A nucleic acid delivery vector, its preparation method and application

Abstract

This invention discloses a nucleic acid delivery vector, its preparation method, and its applications, belonging to the field of drug delivery vector technology. The nucleic acid delivery vector provided by this invention is based on the LNP structure, retaining key lipids or lipid-like substances unchanged. By altering the surface composition of the LNP, it can effectively reduce the accumulation of LNP in the liver, achieving effective in-situ mRNA delivery and protein expression; thereby significantly improving LNP organ toxicity, reducing mRNA drug side effects, and enhancing mRNA drug tolerance; it achieves in-situ targeting and high nucleic acid expression by promoting in-situ uptake of the vector and enhancing endosome escape; by optimizing 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, sonication of the application site can also promote the in-situ expression level of the nucleic acid delivery vector.

Description

Technical Field

[0001] This invention relates to the field of drug delivery carrier technology, and in particular to a nucleic acid delivery carrier, its preparation method, and its application. Background Technology

[0002] Messenger RNA (mRNA) is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. The development of mRNA vaccines has been significantly driven by infectious diseases caused by RNA viruses. Theoretically, all diseases treated with protein therapies can be addressed with mRNA therapies, and mRNA drugs can enter the cytoplasm and express specific proteins intracellularly, a feature unmatched by traditional protein drugs. Therefore, mRNA holds broad application prospects in fields such as vaccines, tumor immunotherapy, protein replacement, Cas9 gene editing, in vivo CAR-T therapy, and antibody drugs.

[0003] Safe and efficient delivery is one of the biggest challenges in developing mRNA therapies and a major technical hurdle restricting the development of mRNA drugs. On the one hand, mRNA is easily degraded when exposed to the in vivo environment alone; on the other hand, its large molecular weight and negative charge make it difficult to overcome a series of biological barriers to reach intracellular proteins for expression. Therefore, developing effective RNA vaccines and immune adjuvant delivery systems is crucial for mRNA vaccine development.

[0004] In recent years, researchers have explored various materials, such as lipids, lipid-like substances, polymers, peptides, proteins, extracellular vesicles, and viruses, as mRNA delivery systems. Lipid nanoparticles (LNPs), formed by lipids and lipid-like substances with mRNA, have proven to be the best carriers for mRNA delivery and are currently the only mRNA delivery system used clinically. Currently marketed mRNA vaccines all use LNPs as their delivery system; however, compared to mRNA vaccines, mRNA drugs require LNPs to deliver higher doses and exhibit better tolerability.

[0005] LNPs, typically around 100 nanometers in diameter, can effectively load mRNA molecules, protect them from enzymatic degradation, and improve their internalization efficiency. However, studies have shown that long-term use of LNP-mRNA can lead to hepatotoxicity. In-situ targeted therapies, which act directly on the lesion site to achieve therapeutic goals, can significantly reduce the organ toxicity of LNPs and alleviate 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 purpose of this invention is to provide a nucleic acid delivery vector, its preparation method, and its application, in order to solve the problem of hepatotoxicity caused by LNP-mRNA, and to provide an mRNA delivery vector with stronger in situ targeting and higher expression levels.

[0007] To achieve the above objectives, the present invention provides a nucleic acid delivery carrier comprising the following components in molar amounts: 20-80 ionizable lipids, 1-30 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, 15-50 structural lipids, and 0.25-10 PEG-lipids.

[0008] Preferably, ionizable lipids are replaced with equimolar amounts of cationic lipids; cationic lipids are lipid molecules that are positively charged under physiological pH conditions.

[0009] Preferably, the composition includes 30-60% ionizable lipids or cationic lipids, 10-30% 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, 15-40% structural lipids, and 0.25-5% PEG-lipids.

[0010] Preferably, the composition includes 40-60 ionizable lipids or cationic lipids, 10-20 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, 15-40 structural lipids, and 0.5-3 PEG-lipids.

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

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

[0013] Preferably, the ionizable lipids and cationic lipids 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, coccosterol, lycopene, campesterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, ergocalciferol, or campesterol.

[0015] Preferably, the PEG-lipid is a PEG lipid polymer with PEG modified at one end, having a molecular weight of no more than 2000, and is one or more of PEG1000-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.

[0016] Preferably, it also includes active lipids; the molar amount of active lipids accounts for 10-50% of the total molar amount of components; the active lipids are tefloxacin and / or oxytetracycline or their salts; the salts are hydrochloride, hydrobromide, sulfate, aminosulfonate, phosphate, nitrate, acetate, propionate, succinate, glycolate, stearate, lactate, malate, tartrate, citrate, ascorbate, pyrate, maleate, hydroxymaleate, phenylacetate, glutamate, benzoate, salicylate, p-aminobenzenesulfonate, 2-acetoxybenzoate, fumarate, toluenesulfonate, methanesulfonate, ethanedisulfonate, oxalate or hydroxyethanesulfonate.

[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 noncoding RNA (lncRNA), microRNA (miRNA), small activating RNA (saRNA), multi-coding nucleic acid (MCNA), polymerized coding nucleic acid (PCNA), guide RNA (gRNA), CRISPR RNA (crRNA), and ribozymes.

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

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

[0020] Preferably, the nitrogen-phosphorus molar ratio of the prepared nucleic acid delivery vector is 3 to 12:1; more 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; dialysis is performed using 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 trihydrogen citrate; preferably sodium citrate and potassium citrate.

[0023] The application of the nucleic acid delivery carrier described above in the preparation of nucleic acid drugs, wherein the nucleic acid drug uses the nucleic acid delivery carrier encapsulating nucleic acid as the active ingredient; it 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, flavoring agents, 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, a topical preparation, or an injectable preparation.

[0025] Nucleic acid drugs target mammals.

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

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

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

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

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

[0031] Preferably, the duty cycle of the ultrasonic action is 40-80%.

[0032] Therefore, the nucleic acid delivery vector, its preparation method, and its application provided by this invention have the following specific technical effects:

[0033] (1) The nucleic acid delivery vector provided by the present invention is based on the LNP structure, retains the key lipids or lipids unchanged, and by changing the surface composition of LNP, it can effectively reduce the accumulation of LNP in the liver, realize effective in situ delivery of mRNA and protein expression; thereby achieving 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 acid by promoting the in situ uptake of the vector and enhancing the escape of endosomal components. By optimizing each component and parameter 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 can also promote the in situ expression level of the nucleic acid delivery vector by sonicating the application part.

[0036] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 These are animal imaging images and fluorescence intensity statistics of mice injected with different groups of mRNA-LNP in the efficacy test 1 of this invention; where A is animal imaging image of mRNA-LNP mice prepared in injection groups 1-3; B is the statistical result of fluorescence intensity in the muscle and liver of mice in A; C is animal imaging image of mRNA-LNP mice prepared in injection groups 2 and 8; D is the statistical result of fluorescence intensity in the muscle and liver of mice in C; E is animal imaging image of mRNA-LNP mice prepared in injection groups 8-11; F is the statistical result of fluorescence intensity in the muscle and liver of mice in E; G is the statistical result of fluorescence intensity in the muscle and liver of mRNA-LNP mice prepared in injection groups 10 and 12-14. Animal imaging images; H shows the statistical results of fluorescence intensity in the muscle and liver of mice in group G; I shows the animal imaging images of mice prepared with Moderna's commercially available mRNA-1273 (control) and mRNA-LNP (optimized) prepared in injection group 10; J shows the statistical results of fluorescence intensity in the muscle of mice in group I; K shows the statistical results of fluorescence intensity in the liver of mice in group I; L shows the animal imaging images of mice prepared with Moderna's commercially available mRNA-1273 (control) and mRNA-LNP (optimized) prepared in injection group 10; M shows the statistical results of Fluc fluorescence intensity in the liver of mice in group L; N shows the statistical results of DID (LNP distribution) fluorescence intensity in the liver of mice in group L.

[0039] Figure 2These are animal imaging images and fluorescence intensity statistics of mRNA-LNP mice prepared in injection groups 1, 10, and 15-18 in the efficacy test 1 of this invention; where A is animal imaging image of mRNA-LNP mice prepared in injection groups 1, 10, and 15-18; and B is the fluorescence intensity statistics of mRNA-LNP mice prepared in injection groups 1, 10, and 15-18.

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

[0041] Figure 4 These are animal imaging images, fluorescence intensity statistics, and total isolated protein crown statistics for mRNA-LNP (optimized) and Moderna commercial prescription mRNA-1273 (control) prepared in injection group 10 in the efficacy test 3 of this invention; where A is an animal imaging image; B is the fluorescence intensity statistics; and C is the total isolated protein crown statistics.

[0042] Figure 5 The results of expression detection of mRNA-LNP prepared by adding different active lipids in test 4 of the present invention in HeLa cells and 293T cells; where A is HeLa cells; B is 293T cells;

[0043] Figure 6 These are the in situ expression results from efficacy tests 5 and 6 of this invention; where A is an animal imaging photograph of mRNA-LNP mice prepared in injection groups 21, 23, and 28; B is the statistical result of the relative expression level of mice in A; C is an animal imaging photograph of mRNA-LNP mice prepared in injection groups 23 and 28 and after storage for 7 days; D is the statistical result of the relative expression level in C; E is an animal imaging photograph of mRNA-LNP mice prepared in injection groups 21-26; F is the statistical result of the relative expression level of mice in E; G is an animal imaging photograph of mRNA-LNP mice prepared in injection groups 21, 27-31; and H is the statistical result of the relative expression level of mice in G.

[0044] Figure 7 These are the results of laser confocal microscopy and lysosome colocalization in test 6 of the present invention; where A is the laser confocal microscopy image; and B is the lysosome colocalization result.

[0045] Figure 8These are animal imaging images and expression level statistics of mice after injection of mRNA-LNP prepared with different sodium citrate concentrations in test 7 of this invention; where A is animal imaging image of mice after injection of mRNA-LNP prepared with different sodium citrate concentrations in groups 10 and 32-33; B is the fluorescence intensity statistics in A; C is animal imaging image of mice injected with mRNA-LNP prepared with different nitrogen-phosphorus ratios in groups 10 and 34-35; D is animal imaging image of mice injected with mRNA-LNP prepared with mRNA ...

[0046] Figure 9 This is the result of the in situ expression assessment of mRNA-LNP under different ultrasound parameters in the efficacy test 8 of this invention; where A represents the particle size of mRNA-LNP (optimized - 150mM) and Moderna commercial formulation mRNA-1273 (control) prepared in injection groups 10 (optimized) and 32 (with and without ultrasound assistance); B represents the encapsulation efficiency of mRNA-LNP (optimized - 150mM) and Moderna commercial formulation mRNA-1273 (control) prepared in injection groups 10 (optimized) and 32 (with and without ultrasound assistance); C represents the cell transfection efficiency of mRNA-LNP (optimized - 150mM) and Moderna commercial formulation mRNA-1273 (control) prepared in injection groups 10 (optimized) and 32 (with and without ultrasound assistance); D represents the particle size of mRNA-LNP (optimized - 150mM) prepared in injection groups 10 (optimized) and 32 (with and without ultrasound assistance). Animal imaging images of mice treated with mRNA-LNP (optimized -150mM) at different times after injection (0mM) and Moderna commercial prescription mRNA-1273 (control); E shows the statistical results of relative muscle expression in D; F shows animal imaging images of mice treated with mRNA-LNP (optimized -150mM) at different times after injection (group 32); G shows the statistical results of relative muscle expression in F; H shows animal imaging images of mice treated with mRNA-LNP (optimized -150mM) at different ultrasound frequencies; I shows the statistical results of relative muscle expression in H; J shows animal imaging images of mice treated with mRNA-LNP (optimized -150mM) at different ultrasound intensities; K shows the statistical results of relative muscle expression in J; L shows animal imaging images of mice treated with mRNA-LNP (optimized -150mM) at different ultrasound duty cycles; M shows the statistical results of relative muscle expression in L. Detailed Implementation

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

[0048] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all descriptions of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0049] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail in the embodiments are all conventional techniques in the art.

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

[0051]

[0052] Example 1

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

[0054] (1) Prepare an aqueous phase by dissolving the mRNA encoding luciferase in a sodium citrate solution with a pH of 4 and a concentration of 50 mM, so that the concentration of the mRNA encoding luciferase in the solution is 170 ng / μL.

[0055] An organic phase was prepared by dissolving each lipid in anhydrous ethanol at a molar ratio to achieve a lipid-to-mRNA ratio (nitrogen / phosphorus ratio, N / P) of 6:1. The composition information of each group in the organic phase is shown in Table 1.

[0056] Table 1 Organic phase composition information for different treatments

[0057]

[0058]

[0059] (2) The aqueous phase and organic phase prepared in step (1) were rapidly mixed and encapsulated using a fishbone-type microfluidic system at a volume ratio of 3:1. Then, all the encapsulated products were dialyzed overnight in PBS buffer at 4°C and pH=7.4 to restore the pH to neutral (pH~7), thus obtaining lipid nanoparticles (mRNA-LNP) encapsulated with luciferase mRNA.

[0060] Effect Test 1

[0061] The in situ expression detection of the mRNA-LNP prepared in Example 1 is as follows:

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

[0063] (2) The mRNA-LNP prepared in each group in Example 1 was adjusted to a concentration of 0.5 μg / μL using PBS. Then, 100 μL was injected into the leg muscle of Balb / C mice (female; 7 weeks old; weight 22g). Three biological replicates were set up for each group. At 6 h after injection, the distribution and expression of luciferase in mice were detected by a small animal imaging instrument, and the expression site and fluorescence value 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 significantly reduced the expression of mRNA-LNP in the liver, with the mRNA-LNP prepared at a molar ratio of 10 showing the most significant reduction in liver expression. Compared with mRNA-LNP prepared by other PEG lipids, mRNA-LNP prepared by PEG 1000-DMG significantly reduced its expression in the liver. Cholesterol content had no significant effect on liver expression.

[0065] Table 2. Statistical table of encapsulation efficiency, particle size, PDI, and potential data.

[0066]

[0067] The mRNA-LNP prepared by the combination of low molecular weight PEG lipids (molecular weight <1500) and high content neutral lipid DOPE (groups 8-14 and 16, 18-20) showed a significant inhibitory effect on liver targeting of LNP, achieving in situ targeting of mRNA-LNP. Furthermore, the combination also showed a significant inhibitory effect on liver targeting of other ionizable lipids (groups 16, 18), indicating that the combination has universality 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 detailed below:

[0070] mRNA-LNP, prepared in a molar ratio of Group 10, was used as a solvent in PBS to prepare solutions with concentrations of 0.2 mg / kg, 0.5 mg / kg, and 1 mg / kg. Thirty-five Balb / C mice (female; 7 weeks old; weighing 22 g) were randomly divided into 7 groups. Three groups were randomly selected and injected intramuscularly into the leg with 100 μL of the 0.2 mg / kg, 0.5 mg / kg, and 1 mg / kg solutions, respectively. Three more groups were randomly selected and injected with an equal volume of mRNA-LNP prepared in the molar ratio of Group 1. The remaining group was injected with an equal volume of PBS as a control group. Injections were given once every week for a total of 3 times. Twenty-four hours after the third injection, the levels of the hepatotoxic marker ALT and the inflammatory marker IL-1β in the blood were detected by ELISA. The results are as follows: Figure 3 As shown, the mRNA-LNP prepared at a molar ratio of Group 10 exhibited low hepatotoxicity upon intramuscular injection, especially at high doses (1 mg / kg), where the blood ALT level was significantly lower than that of the control group. Furthermore, the mRNA-LNP in the control group induced severe inflammation and a significant increase in IL-1β secretion in mice during in vivo circulation, while the mRNA-LNP prepared at a molar ratio of Group 10 did not cause hepatotoxicity or inflammatory responses. Therefore, the nanoparticle composition provided by this invention significantly improves the safety of use.

[0071] Effect Test 3

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

[0073] mRNA-LNPs were prepared using the method described in Example 1, at the molar ratio shown in Group 10, except that 0.1 mol% DiD probe was added to the lipids during the preparation process. The probe binds to phospholipids and exhibits red fluorescence. The prepared mRNA-LNPs were dissolved in PBS to form a solution with a concentration of 0.1 mg / mL. 100 μL of this solution was then injected intramuscularly into the leg muscles of Balb / C mice (female; 7 weeks old; weighing 22 g) as the optimization group. An equal volume of mRNA-LNPs prepared in Group 1 at the same concentration was injected as the control group. Blood samples were collected from the tail vein at 2 h and 4 h post-injection and immediately dissolved in 1% SDS solution. Standard curves were plotted using blood samples from the optimization and control groups to detect the distribution of LNPs in the blood. Six h after injection, the mice were dissected, and the heart, liver, spleen, lung, and kidney were harvested for fluorescence imaging to detect the distribution of LNPs in the organs. The results are as follows: Figure 4 As shown, compared with the control group, the optimized group significantly reduced the distribution of LNP in blood and organs.

[0074] The mRNA-LNP prepared by the optimized group was co-incubated with mouse serum at 37°C, and then the protein crown content was quantified using the BCA method. The results are as follows: Figure 4As shown, the optimized group exhibited significantly higher adsorption of protein corona than the control group. DIA analysis of the quantitative protein corona components yielded results shown in Table 3, identifying 20 enhanced expression proteins, indicating that the in-situ targeted mRNA-LNP formulation prepared in this invention adsorbed increased levels of immunoglobulins 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 related protein corona, thereby rapidly clearing LNPs entering the circulation.

[0075] Table 3. Results of quantitative protein composition analysis

[0076]

[0077] Example 2

[0078] An in-situ targeting vector was constructed to encapsulate mRNA using a composition with added active lipids, as detailed below:

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

[0080] Table 4 Organic phase composition information for 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 part (2) of Example 1, that is, to obtain mRNA-LNP with added active lipids.

[0086] Effect Test 4

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

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

[0089] Effect Test 5

[0090] The in situ expression detection of the mRNA-LNP prepared in Example 2 is as follows:

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

[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 levels of the mRNA-LNPs prepared in each group in Example 2 were tested, as follows:

[0095] (1) After storing the mRNA-LNP prepared in Example 2 at 4°C for 30 days, the concentration of mRNA-LNP prepared in each group in Example 2 was adjusted to 50 ng / μL with PBS. Then, 100 μL was injected intramuscularly into the leg of Balb / C mice (female; 7 weeks old; weight 22 g). Five biological replicates were set up for each group. Six hours after injection, the distribution and expression of luciferase in mice were detected by a small animal imaging instrument, and the expression site and fluorescence value were recorded by photograph. The control group was the mice that were injected intramuscularly immediately after preparation using the same method.

[0096] Further optimization of the added content (10%-50% molar percentage of the total of the aforementioned four components) and verification of mRNA-LNP stability - by storing at 4 degrees Celsius for 7 days, intramuscular injection was performed to detect LNP expression levels and compare them with the expression levels 7 days prior to observe stability.

[0097] (2) CY5 fluorescently labeled RNA was transfected into HeLa cells, and laser confocal imaging was used to localize the RNA and lysosomes, verifying that the addition of gatifloxacin could enhance the efficiency of endosome escape.

[0098] 20% (v / v) of CY5-mRNA-LNP (solvent: anhydrous ethanol) was added to the aqueous phase prepared in Example 1 (1). The mixture was rapidly mixed using a fishbone microfluidic system. After transfecting HELA cells for 6 hours, the cell nuclei and lysosomes were stained and observed under a laser confocal microscope. The lysosomes and mRNA were co-located using ImageJ software (red and green fluorescence) to assess the escape of integrities from the cells.

[0099] The results are as follows Figure 6 and Figure 7 As shown, by Figure 6 It can be seen that LNPs with added active lipid gatifloxacin hydrochloride are more stable than those with oxytetracycline hydrochloride (stable at 4℃), indicating that the addition of active lipid gatifloxacin hydrochloride can significantly promote and stabilize in situ expression. Figure 7 It can be seen that the liver did not express gatifloxacin hydrochloride with the addition of 20% active lipid, but the in situ expression by promoting endosome escape was 200% that of the control group, indicating that the addition of gatifloxacin hydrochloride with active lipid promotes in situ expression by promoting endosome escape.

[0100] Example 3

[0101] An in-situ targeting vector was constructed to encapsulate mRNA using a lipid nanoparticle composition, as detailed below:

[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 group of the organic phase is shown in Table 5.

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

[0104] Table 5. Organic phase composition information for each group

[0105]

[0106]

[0107] Effect Test 7

[0108] The in situ expression of each mRNA-LNP prepared in Example 3 was detected using the method described in Effect Test 1. The results are as follows: Figure 8 As shown, a citrate concentration of 150 mM promotes in situ expression, and LNPs are more stable when N / P = 6.

[0109] Effect Test 8

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

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

[0112] (2) Intramuscular injection was performed using the method described in Effect Test 1. Two hours after the injection, an ultrasonic physiotherapy device was applied to the injected muscle site. The parameters of the ultrasonic physiotherapy device were set as follows: frequency 1Hz, intensity 1W / cm². 2 The duty cycle was 80%, and the treatment time was 10 min. The promoting effect of ultrasound on the in situ expression of mRNA-LNP prepared in groups 1, 10 molar ratio, and 32 was evaluated. The results are as follows: Figure 9 As shown, only group 32 (optimized -150mM) combined with sonication showed an expression level of 200% of the original level. This demonstrates the effectiveness of constructing a stable mRNA-LNP structure.

[0113] (3) Using the same method as in (2), except that the ultrasound treatment time was 1h, 2h, 3h and 4h after injection, and the results were as follows: Figure 9 As shown, ultrasound was used to promote mRNA expression 1-3 hours after injection, but no enhancement effect was observed 4 hours after injection. This is presumably because by 4 hours after injection, no mRNA-LNP was in situ in the tissue fluid, and it had already entered muscle cells or entered the bloodstream through circulation. Therefore, ultrasound could not promote expression.

[0114] (4) Using the same method as in (2), except that the ultrasonic frequency is set to 1Hz and 3Hz respectively, the duty cycle is 80%, and the intensity is 1W / cm. 2 The action time was 10 minutes, and the results were as follows: Figure 9 As shown, 3Hz significantly enhanced mRNA expression, reaching 500% of the control group.

[0115] (5) Using the same method as in (2), except that the ultrasonic intensity is set to 0.5 W / cm. 2 1W / cm 2 and 2W / cm 2 The operating frequency was 3Hz, the duty cycle was 80%, and the operating time was 10 minutes. The results are as follows: Figure 9 As shown, 0.5-1W / cm 2 The promoting effect gradually increases, reaching 2W / cm 2 The expression of mRNA-LNP was significantly reduced. This indicates that low pulse intensity can promote the expression of mRNA-LNP, while increasing pulse intensity may disrupt the LNP structure, thereby reducing the promotion of mRNA-LNP expression.

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

[0117] Therefore, the nucleic acid delivery vector provided by this invention is based on the LNP structure, retaining key lipids or lipid-like substances unchanged. By altering the surface composition of LNP, the accumulation of LNP in the liver can be effectively reduced, achieving effective in-situ mRNA delivery and protein expression. This results in significantly improving LNP organ toxicity, reducing mRNA drug side effects, and enhancing mRNA drug tolerance. In-situ targeting and high nucleic acid expression are achieved by promoting in-situ uptake of the vector and enhancing endosome escape. Optimization of various components and parameters or the addition of active lipids can enhance the in-situ expression level and stability of the prepared nucleic acid delivery vector. After use, sonication of the application site 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 and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions 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 components include the following molar amounts: 20-80 ionizable lipids or cationic lipids, 1-30 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, 15-50 structural lipids, and 0.25-10 PEG-lipids. Cationic lipids are lipid molecules that carry a positive charge under physiological pH conditions; The ionizable lipids and cationic lipids are one or more of SM102, ALC0315, CKK-E12, 5A2-SC8, DLin-MC3-DMA and C12-200; PEG-lipids are PEG lipid polymers with PEG modified at one end, with a molecular weight not greater than 2000, and are 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. It also includes active lipids; the molar amount of active lipids accounts for 10-50% of the total molar amount of components; the active lipids are gatifloxacin and / or oxytetracycline or their salts; the salts are hydrochloride, hydrobromide, sulfate, aminosulfonate, phosphate, nitrate, acetate, propionate, succinate, glycolate, stearate, lactate, malate, tartrate, citrate, ascorbate, pyrate, maleate, hydroxymaleate, phenylacetate, glutamate, benzoate, salicylate, p-aminobenzenesulfonate, 2-acetoxybenzoate, fumarate, toluenesulfonate, methanesulfonate, ethanedisulfonate, oxalate or hydroxyethanesulfonate.

2. The nucleic acid delivery vector according to claim 1, characterized in that: The structural lipids are one or more of the following: cholesterol, sitosterol, coccosterol, lycopene, rapeseed sterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, alfalfa sterol, or ergocalciferol.

3. The nucleic acid delivery vector according to claim 1, characterized in that: Nucleic acid is one or more of ASO, RNA, and DNA; RNA is one or more of interfering RNA, short hairpin RNA, antisense RNA, messenger RNA, modified messenger RNA, long noncoding RNA, microRNA, small activating RNA, polynucleotide, guide RNA, CRISPR RNA, and ribozyme.

4. A method for preparing a nucleic acid delivery vector as described in any one of claims 1 to 3, characterized in that: Nucleic acid is dissolved in a salt solution to obtain an aqueous phase, and other components are dissolved in anhydrous ethanol to obtain an organic phase. The aqueous and organic phases are then rapidly mixed and dialyzed overnight.

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

6. The use of a nucleic acid delivery vector as described in any one of claims 1 to 3 in the preparation of nucleic acid drugs, characterized in that: Nucleic acid drugs use nucleic acid delivery carriers that encapsulate nucleic acids as active ingredients; they 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, flavoring agents, stabilizers, antioxidants, preservatives, and pyrogen-free water; cellulose and its derivatives include sodium carboxymethyl cellulose and cellulose acetate.

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