanoparticles, in addition to the above three components, other components such as phospholipids, small molecule drugs, polypeptides, etc. can also be incorporated.

[0102] After the lipid nanoparticles are loaded with nucleic acid, the usage method is to be used in combination with microbubbles and ultrasound.

[0103] The microbubble structure is a bubble structure formed by lipid - encapsulated gas. The gas component in the microbubble structure is fluorocarbon gas or fluorosulfur gas, preferably perfluoropropane, perfluorobutane, sulfur hexafluoride; the lipid component in the microbubble structure is phospholipid or phospholipid derivative; the microbubble is preferably a phospholipid - based ultrasound contrast agent approved for clinical use to enhance imaging.

[0104] The lipid nanoparticles and microbubbles need to be injected simultaneously. The injection methods include intravenous injection, intramuscular injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, intradermal injection. The injection can be carried out by drip infusion or bolus injection.

[0105] After injection or simultaneously with injection, ultrasound needs to be applied to the target - targeted tissue. The working frequency of the ultrasound 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.

[0106] The dose of microbubbles is 5 × 10 6 microbubbles per gram of body weight, and the dose of lipid nanoparticles is 0.1 μg of mRNA per gram of body weight. After the two are mixed, an appropriate amount of normal saline is added to make the total volume 250 μL.

[0107] By applying ultrasound to the target tissue, the ultrasound can interact with the microbubbles in this area to produce stable cavitation, change the cell permeability, so that the lipid nanoparticles loaded with nucleic acid can enter the cells, realizing the expression of nucleic acid drugs, while in the parts where ultrasound is not applied, the nucleic acid drugs are rarely effective or ineffective.

[0108] The target - targeted tissues include extra - hepatic tissue organs such as the heart, blood vessels, brain, skeletal muscle, tumor, eyes, bladder, pancreas, kidneys, etc.

[0109] The delivered nucleic acids include mRNA, DNA, plasmids, sgRNA, circular RNA, siRNA, microRNA, etc. They can target endogenous targets through siRNA, microRNA, sgRNA, etc., and can also achieve the expression of exogenous proteins with therapeutic functions through mRNA, DNA, plasmids, circular RNA, etc.

[0110] Example 1

[0111] First, MC3, DSPC (distearoyl phosphatidylcholine), and cholesterol were mixed at a molar ratio of 50:10:38.5, and then combined with 8 PEG lipids (DMPE-PEG2000, DSG-PEG2000, DPPE-PEG2000, DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG5000, DOPE-PEG2000) respectively, with the molar percentage of PEG lipids being 10%.

[0112] They were respectively loaded with mRNA expressing the luciferase reporter gene, and the preparation method was as follows:

[0113] The above PEG lipids were all dissolved in ethanol at a concentration of 20 mM to prepare stock solutions. Then all the lipids were mixed according to the ratio, and the mRNA 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 rapidly 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.

[0114] The obtained lipid nanoparticles loaded with luciferase mRNA were intravenously injected into mice at a dose of 0.1 mg / kg mRNA. 3 h after injection, the luciferase substrate was injected into the mice. After 2 minutes, the mice were sacrificed, and the tumors and each major organ were taken out and subjected to bioluminescence imaging respectively to quantitatively analyze the luminescence intensity of each tissue and organ.

[0115] The results are as Figure 1As shown in b-d, it can be seen that for PEG lipids with a C18 hydrophobic chain (DSG-PEG2000, DSPE-PEG2000, DSPE-PEG5000), the mRNA expression levels in the liver and spleen are significantly reduced compared to other lipids. When the C18 hydrophobic chain is an unsaturated chain (DOPE-PEG2000), the mRNA expression levels in the liver and spleen are also relatively high. The longer the hydrophobic chain, the lower the mRNA expression levels in the liver and spleen (DMPE-PEG2000 > DPPE-PEG2000 > DSPE-PEG2000). The longer the PEG chain, the lower the mRNA expression levels in the liver and spleen (DSPE-PEG1000 > DSPE-PEG2000 > DSPE-PEG5000).

[0116] Based on this, it can be concluded that in order to achieve low mRNA expression levels in the liver and spleen, two saturated long hydrophobic chains (at least 18 carbons) should be selected, the molecular weight of the PEG chain should be greater than 1000, and preferably the PEG lipids are DSG-PEG2000, DSPE-PEG2000, DSPE-PEG5000.

[0117] Example 2

[0118] In this example, the effects of the doping ratios of DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000 on the mRNA expression levels in the liver and spleen were further explored, with DMG-PEG2000 as a control.

[0119] The molar ratios of MC3, DSPC, and cholesterol were fixed at 50:10:38.5, and the molar percentages of the PEG lipids were changed (1.5%, 5%, 7.5%, 10%). The experimental method was referred to Example 1.

[0120]

[0121] The results are as Figure 2 shown. The doping ratio of DMG-PEG2000 has little effect on the mRNA expression levels in the liver and spleen. However, for DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000, the higher the doping ratio, the lower the mRNA expression levels in the liver and spleen. In order to achieve lower mRNA expression levels in the liver and spleen, the doping ratio of the PEG lipids should be higher than 5%.

[0122] Example 3

[0123] In this example, the mRNA expression levels in each organ at different time points were further studied.

[0124] 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.

[0125] The results are as Figure 3 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 very 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 doping ratio of 10% were very low in the liver and spleen at each time point.

[0126] Example 4

[0127] This example investigated the effects of different ionizable lipids on the mRNA expression levels in various organs.

[0128] In addition to MC3, three representative ionizable lipids, SM102, cKK-E12, and C12-200, were selected as ionizable lipids. The molar ratio of ionizable lipid, DSPC, and cholesterol was fixed at 50:10:38.5, and 10% DSPE-PEG2000 was selected as the PEG lipid, with 1.5% DMG-PEG2000 as the control. The experimental method was referred to Example 1.

[0129]

[0130] The results are as Figure 4 shown. It can be seen that at 3 h after injection, compared with 1.5% DMG-PEG2000, the use of 10% DSPE-PEG2000 could significantly reduce the mRNA expression in the major organs of the heart, liver, spleen, lung, and kidney. It was shown that the selection of polyethylene glycol lipid to shield nucleic acid expression in organs was a general strategy and was not affected by the type of ionizable lipid.

[0131] Example 5 investigated the mRNA expression of lipid nanoparticles in the ultrasound irradiation area

[0132] The four different lipid nanoparticles prepared in Example 3 were respectively mixed with microbubbles (provided by Beijing Feiruida Medical Technology Co., Ltd., mainly composed of phospholipids encapsulating perfluoropropane gas) (the dose of microbubbles was 5 × 10 per gram of body weight 6Microbubbles, with the dose of mRNA in lipid nanoparticles being 0.1 μg per gram of body weight, were mixed, and an appropriate amount of normal saline was added to make the total volume 250 μL. Then, they were infused via the tail vein of mice. At the same time, ultrasound was applied to the right leg of the mice (ultrasound frequency: 1 MHz, sound pressure: 0.4 MPa). After ultrasound, bioluminescence imaging was used to continuously monitor for 7 days.

[0133] 30 days after the first administration, when the bioluminescence signal disappeared, four different lipid nanoparticles were separately injected with microbubbles for the second time. At the same time, ultrasound was applied to the right leg of the mice. After ultrasound, bioluminescence imaging was used to continuously monitor for 7 days.

[0134] The results are as Figure 5 shown. When using the control group lipid nanoparticles (1.5% DMG-PEG2000), not only was there a high mRNA expression level at the site where ultrasound was applied after injection, but also there was a high-intensity mRNA expression level throughout the body in the first two days. However, when using lipid nanoparticles with 10% doping ratios of DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000, the mRNA expression was almost only in the right leg where ultrasound was applied, while the mRNA expression levels throughout the body were very low. This result indicates that lipid nanoparticles with 10% DSG-PEG2000, DSPE-PEG2000, and DSPE-PEG5000 not only show an obvious inhibitory effect on off-target effects but also can achieve specific expression of mRNA at the ultrasound site.

[0135] It can be seen that lipid nanoparticles formed by combining PEG lipids (DSG-PEG2000, DSPE-PEG2000, DSPE-PEG5000) containing saturated C18 hydrophobic chains at a 10% ratio with MC3, DSPC, and cholesterol can achieve specific expression of mRNA at the ultrasound site while inhibiting off-target in various organs.

[0136] Example 6

[0137] In this example, the components of the lipid nanoparticles were further optimized (fixing the PEG lipid to 10% DSPE-PEG2000). Different components of lipid nanoparticles were separately mixed with microbubbles and infused via the tail vein of mice. At the same time, ultrasound was applied to the right leg of the mice. One day later, bioluminescence imaging was used to quantitatively analyze the luminescence signal of the right leg.

[0138]

[0139] All lipids were mixed in ethanol at the ratios shown in the table above, with a total concentration of 20 mM. The 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 rapidly 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 mRNA-loaded lipid nanoparticles.

[0140] As Figure 6 shown, 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. Among the three-component lipid nanoparticles without cholesterol, changing the ratio of DSPC did not significantly improve the mRNA expression level. Among the three-component lipid nanoparticles without DSPC, when the cholesterol ratio was about 45%, the mRNA expression level was significantly increased. Thus, when the ratio of 10% DSPE-PEG2000, 45% MC3, and 45% cholesterol was used, the mRNA expression level was the highest.

[0141] Example 7 investigated the effect of different ionizable lipids on mRNA expression at the ultrasound site

[0142] In addition to MC3, four other ionizable lipids were selected in this example: SM102, ALC0315, cKK-E12, and C12-200.

[0143]

[0144] All lipids were mixed in ethanol at the ratios shown in the table above, with a total concentration of 20 mM. The 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 rapidly 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 mRNA-loaded lipid nanoparticles.

[0145] The lipid nanoparticles with different components described above were respectively mixed with microbubbles (the dose of microbubbles was 5 × 10 6 microbubbles per gram of body weight, and the dose of lipid nanoparticles was 0.1 μg of mRNA per gram of body weight. After mixing the two, an appropriate amount of normal saline was added to make the total volume 250 μL), and the mixture was infused via the tail vein of the mice. At the same time, ultrasound was applied to the right leg of the mice, and the luminescence signal of the right leg was quantified by bioluminescence imaging 1 day later.

[0146] As shown Figure 7 in the figure, except for MC3, the lipid nanoparticles obtained from other ionizable lipids can achieve specific expression of mRNA at the sonication site, and the overall mRNA expression level of the SLNP group without DSPC is higher than that of the LNP group.

[0147] Example 8 examines the particle sizes of MC3-LNP and MC3-SLNP

[0148] The particle sizes and morphological characteristics of the MC3-LNP and MC3-SLNP obtained in Example 7 were observed by cryo-electron microscopy, respectively.

[0149] The results are as Figure 8 shown. The average particle size of the lipid nanoparticles of MC3-LNP is 14.23 nm, which is smaller than the average particle size of 69.08 nm of MC3-LNP.

[0150] Example 9

[0151] This example examines the feasibility of local injection.

[0152] MC3-LNP or MC3-SLNP was mixed with microbubbles respectively (the dose of microbubbles was 7×10 7 microbubbles, and the dose of lipid nanoparticles was 0.5 μg of mRNA. After mixing the two and adding an appropriate amount of normal saline to a total volume of 25 μL), it was directly injected into the skeletal muscle of mice, and then ultrasound was applied to the injection site.

[0153] Four groups were set for injecting MC3-SLNP: (1) only injecting MC3-SLNP; (2) injecting MC3-SLNP and microbubbles simultaneously, but without ultrasound; (3) only injecting MC3-SLNP and applying ultrasound after injection; (4) injecting MC3-SLNP and microbubbles simultaneously and applying ultrasound after injection.

[0154] Two groups were set for injecting MC3-LNP: only injecting MC3-SLNP; injecting MC3-SLNP and microbubbles simultaneously and applying ultrasound after injection.

[0155] As Figure 9 shown, the combination of MC3-SLNP, microbubbles and ultrasound can achieve efficient expression of mRNA in the skeletal muscle of mice, and the expression levels of mRNA are relatively low when lacking ultrasound or microbubbles. Although MC3-LNP can also achieve efficient expression of mRNA in the skeletal muscle, it will also cause a large amount of off-target expression in the liver and spleen.

[0156] Example 10 examines the delivery of other nucleic acid drugs by MC3-SLNP

[0157] MC3-SLNP and MC3-LNP loaded with plasmid DNA capable of expressing luciferase were prepared separately.

[0158] All lipids were mixed in ethanol in a certain proportion, with a total concentration of 20 mM. The plasmid DNA expressing luciferase (containing the luciferase reporter gene driven by the 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 rapidly 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.

[0159] After being mixed with microbubbles respectively, they were intravenously infused, and ultrasound was applied to the right leg of the mice simultaneously.

[0160] As Figure 10 shown in a-b, MC3-SLNP can achieve the expression of plasmid DNA more efficiently and without obvious off-target. In addition, MC3-SLNP has higher safety than MC3-LNP. When the injection dose of DNA is 2.5 mg / kg, all mice injected with MC3-LNP died two days later, while all mice injected with MC3-SLNP survived ( Figure 10 c). This indicates the high safety and effectiveness of the ternary formulation of SLNP of the present invention.

[0161] Example 11 Investigating the Expression of SLNP Combinations in Different Organs and Tissues

[0162] The delivery effect of SLNP was evaluated using Ai9 mice. The expression of red fluorescent protein tdTomato can be achieved by delivering mRNA expressing Cre recombinase in Ai9 mouse cells, so that the expression of mRNA can be indicated by the fluorescence protein situation.

[0163] 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, with 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 mixture solution and the mRNA solution were rapidly 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.

[0164] Prepare MC3-SLNP loaded with Cre mRNA, mix it with microbubbles (the dose of microbubbles is 5 × 10 per gram of body weight 6 microbubbles, and the dose of lipid nanoparticles is 0.5 μg of mRNA per gram of body weight. After mixing the two and adding an appropriate amount of normal saline to a total volume of 250 μL), perform intravenous drip while applying ultrasound. The process is as Figure 11 shown in a.

[0165] When irradiating the mouse leg with ultrasound (ultrasound frequency is 1 MHz, sound pressure is 0.4 MPa), specific expression of red fluorescent protein in skeletal muscle can be achieved ( Figure 11 b).

[0166] When irradiating the right brain of the mouse with ultrasound (ultrasound frequency is 1 MHz, sound pressure is 0.2 MPa), specific expression of red fluorescent protein can be observed in the right brain ( Figure 11 c).

[0167] When irradiating the left carotid artery of the mouse with ultrasound (ultrasound frequency is 3.2 MHz, sound pressure is 0.2 MPa), specific expression of red fluorescent protein can be achieved in the left carotid artery, while there is no expression in the right carotid artery without ultrasound irradiation ( Figure 11 d).

[0168] When irradiating the mouse heart with ultrasound (ultrasound frequency is 3.2 MHz, sound pressure is 0.3 MPa), specific expression of red fluorescent protein can be observed in the heart ( Figure 11 e).

[0169] Example 12 examines the application of SLNP combination in gene editing

[0170] To evaluate the application of SLNP combination in treatment, in this example, SLNP was used to simultaneously load mRNA expressing Cas9 nuclease and small guide RNA (gRNA) targeting the VCAM1 gene, and its gene editing effect on the VCAM1 gene in vascular endothelial cells was evaluated.

[0171] To prepare SLNP for gene editing, DSPE-PEG2000, MC3, and cholesterol were mixed in ethanol at a molar ratio of 10:45:45, with a total concentration of 20 mM. The mRNA expressing Cas9 nuclease and the gRNA targeting the VCAM1 gene were dissolved in 10 mM citrate buffer (pH 4.0) at a mass ratio of 2:1, with a total concentration of 0.1 μg / μL. Then, the lipid mixed solution and the RNA solution were rapidly 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.

[0172] The obtained lipid nanoparticles for VCAM1 gene editing were mixed with microbubbles (the dose of microbubbles was 5 × 10 6 microbubbles per gram of body weight, and the dose of lipid nanoparticles was 2.5 μg of RNA per gram of body weight. After mixing the two and adding an appropriate amount of physiological saline to a total volume of 250 μL), they were intravenously infused through the tail vein. Ultrasound was applied to the left carotid artery of the mice, and the acoustic pressures of the ultrasound were selected as 0.2 MPa, 0.3 MPa, and 0.4 MPa respectively. One week after the experiment, the mice were sacrificed, the carotid arteries of the mice were removed, and the vascular endothelial cells of the mice were isolated. The gene editing effect of the VCAM1 gene in the endothelial cells was analyzed by next-generation sequencing.

[0173] As Figure 12 shown, compared with the right carotid artery (RCA) without applying ultrasound, the left carotid artery achieved efficient editing of the VCAM1 gene in vascular endothelial cells at three different acoustic pressures, and the highest editing efficiency could reach more than 20, while almost no editing was detected in the RCA.

[0174] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. An ultrasound-responsive lipid nanoparticle, characterized in that, The raw materials for preparing the lipid nanoparticles are as follows: 1) Polyethylene glycol lipids; 2) Ionizable cationic lipids; 3) Steroids; The molar percentage of the polyethylene glycol lipids is 5% - 20%, the molar percentage of the ionizable cationic lipids is 5% - 90%, and the molar percentage of the steroids is 15% - 70%; 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; The polyethylene glycol lipids are selected from one or more of DSPE-PEG2000, DSPE-PEG5000, and DSG-PEG2000; The ionizable cationic lipids are selected from Dlin-MC3-DMA, SM102, ALC0315, cKK-E12, or C12-200; The steroids include sitosterol, stigmasterol, lanosterol, ergosterol, fucosterol, or cholesterol.

2. Use of the lipid nanoparticles according to claim 1 in the preparation of an ultrasound-targeted drug.

3. An ultrasound-targeted drug, characterized in that, Comprising the lipid nanoparticles according to claim 1 and ultrasound microbubbles.

4. The ultrasound-targeted drug according to claim 3, wherein The gas component in the ultrasound microbubbles is a fluorocarbon gas or a fluorosulfur gas.

5. The ultrasound-targeted drug according to claim 3, characterized in that, The gas component in the ultrasound microbubbles is perfluoropropane, perfluorobutane, or sulfur hexafluoride.

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