Nucleic acid delivery carrier as well as preparation method and application thereof

Abstract

The invention discloses a nucleic acid delivery carrier as well as a preparation method and application thereof, and belongs to the technical field of drug delivery carriers. The nucleic acid delivery carrier provided by the invention is based on an LNP structure, key lipids or lipidoids are kept unchanged, accumulation of the LNP in the liver can be effectively reduced by changing the surface composition of the LNP, and effective mRNA in-situ delivery and protein expression are realized; further, the effects of greatly improving the LNP organ toxicity, relieving the mRNA drug side effect and improving the mRNA drug tolerance are achieved; in-situ targeting and high expression of nucleic acid are realized by promoting in-situ intake of the carrier and enhancing escape of endosome; the in-situ expression level and stability of the prepared nucleic acid delivery carrier can be enhanced by optimizing the components and parameters or adding active lipid; after use, ultrasound is performed on the application part, and the in-situ expression level of the nucleic acid delivery vector can also be promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery vectors, and in particular to a nucleic acid delivery vector and a preparation method and application thereof. Background Art

[0002] Messenger RNA (mRNA), also known as messenger RNA, is a type of single-stranded RNA transcribed from a single strand of DNA. Infectious diseases caused by RNA viruses have significantly driven the development of mRNA vaccines. In theory, all diseases treated with proteins can be addressed with mRNA therapies. Furthermore, mRNA drugs can enter the cytoplasm and express specific proteins within the cell, a feat unmatched by traditional protein drugs. Therefore, mRNA holds broad application prospects in vaccines, tumor immunotherapy, protein replacement, Cas9 gene editing, in vivo Car-T therapy, and antibody drugs.

[0003] Safe and effective delivery is one of the biggest challenges in developing mRNA therapeutics and a technical obstacle that hinders the development of mRNA drugs. On the one hand, mRNA is easily degraded when exposed to the body's environment alone. On the other hand, its large molecular weight and negative charge make it difficult for it to pass through a series of biological barriers to reach cells and express proteins. Therefore, developing an effective RNA vaccine and immune adjuvant delivery system is key to mRNA vaccine research and development.

[0004] In recent years, people have tried to use various materials, such as lipids, lipidoids, polymers, peptides, proteins, extracellular vesicles, and viruses, as delivery systems for mRNA. Lipid nanoparticles (LNPs), formed by lipids and lipidoids and mRNA, have been proven to be the best carriers for delivering mRNA and are currently the only mRNA delivery system used in clinical practice. Currently available mRNA vaccines all use LNPs as a delivery system. However, compared to mRNA vaccines, mRNA drugs require LNPs to have higher delivery doses and better tolerability.

[0005] LNPs, typically around 100 nanometers in diameter, can effectively load mRNA molecules, protecting them from enzymatic degradation and improving their internalization efficiency. However, studies have shown that long-term use of LNP-mRNA can cause hepatotoxicity. In situ targeted drugs, which primarily act directly at the site of disease, can significantly reduce the organ toxicity of LNPs and mitigate the side effects and tolerability of mRNA drugs, providing strong support for the application of LNP / mRNA in areas such as protein replacement therapy. Summary of the Invention

[0006] The present application aims to provide a nucleic acid delivery carrier, a preparation method and application thereof, and to provide an mRNA delivery carrier with stronger in-situ targeting and higher expression level to solve the problem of liver toxicity caused by LNP-mRNA.

[0007] To achieve the above-mentioned purpose, the present application provides a nucleic acid delivery carrier comprising the following components in molar parts: ionizable lipid 20-80, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine 1-30, structural lipid 15-50, PEG-lipid 0.25-10.

[0008] Preferably, the ionizable lipid is replaced with an equimolar amount of cationic lipid; the cationic lipid is a lipid molecule with positive charge under physiological pH conditions.

[0009] Preferably, the ionizable lipid or cationic lipid is 30-60, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine is 10-30, structural lipid is 15-40, and PEG-lipid is 0.25-5.

[0010] Preferably, the ionizable lipid or cationic lipid is 40-60, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine is 10-20, structural lipid is 15-40, and PEG-lipid is 0.5-3.

[0011] Preferably, the ionizable lipid or cationic lipid is 45-55, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine is 13-16, structural lipid is 35-50, and PEG-lipid is 0.5-2.5.

[0012] Preferably, the molar ratio of ionizable lipid or cationic lipid: neutral lipid: cholesterol: PEG-lipid = 50:16:38.5:1.5.

[0013] Preferably, the ionizable lipid and cationic lipid are one or more of SM102, ALC0315, CKK-E12, 5A2-SC8, DLin-MC3-DMA and C12-200.

[0014] Preferably, the structural lipid is one or more of cholesterol, sitosterol, coprostanol, friedelin, brassicasterol, ergosterol, tomatidine, ursolic acid, alpha-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol.

[0015] Preferably, the PEG-lipid is a PEG-lipid polymer modified with PEG at one end, with a molecular weight of no more than 2000, such as one or more of PEG 1000-DMG, PEG 1200-DMG, PEG 2000-DMG, PEG 1500-DMG, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0016] Preferably, the active lipid is also included; the molar fraction of the active lipid is 10-50% of the total molar fraction; the active lipid is sitafloxacin and / or terramycin or a salt thereof; the salt is hydrochloride, hydrobromide, sulfate, sulfamate, phosphate, nitrate, acetate, propionate, succinate, glycolate, stearate, lactate, malate, tartrate, citrate, ascorbate, pamoate, maleate, hydroxymaleate, phenylacetate, glutamate, benzoate, salicylate, p-aminobenzenesulfonate, 2-acetoxybenzoate, fumarate, toluenesulfonate, methanesulfonate, ethanedisulfonate, oxalate, or isethionate.

[0017] Preferably, the nucleic acid is one or more of ASO, RNA, and DNA; the RNA is one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), and ribozyme.

[0018] Preferably, the nucleic acid delivery carrier is a nanoparticle or a liposome; the liposome includes a lipid bilayer.

[0019] A method for preparing the nucleic acid delivery carrier as described above, dissolving the nucleic acid in a salt solution to obtain an aqueous phase, dissolving other components in anhydrous ethanol to obtain an organic phase, rapidly mixing the aqueous phase and the organic phase, and dialyzing overnight.

[0020] Preferably, the nucleic acid delivery carrier prepared has a nitrogen-phosphorus molar ratio of 3-12:1; preferably, the nitrogen-phosphorus molar ratio is 6:1.

[0021] Preferably, the salt solution is citrate, sodium acetate, or CitPhos, with a concentration of 100-200 mmol / L and pH=4; the dialysis uses PBS buffer.

[0022] Citrates include cations based on alkali metals and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc., and can also be hydrogen citrate, dihydrogen citrate and tricitrate; preferably, sodium citrate and potassium citrate.

[0023] A use of the nucleic acid delivery vector as described above in the preparation of a nucleic acid drug, wherein the nucleic acid drug has the nucleic acid delivery vector encapsulating the nucleic acid as an active ingredient; the nucleic acid drug may also include a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier includes cellulose and its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavorings, stabilizers, antioxidants, preservatives, inert excipients and pyrogen-free water; cellulose and its derivatives include sodium carboxymethyl cellulose, sodium ethyl cellulose and cellulose acetate.

[0024] Preferably, the nucleic acid drug is an oral preparation, an external preparation or an injectable preparation.

[0025] The target of nucleic acid drugs is mammals.

[0026] Preferably, auxiliary ultrasound after administration can increase the expression level of nucleic acid.

[0027] Preferably, the ultrasound time is 1 to 4 hours after injection.

[0028] Preferably, the ultrasonic time is 2 hours.

[0029] Preferably, the ultrasonic action frequency is 1 to 3 Hz.

[0030] Preferably, the ultrasonic intensity is 0.5 to 1 W / cm 2 .

[0031] Preferably, the duty cycle of the ultrasound action is 40 to 80%.

[0032] Therefore, the present invention provides a nucleic acid delivery vector and its preparation method and application, and its specific technical effects are as follows:

[0033] (1) The nucleic acid delivery vector provided by the present invention is based on the LNP structure, retaining key lipids or lipidoids unchanged. By changing the surface composition of LNP, it can effectively reduce the accumulation of LNP in the liver, achieve effective in situ mRNA delivery and protein expression, and thus achieve the effects of significantly improving LNP organ toxicity, reducing mRNA drug side effects, and improving mRNA drug tolerance;

[0034] (2) The nucleic acid delivery vector provided by the present invention achieves in situ targeting and high expression of nucleic acids by promoting the in situ uptake of the vector and enhancing endosomal escape; by optimizing the components and parameters or adding active lipids, the in situ expression level and stability of the prepared nucleic acid delivery vector can be enhanced;

[0035] (3) After use, the nucleic acid delivery vector provided by the present invention is subjected to ultrasound on the administered portion, which can also promote the in situ expression level of the nucleic acid delivery vector.

[0036] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. 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 any creative labor.

[0038] Figure 1 These are the animal imaging instrument detection photos and fluorescence intensity statistical results of mice injected with different groups of mRNA-LNP in the effect test 1 of the present invention; wherein A is the animal imaging instrument detection photo of the mRNA-LNP mice prepared by injection groups 1 to 3; B is the statistical result of the fluorescence intensity of the muscles and livers of the mice in A; C is the animal imaging instrument detection photo of the mRNA-LNP mice prepared by injection groups 2 and 8; D is the statistical result of the fluorescence intensity of the muscles and livers of the mice in C; E is the animal imaging instrument detection photo of the mRNA-LNP mice prepared by injection groups 8 to 11; F is the statistical result of the fluorescence intensity of the muscles and livers of the mice in E; G is the mRNA-LNP mice prepared by injection groups 10 and groups 12 to 14. Animal imaging detection photos; H is the statistical result of fluorescence intensity of muscle and liver of mice in G; I is the animal imaging detection photo of mice injected with Moderna's commercial prescription mRNA-1273 (control) and mRNA-LNP (optimized) prepared by injection group 10; J is the statistical result of fluorescence intensity of muscle of mice in I; K is the statistical result of fluorescence intensity of liver of mice in I; L is the animal imaging detection photo of mice injected with Moderna's commercial prescription mRNA-1273 (control) and mRNA-LNP (optimized) prepared by injection group 10; M is the statistical result of Fluc fluorescence intensity of mouse liver in L; N is the statistical result of DID (LNP distribution) fluorescence intensity of mouse liver in L;

[0039] Figure 2These are the animal imaging instrument detection photos and fluorescence intensity statistical results of the mRNA-LNP mice prepared by injection groups 1, 10, and 15-18 in the efficacy test 1 of the present invention; wherein A is the animal imaging instrument detection photo of the mRNA-LNP mice prepared by injection groups 1, 10, and 15-18; B is the fluorescence intensity statistical result of the mRNA-LNP mice prepared by injection groups 1, 10, and 15-18;

[0040] Figure 3 These are the test results of hepatotoxicity and IL-1β levels in the blood after different dosages in the efficacy test 2 of the present invention; wherein A is the test result of ALT level; B is the test result of AST level; and C is the test result of IL-1β level.

[0041] Figure 4 These are the animal imaging test photos, fluorescence intensity statistical results, and isolated protein crown total amount statistical results after injection of the mRNA-LNP (optimized) prepared in Group 10 and Moderna's commercially available mRNA-1273 (control) in efficacy test 3 of the present invention; wherein A is the animal imaging test photo; B is the fluorescence intensity statistical result; C is the isolated protein crown total amount statistical result;

[0042] Figure 5 These are the expression test results of mRNA-LNP prepared by adding different active lipids in effect test 4 of the present invention in Hela cells and 293t cells; A represents Hela cells; B represents 293t cells;

[0043] Figure 6 These are the in situ expression results in effect test 5 and effect test 6 of the present invention; wherein A is an animal imaging instrument detection photo of mRNA-LNP mice prepared in injection groups 21, 23, and 28; B is the statistical result of the relative expression level of the mice in A; C is an animal imaging instrument detection photo of mRNA-LNP mice prepared in injection groups 23 and 28 and stored for 7 days; D is the statistical result of the relative expression level in C; E is an animal imaging instrument detection photo of mRNA-LNP mice prepared in injection groups 21-26; F is the statistical result of the relative expression level of the mice in E; G is an animal imaging instrument detection photo of mRNA-LNP mice prepared in injection groups 21, 27-31; H is the statistical result of the relative expression level of the mice in G;

[0044] Figure 7 These are the laser confocal microscopy images and lysosome co-localization results in effect test 6 of the present invention; A is the laser confocal microscopy image; B is the lysosome co-localization result;

[0045] Figure 8are the animal imaging instrument detection photos of mice after injection of mRNA-LNPs prepared with different sodium citrate concentrations in effect test 7 of the application and the expression amount statistical results of mice; wherein A is the animal imaging instrument detection photo of mice after injection of mRNA-LNPs prepared with different sodium citrate concentrations in group 10, group 32-33; B is the fluorescence intensity statistical result in A; C is the animal imaging instrument detection photo of mice after injection of mRNA-LNPs with different nitrogen-phosphorus ratios in group 10, group 34-35; D is the animal imaging instrument detection photo of mice after injection of mRNA-LNPs prepared in group 10, group 34-35 after storage at 4°C for 30 days; E is the particle size statistical result in C, D; F is the PDI statistical result in C, D; G is the fluorescence intensity statistical result in C, D;

[0046] Figure 9 are the in situ expression investigation results of mRNA-LNPs under different ultrasound action parameters in effect test 8 of the application; wherein A is the particle size of mRNA-LNPs (optimization-150mM) prepared in group 10 (optimization), group 32 and Moderna commercial prescription mRNA-1273 (control) with ultrasound assistance and without ultrasound assistance; B is the encapsulation rate of mRNA-LNPs (optimization-150mM) prepared in group 10 (optimization), group 32 and Moderna commercial prescription mRNA-1273 (control) with ultrasound assistance and without ultrasound assistance; C is the cell transfection efficiency of mRNA-LNPs (optimization-150mM) prepared in group 10 (optimization), group 32 and Moderna commercial prescription mRNA-1273 (control) with ultrasound assistance and without ultrasound assistance; D is the animal imaging instrument detection photo of mRNA-LNPs (optimization-150mM) prepared in group 10 (optimization), group 32 and Moderna commercial prescription mRNA-1273 (control) with ultrasound assistance and without ultrasound assistance; E is the relative expression amount statistical result of muscle in D; F is the animal imaging instrument detection photo of mice after ultrasound treatment at different times after injection of mRNA-LNPs (optimization-150mM) prepared in group 32; G is the relative expression amount statistical result of muscle in F; H is the animal imaging instrument detection photo of mice after injection of mRNA-LNPs (optimization-150mM) prepared in group 32 treated with different ultrasound action frequencies; I is the relative expression amount statistical result of muscle in H; J is the animal imaging instrument detection photo of mice after injection of mRNA-LNPs (optimization-150mM) prepared in group 32 treated with different ultrasound action intensities; K is the relative expression amount statistical result of muscle in J; L is the animal imaging instrument detection photo of mice after injection of mRNA-LNPs (optimization-150mM) prepared in group 32 treated with different ultrasound action duty cycles; M is the relative expression amount statistical result of muscle in L. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0048] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0049] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources; the methods and steps not described in detail in the examples are conventional techniques in the art.

[0050] The full Chinese and English names of the abbreviations used in the examples are shown in the following table:

[0051]

[0052] Example 1

[0053] The in situ targeting vector encapsulating mRNA (mRNA-LNP) was constructed as follows:

[0054] (1) Prepare the aqueous phase and dissolve the mRNA encoding luciferase in a 50 mM sodium citrate solution at pH 4 to a concentration of 170 ng / μL of the mRNA encoding luciferase.

[0055] To prepare the organic phase, each lipid component was dissolved in anhydrous ethanol at a molar ratio such that the ionized lipid:mRNA ratio (nitrogen to phosphorus ratio, N / P) was 6:1. The composition of each organic phase component is shown in Table 1.

[0056] Table 1 Composition information of organic phase under different treatments

[0057]

[0058]

[0059] (2) The aqueous phase and the organic phase prepared in step (1) were rapidly mixed and encapsulated by a fishbone microfluidic system at a volume ratio of 3:1, and then the entire mixed and encapsulated product was dialyzed overnight in a PBS buffer at 4°C and pH = 7.4 to restore the pH to neutral (pH ~ 7), thereby obtaining lipid nanoparticles (mRNA-LNPs) encapsulating luciferase mRNA.

[0060] Effect Test 1

[0061] The mRNA-LNP prepared in Example 1 was subjected to in situ expression detection, as follows:

[0062] (1) The particle size, PDI, and encapsulation efficiency of the mRNA-LNP prepared in Example 1 were analyzed using a particle size analyzer and an RNA fluorescence (RiboGreen) quantitative detection kit, respectively. The results are shown in Table 1.

[0063] (2) The mRNA-LNP prepared in each group of Example 1 was adjusted to a concentration of 0.5 μg / μL using PBS, and then 100 μL was injected into the leg muscles of Balb / C mice (female; 7 weeks old; weight 22 g). Three biological replicates were set up for each group. The distribution and expression of luciferase in the mice were detected by small animal imaging at 6 h after injection, and the expression sites and fluorescence values ​​were recorded by taking pictures.

[0064] The results are as follows Figure 1 、 Figure 2 As shown in Table 2, intramuscular injection of mRNA-LNP prepared by replacing DSPC with DOPE can significantly reduce the expression of mRNA-LNP in the liver, among which the mRNA-LNP prepared with a molar ratio of group 10 has the most significant reduction in liver expression; compared with mRNA-LNP prepared with other PEG lipids, mRNA-LNP prepared with PEG 1000-DMG can significantly reduce liver expression; cholesterol content has no significant effect on liver expression.

[0065] Table 2 Statistics of encapsulation efficiency, particle size, PDI and potential data

[0066]

[0067] The mRNA-LNPs prepared by the combination of low molecular weight PEG lipids (molecular weight <1500) and high content neutral lipid DOPE (groups 8-14 and groups 16, 18-20) had a significant inhibitory effect on the liver targeting of LNP, achieving in situ targeting of mRNA-LNP. In addition, other groups of ionizable lipids (groups 16, 18) under this combination also had a significant inhibitory effect on liver targeting, indicating that this combination has universal applicability for achieving in situ targeting.

[0068] Effect Test 2

[0069] The mRNA-LNP prepared in Example 1 was subjected to toxicity and inflammation tests, as follows:

[0070] The mRNA-LNP prepared at the molar ratio of group 10 was prepared with PBS as solvent into solutions with concentrations of 0.2 mg / kg, 0.5 mg / kg and 1 mg / kg, respectively. 35 Balb / C mice (female; 7 weeks old; weighing 22 g) were randomly divided into 7 groups. Three groups of mice were randomly selected and injected with 100 μL of 0.2 mg / kg, 0.5 mg / kg and 1 mg / kg solutions into the leg muscles, and then three groups were randomly selected to be injected with the same amount of mRNA-LNP prepared at the molar ratio of group 1. The remaining group was injected with the same amount of PBS solvent as the control group. The injections were performed once every other week for a total of 3 times. 24 hours after the third injection, the levels of hepatotoxicity indicator ALT and inflammatory indicator IL-1β in the blood were detected by ELISA. The results are as follows: Figure 3 As shown, mRNA-LNP prepared at a molar ratio of Group 10 had low hepatotoxicity upon intramuscular injection, particularly at a high dose (1 mg / kg), where ALT levels in the blood were significantly lower than in the control group. Furthermore, the control group's mRNA-LNP circulated in vivo, leading to severe inflammation in mice and a significant increase in IL-1β secretion. However, the mRNA-LNP prepared at a molar ratio of Group 10 did not induce hepatotoxicity or inflammatory reactions. Therefore, the nanoparticle composition provided by the present invention significantly improves safety in use.

[0071] Effect Test 3

[0072] The in vivo pharmacokinetic study of the mRNA-LNP prepared in Example 1 was conducted as follows:

[0073] The preparation method of Example 1 was used to prepare mRNA-LNP at the molar ratio shown in Group 10, except that 0.1 mol% DiD probe was added to the lipid during the preparation process. The probe was bound to the phospholipid and had red fluorescence. The prepared mRNA-LNP was prepared into a solution with a concentration of 0.1 mg / mL using PBS as a solvent, and then 100 μL of the solution was injected into the leg muscle of Balb / C mice (female; 7 weeks old; weighing 22 g) as the optimization group, and the control group was injected with an equal amount of mRNA-LNP prepared in Group 1 at the same concentration. Blood was collected from the tail vein 2h and 4h after the injection, and immediately dissolved in a 1% SDS solution. The blood of the optimized group and the control group mice was used as a standard curve to detect the distribution of LNP in the blood. 6h after the injection, the mice were dissected, and the heart, liver, spleen, lung, and kidney were taken for fluorescence imaging to detect the distribution of LNP in the organs. The results are shown in Figure 2. Figure 4 As shown in the figure, compared with the control group, the optimized group significantly reduced the distribution of LNP in the blood and organs.

[0074] The mRNA-LNP prepared in the optimized group was co-incubated with mouse serum at 37°C, and then the protein content was quantified using the BCA method. Figure 4As shown in Table 3, the protein corona adsorbed by the optimized group was significantly higher than that of the control group. The quantitative protein corona components were analyzed using DIA, and the results are shown in Table 3. A total of 20 proteins with enhanced expression were identified, indicating that the mRNA-LNP in situ targeted formulation prepared by the present invention increased the adsorption of immune and albumin. Therefore, it is speculated that this may be because the surface of the mRNA-LNP prepared in the optimized group can adsorb a large amount of clearance-related protein corona, thereby enabling the rapid clearance of LNPs entering the circulation.

[0075] Table 3 Quantitative protein corona component analysis results

[0076]

[0077] Example 2

[0078] The composition with added active lipids was used as a raw material to construct an in situ targeted vector to encapsulate mRNA, as follows:

[0079] The aqueous phase and the organic phase were prepared by the method in part (1) of Example 1, except that the composition information of each group of organic phases is shown in Table 4.

[0080] Table 4 Composition information of organic phase under different treatments

[0081]

[0082]

[0083] The structural formula of gatifloxacin hydrochloride is

[0084] The structural formula of oxytetracycline hydrochloride is

[0085] The encapsulation method is exactly the same as that in part (2) of Example 1, that is, mRNA-LNP with added active lipids is obtained.

[0086] Effect Test 4

[0087] The expression of the mRNA-LNP prepared in Example 2 was detected as follows:

[0088] The mRNA-LNP prepared in each group of Example 2 was adjusted to a concentration of 0.1 mg / mL using PBS, and 1 μL was added to activated 293t (human renal epithelial cells) and HeLa (human cervical cancer cells) cells, respectively. The cells were incubated at 37°C under 5% CO2 conditions. The results were obtained using a luciferase reporter gene assay reagent, and 10 compounds that simultaneously enhanced expression were identified, such as Figure 5 .

[0089] Effect Test 5

[0090] The mRNA-LNP prepared in Example 2 was subjected to in situ expression detection, as follows:

[0091] The mRNA-LNP prepared in each group of Example 2 was adjusted to a concentration of 50 ng / μL using PBS, and then 100 μL was injected into the leg muscle of Balb / C mice (female; 7 weeks old; weight 22 g). Five biological replicates were set up for each group. The substrate was injected 6 hours later, and the distribution and expression of luciferase in the mice were detected by small animal imaging. The expression sites and fluorescence values ​​were recorded by photographing.

[0092] The results are as follows Figure 6 As shown, the addition of 20% gatifloxacin can form stable LNPs and significantly promote the expression efficiency of in situ targeted LNPs, indicating that the addition of active lipids can promote mRNA-LNP expression.

[0093] Effect Test 6

[0094] The stability and expression level of the mRNA-LNP prepared in each group of Example 2 were tested as follows:

[0095] (1) After the mRNA-LNP prepared in Example 2 was stored at 4°C for 30 days, the mRNA-LNP prepared in each group of Example 2 was adjusted to a concentration of 50 ng / μL with PBS. 100 μL of the mRNA-LNP was then injected intramuscularly into the leg muscles of Balb / C mice (female, 7 weeks old, weighing 22 g). Five biological replicates were set up for each group. 6 hours after injection, the distribution and expression of luciferase in the mice were detected using a small animal imaging device, and the expression sites and fluorescence values ​​were photographed and recorded. The mRNA-LNP prepared in the same manner immediately after preparation served as the control group.

[0096] The added content was further optimized (10%-50% molar ratio of the sum of the above four components) and the stability of the mRNA-LNP was verified by intramuscular injection after storage at 4 degrees Celsius for 7 days, and the LNP expression level was detected and compared with the expression level 7 days ago to observe the stability.

[0097] (2) CY5 fluorescently labeled RNA was transfected into HeLa cells, and laser confocal microscopy was used to co-localize RNA with lysosomes to verify that the addition of gatifloxacin could enhance the efficiency of endosomal escape:

[0098] To the aqueous phase prepared in (1) of Example 1, 20% volume fraction of CY5-mRNA-LNP at a concentration of 17.7 mM (solvent: anhydrous ethanol) was added, and rapid mixing was performed by fishbone microfluidics. After 6 h of transfection into HELA cells, the cell nuclei and lysosomes were stained and then observed under a laser confocal microscope. The lysosomes and mRNA (red and green fluorescence) were co-localized using Image J software to evaluate the escape of endosomal inclusions in the cells.

[0099] The results are as follows Figure 6 and Figure 7 As shown by Figure 6 It can be seen that compared with oxytetracycline hydrochloride, LNPs with added active lipid gatifloxacin hydrochloride are more stable (can be stored stably at 4°C), indicating that the addition of active lipid gatifloxacin hydrochloride can significantly promote in situ expression and stability. Figure 7 It can be seen that when 20% active lipid gatifloxacin hydrochloride was added, there was no expression in the liver, and the in situ expression by promoting endosomal escape was 200% of the control group, indicating that the addition of active lipid gatifloxacin hydrochloride promoted in situ expression by promoting endosomal escape.

[0100] Example 3

[0101] The lipid nanoparticle composition is used as a raw material to construct an in situ targeted carrier to encapsulate mRNA, as follows:

[0102] The aqueous and organic phases were prepared using the method described in part (1) of Example 1, except that the concentrations of sodium citrate in the aqueous phase were 50 mM, 150 mM, and 300 mM, respectively. The composition of each organic phase is shown in Table 5.

[0103] The encapsulation method is exactly the same as that in part (2) of Example 1, i.e., mRNA-LNP is obtained.

[0104] Table 5 Information on the composition of the organic phase in each group

[0105]

[0106]

[0107] Effect Test 7

[0108] The mRNA-LNPs prepared in Example 3 were tested for in situ expression using the method described in Effect Test 1. Figure 8 As shown, it was shown that citrate concentration of 150 mM promoted in situ expression, and LNP was more stable when N / P=6.

[0109] Effect Test 8

[0110] The effect of ultrasound assistance on the in situ expression of mRNA-LNP prepared in Example 3 was investigated as follows:

[0111] (1) 1 μg of luciferase mRNA-encapsulated LNP was added to 500 μL PBS or cell culture medium and ultrasonically treated in 24-cell culture plates. The particle size, encapsulation efficiency and cell transfection efficiency before and after ultrasonic treatment were detected. The results are as follows: Figure 9 Only group 32 (optimized - 150 mM) remained stable after ultrasonic treatment, with no significant changes in properties, demonstrating that increasing the citrate concentration in the aqueous phase enhanced the stability of LNPs.

[0112] (2) The method in effect test 1 was used for intramuscular injection. Then, 2 hours after the injection, the ultrasonic therapy device was applied to the injected muscle. The parameters of the ultrasonic therapy device were set to: frequency 1 Hz, intensity 1 W / cm 2 The effect of ultrasound on the in situ expression of mRNA-LNP prepared by group 1, group 10 and group 32 was evaluated. Figure 9 As shown, only group 32 (optimized - 150 mM) combined with ultrasound showed an expression level of 200% of the original, demonstrating the effectiveness of constructing a stable mRNA-LNP structure.

[0113] (3) The same method as in part (2) was used, except that the ultrasound exposure time was 1 h, 2 h, 3 h, and 4 h after injection. The results were as follows: Figure 9 As shown in the figure, it was found that combined use of ultrasound 1-3 hours after injection could promote mRNA expression, but there was no enhancement effect 4 hours after injection. It was speculated that at 4 hours after injection, there was no mRNA-LNP in the tissue fluid in situ, and it had entered the muscle cells or entered the blood through circulation, so combined use of ultrasound could not promote expression.

[0114] (4) The same method as in part (2) was used, except that the ultrasound frequencies were set to 1 Hz and 3 Hz, the duty cycle was 80%, and the intensity was 1 W / cm 2 , the action time is 10 minutes, the results are as follows Figure 9 As shown, 3 Hz can significantly enhance mRNA expression, which is 500% of the control group.

[0115] (5) The same method as in part (2) was used, except that the ultrasound intensity was set to 0.5 W / cm 2 , 1W / cm 2 and 2W / cm 2 , the action frequency is 3Hz, the duty cycle is 80%, the action time is 10 minutes, and the results are as follows Figure 9 As shown, 0.5-1W / cm 2 The promotion effect gradually increases, and at 2W / cm 2 This indicates that low pulse intensity can promote the expression of mRNA-LNP, and increasing pulse intensity may destroy the LNP structure and thus reduce the expression of mRNA-LNP.

[0116] (6) The same method as in part (2) was used, except that the ultrasound duty cycle was set to 40% and 80%, the action frequency was 3 Hz, and the intensity was 1 W / cm 2 , the action time is 10 minutes, the results are as follows Figure 9 As shown, increasing the duty cycle can significantly enhance expression.

[0117] Therefore, the nucleic acid delivery vector provided by the present invention is based on the LNP structure, retaining key lipids or lipidoids unchanged. By changing the surface composition of LNP, the accumulation of LNP in the liver can be effectively reduced, and effective in situ mRNA delivery and protein expression can be achieved; thereby achieving the effects of significantly improving LNP organ toxicity, reducing mRNA drug side effects, and improving mRNA drug tolerance; in situ targeting and high expression of nucleic acids are achieved by promoting the in situ uptake of the vector and enhancing endosomal escape; by optimizing the various components and parameters or adding active lipids, the in situ expression level and stability of the prepared nucleic acid delivery vector can be enhanced; after use, ultrasound of the application part can also promote the in situ expression level of the nucleic acid delivery vector.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nucleic acid delivery vector, characterized in that The invention comprises the following components in molar parts: 20-80 of ionizable lipid, 1-30 of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, 15-50 of structural lipid and 0.25-10 of PEG-lipid.

2. A nucleic acid delivery vector according to claim 1, characterized in that: The ionizable lipids are replaced with an equal molar portion of cationic lipids; cationic lipids are lipid molecules that are positively charged at physiological pH.

3. A nucleic acid delivery vector according to claim 2, characterized in that: The ionizable lipid and cationic lipid are one or more of SM102, ALC0315, CKK-E12, 5A2-SC8, DLin-MC3-DMA and C12-200.

4. A nucleic acid delivery vector according to claim 1, characterized in that: The structural lipids are one or more of cholesterol, sitosterol, coprosterol, saposterol, brassicasterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol or campesterol.

5. A nucleic acid delivery vector according to claim 1, characterized in that: PEG-lipid is a PEG lipid polymer modified with PEG at one end, with a molecular weight of no more than 2000, and is one or more of PEG 1000-DMG, PEG 1200-DMG, PEG 2000-DMG, PEG1500-DMG, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

6. A nucleic acid delivery vector according to claim 1, characterized in that: Also includes active lipids; the molar fraction of the active lipids accounts for 10-50% of the molar fraction of the total components; the active lipids are tefloxacin and / or oxytetracycline or their salts; The salt is hydrochloride, hydrobromide, sulfate, sulfamate, phosphate, nitrate, acetate, propionate, succinate, glycolate, stearate, lactate, malate, tartrate, citrate, ascorbate, pamoate, maleate, hydroxymaleate, phenylacetate, glutamate, benzoate, salicylate, p-anilinesulfonate, 2-acetoxybenzoate, fumarate, toluenesulfonate, methanesulfonate, edisylate, oxalate or isethionate.

7. A nucleic acid delivery vector according to claim 1, characterized in that: The nucleic acid is one or more of ASO, RNA and DNA; the RNA is one or more of interfering RNA, small interfering RNA, short hairpin RNA, antisense RNA, messenger RNA, modified messenger RNA, long non-coding RNA, microRNA, small activating RNA, polyencoding nucleic acid, polymeric encoding nucleic acid, guide RNA, CRISPR RNA and ribozyme.

8. A method for preparing the nucleic acid delivery vector according to any one of claims 1 to 7, characterized in that: The nucleic acid is dissolved in a salt solution to obtain an aqueous phase, and the other components are dissolved in anhydrous ethanol to obtain an organic phase. The aqueous phase and the organic phase are quickly mixed and dialyzed overnight.

9. The method for preparing a nucleic acid delivery vector according to claim 8, characterized in that: The saline solution is citrate, sodium acetate or CitPhos, with a concentration of 100-200 mmol / L and a pH of 4; PBS buffer is used for dialysis.

10. Use of the nucleic acid delivery vector according to any one of claims 1 to 7 in the preparation of a nucleic acid drug, characterized in that: Nucleic acid drugs use nucleic acid delivery vectors that encapsulate nucleic acids as active ingredients; they may also include pharmaceutically acceptable carriers; pharmaceutically acceptable carriers include cellulose and its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavorings, stabilizers, antioxidants, preservatives, and pyrogen-free water; cellulose and its derivatives include sodium carboxymethyl cellulose, sodium ethyl cellulose, and cellulose acetate.

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