Preparation method and application of parenchymal hepatic cell targeting gene delivery system

Abstract

The invention discloses a preparation method and application of a hepatic parenchymal cell targeting gene delivery system, and relates to construction and preparation of a hepatic parenchymal cell targeting lipid material GalNAc-PEG2000-DSPE modified MKK4-siRNA (siMKK4) loaded lipid nanoparticle gene delivery system GalNAc-LNP-siMKK4, and the hepatic parenchymal cell targeting gene delivery system can be used for active targeting gene therapy of hepatic failure. The parenchymal hepatic cell targeted gene delivery system selects siMKK4 as a liver regeneration promoting gene therapy drug, has the advantages of clear target spot and capability of accurately silencing a target gene, and can protect siMKK4 from being degraded by extracellular nuclease as a gene delivery carrier, so that the targeted gene delivery system has a good application prospect. N-acetylgalactosamine (GalNAc) is selected as a target head, so that therapeutic genes can be precisely targeted and delivered to parenchymal hepatic cells, the risk of tumorigenesis of other parts of an organism can be reduced while liver regeneration is promoted, and a new thought and a new method are provided for liver regeneration treatment of hepatic failure patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene drug delivery and disease gene therapy, and particularly relates to a preparation method of a liver parenchymal cell-targeted gene delivery system loaded with MKK4-siRNA, which can target liver parenchymal cells, promote liver regeneration, and be used for liver regeneration treatment of liver failure diseases. BACKGROUND

[0002] Acute and chronic liver diseases, including alcoholic liver injury, drug-induced liver injury, liver fibrosis, hepatitis, cirrhosis, fatty liver, and cancer, have become increasingly serious diseases worldwide and are closely related to impaired liver regeneration [Journal of Hepatology. 2023, 79(2): 516-537]. The liver has strong regenerative capacity, but acute or chronic injury can weaken this capacity, leading to liver dysfunction and liver failure. Patients undergoing liver resection, especially those with underlying chronic liver disease, often impair their liver regenerative capacity, which in turn increases the risk of acute liver failure after partial hepatectomy, with a mortality rate of 25%~30% [Mexican Journal of Gastroenterology. 2024, 89(3): 404-417]. Prognosis of surgical resection of the liver, treatment of liver failure, and congenital liver metabolic diseases all depend on liver regeneration. Therefore, promoting liver regenerative capacity is crucial for controlling liver injury and liver failure, and there is an urgent need for intervention strategies to regulate liver regeneration. However, current clinical interventions rely on liver-protective drugs to alleviate postoperative liver pathological damage through anti-inflammatory, detoxification, and cholagogue, and cannot directly promote liver regeneration. So far, there is no drug specifically approved for promoting liver regeneration. Liver parenchymal cells account for 69% of total liver cells and are the main functional cells of the liver, playing a crucial role in liver injury and regeneration. Therefore, therapies that can promote liver parenchymal cell proliferation are crucial for enhancing residual liver regenerative capacity and improving liver function.

[0003] MKK4 is a MAP2 kinase, which is part of the stress-activated protein kinase (SAPK) / mitogen-activated protein kinase (MAPK) signaling network, and plays an important role in cell proliferation and differentiation [Nature Reviews Gastroenterology & Hepatology. 2024, 21: 376]. Inhibition of MKK4 protein plays an important role in promoting liver parenchymal cell proliferation and promoting liver regeneration. Inhibition of MKK4 expression can inhibit the SAPK signaling pathway and promote the downstream pro-regenerative transcriptional program mediated by transcription factors ATF2 and ELK1. Inhibition of MKK4 can also stabilize liver parenchymal cells and inhibit liver parenchymal cell death under liver injury conditions. However, MKK4 is widely present in various tissues and organs of the human body, and if non-specific inhibition of MKK4 expression causes uncontrolled proliferation of cells in other parts, there is a risk of tumor formation. Although MKK4 small molecule inhibitors have been gradually developed, there are problems of target specificity, because MKK4 and MKK7 have similar binding pockets, which also causes MKK4 small molecule inhibitors to bind to MKK7, causing off-target. Therefore, the development of MKK4-siRNA (siMKK4) gene therapy drugs that specifically target liver parenchymal cells and selectively inhibit MKK4 expression is of great significance for liver regeneration.

[0004] Compared with small molecule inhibitors, siRNA has the advantages of clear target and precise silencing of target genes, and has received extensive attention [Signal Transduction and Targeted Therapy. 2020; 5(1): 101-125]. Lipid nanoparticles are ideal carriers for delivering genes, and are usually composed of cationic lipids, helper lipids, and cholesterol. Cationic lipids can form liposome / siRNA complexes with negatively charged siRNA through electrostatic interactions, protecting siRNA from degradation by extracellular nucleases. At the same time, the complex is also easy to bind to the negatively charged cell surface, which is beneficial for the delivery of siRNA into cells through cell membrane fusion or receptor-mediated endocytosis. Lipid nanoparticles have good liver targeting, so many are developed for the treatment of liver diseases [Molecular Therapy. 2024; 32(2): 284-312]. Although lipid nanoparticles have good liver organ targeting, they cannot distinguish between different types of cells in the liver, making it difficult to precisely target liver parenchymal cells, causing liver toxicity, poor gene silencing, and poor liver regeneration.

[0005] Therefore, it is necessary to improve the above-mentioned defects by means of new preparation methods, and the present application selects N-acetylgalactosamine (GalNAc) as a target head to accurately target and deliver the therapeutic gene siMKK4 to liver parenchymal cells, reduce damage to other tissues and organs, and realize specific liver regeneration. SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the present application provides a preparation method of a liver parenchymal cell-targeted gene delivery system and application thereof. The purpose of the present application is to provide a preparation method of a liver parenchymal cell-targeted gene delivery system. The drug delivery system utilizes the easy modification characteristics of the surface of the lipid nanoparticles, and prepares N-acetylgalactosamine (GalNAc)-modified siMKK4-loaded lipid nanoparticles. The liver parenchymal cell-targeted gene delivery system is prepared by an ethanol injection method, and siMKK4 is selected as a liver regeneration-promoting drug. The drug delivery system has the advantages of clear target and accurate silencing of target genes. The lipid nanoparticles protect siMKK4 from degradation by extracellular nucleases as a gene delivery carrier. GalNAc is selected as a target head to accurately target and deliver the gene to liver parenchymal cells. The drug delivery system can reduce the risk of tumor formation in other parts while promoting liver regeneration, and provides a new idea and method for the treatment of liver failure.

[0007] The technical solution adopted by the present application to solve the technical problems comprises the following: A preparation method of a liver parenchymal cell-targeted gene delivery system, wherein DSPE-PEG 2000 -NH2 and GalNAc treated by acylation reaction are used as raw materials, and the two are subjected to an amidation reaction to obtain a liver parenchymal cell-targeted lipid material GalNAc-PEG 2000 -DSPE. 2000 -DSPE, cholesterol, (2,3-dioleyloxypropyl)-trimethylammonium chloride (DOTAP), and distearoylphosphatidylcholine (DSPC) are used to load MKK4-siRNA (siMKK4) by an ethanol injection method, and then a liver parenchymal cell-targeted gene delivery system GalNAc-LNP-siMKK4 is prepared.

[0008] The specific steps of the preparation method of the liver parenchymal cell-targeted gene delivery system are as follows: Step (1): GalNAc is stirred at room temperature until it is completely dissolved in pyridine to obtain a pyridine solution. Butanedioic anhydride and 4-dimethylaminopyridine are added to the pyridine solution, and stirred at a certain reaction temperature for a certain reaction time. The reaction solution is concentrated by reduced pressure distillation, precipitated with ethyl ether, filtered to remove the ethyl ether, and then vacuum dried to obtain GalNAc-COOH.

[0009] Step (2): DSPE-PEG 2000-NH2 is stirred at room temperature until it is completely dissolved in dimethylformamide, and the GalNAc-COOH obtained in step (1) and 1H-benzotriazole-1-yl oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and triethylamine are added, and the mixture is stirred at a certain reaction temperature for a certain reaction time, and the reaction solution is concentrated by distillation under reduced pressure. The concentrate is dissolved in ethanol, and the solution is transferred to a dialysis bag (molecular weight cut-off 1000 Da), and dialyzed in pure water, and the dialysate is collected and freeze-dried to obtain a hepatocyte-targeting lipid material GalNAc-PEG 2000 -DSPE.

[0010] Step (3): GalNAc-PEG 2000 -DSPE, cholesterol, DOTAP and DSPC are dissolved in anhydrous ethanol to obtain a carrier solution, siMKK4 is dissolved in DEPC water, and the siMKK4 solution is added dropwise to the carrier solution under ultrasonic water bath to obtain liquid A; the liquid A is collected in a round-bottom flask, and the ethanol is removed by rotary evaporation under reduced pressure; the rotary evaporated solution is stabilized at room temperature to obtain a hepatocyte-targeting gene delivery system GalNAc-LNP-siMKK4 loaded with siMKK4.

[0011] In the step (1), the molar ratio of GalNAc, succinic anhydride and 4-dimethylaminopyridine is 1:0.5:0.1-1:5:2, the reaction temperature is 0-100 ℃, and the reaction time is 0.1-24 h.

[0012] In the step (2), the molar ratio of DSPE-PEG 2000 -NH2, GalNAc-COOH, PyBOP and triethylamine is 1:0.5:0.5:0.5-1:7.5:7.5:12.5, the reaction temperature is 0-100 ℃, and the reaction time is 0.1-24 h.

[0013] In the step (3), the molar ratio of GalNAc-PEG 2000 -DSPE, cholesterol, DOTAP and DSPC is 0.5:40:50:10-10:40:50:0.5; the ultrasonic time is 0.1-3 h, the ultrasonic temperature is 0-60 ℃; the rotary evaporation time is 0.1-12 h, the rotary evaporation temperature is 25-60 ℃; and the stabilization time is 0.1-6 h.

[0014] The hepatocyte-targeting gene delivery system is used for preparing a liver regeneration drug for treating liver failure diseases.

[0015] The beneficial effects of the present application are: 1. The present application first uses siMKK4 as a regenerative drug, and uses a liver parenchymal cell-targeting lipid material GalNAc-PEG 2000 -DSPE to prepare a gene-loaded nanoparticle GalNAc-LNP-siMKK4, which can load siMKK4 and target delivery to liver parenchymal cells, enhance the regenerative effect of siMKK4, reduce the toxic side effects on other organs and cells, and reduce the risk of siMKK4 tumor formation in other parts.

[0016] 2. The activity evaluation experiment results of the obtained liver parenchymal cell-targeting gene delivery system GalNAc-LNP-siMKK4 also show that the drug delivery system can realize targeted delivery to liver parenchymal cells, and exhibit high in vitro and in vivo liver regeneration promoting effects. Moreover, the treatment does not cause damage to other major organs, and has a good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Fig. 1 is a transmission electron microscope image of GalNAc-LNP-siMKK4 (scale: 100 nm), and Fig. 1b is a particle size distribution diagram of GalNAc-LNP-siMKK4; Figure 2 Fig. 2 is a targeting experiment of GalNAc-LNP-siMKK4 (scale: 20 μm); Figure 3 Fig. 3 is the proliferation promotion situation of AML-12 cells in different treatment groups (scale: 20 μm); Figure 4 Fig. 4 is the liver regeneration promotion situation of mice in different treatment groups (20x); Figure 5 Fig. 5 is an H&E staining diagram of the main organs of mice after treatment (20x). DETAILED DESCRIPTION

[0018] The specific embodiments of the present application are further described below through examples. The following examples are used to illustrate the present application, but are not used to limit the scope of the present application.

[0019] The present application takes liver parenchymal cells as a specific targeting intervention target, uses the high expression characteristic of asialoglycoprotein receptors on the surface of liver parenchymal cells, takes the ligand GalNAc as a targeting group, and uses a lipid nanoparticle as a drug delivery carrier to prepare a liver parenchymal cell-targeting gene delivery system GalNAc-LNP-siMKK4. The drug delivery system can specifically target liver parenchymal cells, play a role in promoting liver parenchymal cell regeneration by inhibiting MKK4 expression, thereby realizing precise targeting and accumulation, and achieving an anti-liver regeneration treatment effect.

[0020] The present application uses DSPE-PEG 2000-NH2 and GalNAc treated by acylation reaction as raw materials, both of which are obtained by amidation reaction to obtain the targeting material GalNAc-PEG 2000 -DSPE, the obtained targeting material GalNAc-PEG 2000 -DSPE, cholesterol, DOTAP and DSPC are dissolved in anhydrous ethanol to obtain a carrier solution, siMKK4 is dissolved in DEPC water, and the siMKK4 solution is added dropwise into the carrier solution under ultrasonic water bath to obtain liquid A, the ultrasonic time is 20 min, and the ultrasonic temperature is 37℃; liquid A is collected in a round-bottom flask, and the ethanol solution is removed by rotary evaporation under reduced pressure, the rotary evaporation time is 15 min, and the rotary evaporation temperature is 45℃; the solution after rotary evaporation is stabilized at room temperature, and the stabilization time is 30 min, to obtain the siMKK4-loaded targeting lipid nanoparticles GalNAc-LNP-siMKK4.

[0021] Example 1 Preparation and characterization of liver parenchymal cell targeting gene delivery system GalNAc-LNP-siMKK4 Step (1): 20 mg of GalNAc was weighed and stirred at room temperature until completely dissolved in pyridine to obtain a pyridine solution, then succinic anhydride (9 mg, 1.0 eq) and 4-dimethylaminopyridine (5.5 mg, 0.5 eq) were added, and the reaction was carried out at 40℃ for 2 h, then the reaction solution was concentrated by distillation under reduced pressure, precipitated with ethyl ether, filtered to remove ethyl ether, and vacuum dried to obtain GalNAc-COOH.

[0022] Step (2): 100 mg of DSPE-PEG 2000 -NH2 was stirred at room temperature until completely dissolved in dimethylformamide, then GalNAc-COOH (86.7 mg, 3.0 eq) obtained in step (1) and PyBOP (140.5 mg, 3.0 eq) and triethylamine (45.6 mg, 5.0 eq) were added, and the reaction was stirred at a reaction temperature of 37℃ for 0.5 h, then the reaction solution was concentrated by distillation under reduced pressure and dissolved in ethanol, then the solution was transferred to a dialysis bag (molecular weight cut-off 1000 Da) and dialyzed with pure water, and the dialysate was freeze-dried to obtain the liver parenchymal cell targeting lipid material GalNAc-PEG 2000 -DSPE.

[0023] Step (3): GalNAc-PEG 2000 -DSPE, cholesterol, DOTAP and DSPC (molar ratio 5:40:50:5) were dissolved in anhydrous ethanol to obtain a carrier solution, siMKK4 was dissolved in DEPC water, and the siMKK4 solution was added dropwise into the carrier solution under ultrasonic water bath to obtain liquid A, the ultrasonic time was 20 min, and the ultrasonic temperature was 37℃; liquid A was collected in a round-bottom flask, and the ethanol solution was removed by rotary evaporation under reduced pressure, the rotary evaporation time was 15 min, and the rotary evaporation temperature was 45℃; the solution after rotary evaporation was stabilized at room temperature, and the stabilization time was 30 min, to obtain the siMKK4-loaded targeting lipid nanoparticles GalNAc-LNP-siMKK4.

[0024] The particle size results of the GalNAc-LNP-siMKK4 gene delivery system are shown in Table 1. Figure 1 As shown in Table 1, the particle size of the GalNAc-LNP-siMKK4 gene delivery system is 175.9 ± 0.8 nm, and the distribution coefficient PDI is 0.11 ± 0.01, and the particle size distribution range is narrow (PDI < 0.3), that is, a uniformly dispersed nano drug delivery system is obtained.

[0025] Example 2 Evaluation of the targeting of the liver parenchymal cell-targeted gene delivery system GalNAc-LNP-siMKK4 The targeting was evaluated by GalNAc competitive uptake inhibition experiment. After AML-12 cells were cultured with 0 or 7 μM GalNac for 1 h, the cells were incubated with LNP-siMKK4 and GalNac-LNP-siMKK4 for 6 h, and then fixed with 4% paraformaldehyde solution for 20 min. After the cell nuclei were stained with DAPI (0.5 g / mL) for 5 min, the cells were observed by CLSM.

[0026] The results of the GalNAc competitive uptake experiment show that Figure 2 after the addition of free GalNAc, the competitive binding of GalNAc to the asialoglycoprotein receptor on the surface of AML-12 cells significantly reduces the uptake of GalNac-LNP-siMKK4 nanoparticles by the cells, but has no effect on the uptake of GalNac-LNP-siMKK4 nanoparticles without the GalNAc targeting head. It can be seen that the uptake of GalNac-LNP-siMKK4 nanoparticles by GalNAc can be competitively inhibited, which verifies that the constructed gene delivery system can achieve active targeting delivery and treatment of AML-12 cells.

[0027] Example 3 In vitro study on the liver regeneration promoting effect of the liver parenchymal cell-targeted gene delivery system GalNAc-LNP-siMKK4 AML-12 cells (5 × 10 4Cells were seeded into 24-well plates at a density of 1 x 104cells / well. After 12 h, cells were treated with CCl4(8 mM) for 4 h, and cells without CCl4treatment were used as a control group. Then, the medium was replaced with medium containing LNP-siNC, LNP-siMKK4, and GalNac-LNP-siMKK4 (equivalent to 50 nM siMKK4) for 6 h. Then, the drug-containing medium was replaced with DMEM / F12 complete medium, and after 48 h of incubation, the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% serum at room temperature for 1 h. The cells were incubated with Ki67 antibody at 4°C overnight. The next day, after washing 3 times with PBST, Alexa Fluor 594-labeled secondary antibody was added and incubated at room temperature for 1 h in the dark. The nuclei were stained with DAPI (0.5 μg / mL) for 5 min, and then observed by CLSM.

[0028] As shown in Figure 3 The in vitro hepatoregeneration-promoting experiment results show that the GalNac-LNP-siMKK4 group has a higher expression of Ki67 and has the best in vitro hepatoregeneration-promoting effect.

[0029] Example 4 In vivo hepatoregeneration-promoting effect of hepatocyte-targeted gene delivery system GalNAc-LNP-siMKK4 Select 5-6 weeks old healthy Kunming mice as experimental animals, and randomly divide them into 5 groups: normal group, CCl4+ PH group, CCl4+ PH+ LNP-siNC, CCl4+ PH+ LNP-siMKK4 and CCl4+ PH+ GalNac-LNP-siMKK4 group. After adapting to the environment for 1 week, the normal group is given the same amount of normal saline for 4 weeks, and the other groups are injected with CCl4 intraperitoneally to construct a liver cirrhosis model. 20% CCl4 oil solution is injected 2 times a week for a total of 4 weeks. On the first day of the fifth week, each group is injected with normal saline, normal saline, LNP-siNC, LNP-siMKK4 and GalNac-LNP-siMKK4 through the tail vein, respectively, wherein the concentration of siNC and siMKK4 is 0.9 mg / kg. On the second day of the fifth week, left lobe resection is performed. On the third day of the fifth week, each group is injected with normal saline, normal saline, LNP-siNC, LNP-siMKK4 and GalNac-LNP-siMKK4 through the tail vein, respectively, wherein the concentration of siNC and siMKK4 is 0.9 mg / kg. On the fifth day of the fifth week, when the treatment is completed, the mice are sacrificed to collect blood, heart, liver, spleen, lung and kidney. The liver regeneration effect is determined by liver photos, H&E staining, Ki67 staining and TUNEL staining. The treatment safety of nanoparticles on major organs (heart, spleen, lung, kidney) is evaluated using H&E staining.

[0030] The in vivo evaluation experiment of promoting liver regeneration shows that after GalNac-LNP-siMKK4 treatment, the liver is red and shiny, and the cirrhosis spots are reduced Figure 4 . H&E staining shows that GalNac-LNP-siMKK4 treatment protects the liver microstructure and significantly reduces the necrotic area of liver cells. Ki67 positive cell signal shows that the number of proliferative cells treated by GalNac-LNP-siMKK4 is 5 times that of the model group and 2 times that of the LNP-siMKK4 group, indicating that it can promote liver regeneration and has better treatment effect than the non-targeted LNP-siMKK4 group. The above results show that the liver parenchymal cell targeted gene delivery system GalNac-LNP-siMKK4 constructed by the application has good effects in reducing cell apoptosis and promoting liver regeneration. From the H&E staining results of heart, spleen, lung and kidney, it can be seen that GalNac-LNP-siMKK4 treatment also does not cause damage to the tissues and organs Figure 5 . Therefore, the GalNac-LNP-siMKK4 constructed by the application not only has excellent liver regeneration promoting effect, but also has good treatment safety, which has important clinical guiding significance for the research of liver regeneration drugs in liver failure treatment.

Claims

1. A method for preparing a liver parenchymal cell-targeted gene delivery system, characterized in that, Includes the following steps: With DSPE-PEG 2000 Using -NH2 and N-acetylgalactosamine (GalNAc) treated with an acylation reaction as raw materials, the two are subjected to an amidation reaction to obtain the liver parenchymal cell-targeting lipid material GalNAc-PEG. 2000 -DSPE, the resulting targeted lipid material GalNAc-PEG 2000 The hepatocyte-targeted gene delivery system GalNAc-LNP-siMKK4 was prepared by loading MKK4-siRNA (siMKK4) onto DSPE, cholesterol, (2,3-dioleoxypropyl)-trimethylammonium chloride (DOTAP), and distearate phosphatidylcholine (DSPC) via ethanol injection.

2. The method for preparing the liver parenchymal cell-targeted gene delivery system according to claim 1, characterized in that, The preparation method of the liver parenchymal cell-targeted gene delivery system includes the following specific steps: Step (1): GalNAc is stirred at room temperature until it is completely dissolved in pyridine to obtain a pyridine solution. Succinic anhydride and 4-dimethylaminopyridine are added to the pyridine solution. The mixture is stirred at a certain reaction temperature for a certain reaction time. The reaction solution is concentrated by vacuum distillation. The solution is precipitated with ice-cold ether. After filtering to remove the ether, it is dried under vacuum to obtain GalNAc-COOH. Step (2): Add DSPE-PEG 2000 -NH2 was stirred at room temperature until completely dissolved in dimethylformamide. GalNAc-COOH obtained in step (1), 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) and triethylamine were added. The reaction was stirred at a certain temperature for a certain time. The reaction solution was concentrated by vacuum distillation. The concentrate was dissolved in ethanol and the solution was transferred to a dialysis bag (molecular weight cutoff 1000 Da). Dialysis was performed in pure water. The dialysate was collected and freeze-dried to obtain the liver parenchymal cell-targeting lipid material GalNAc-PEG. 2000 -DSPE; Step (3): Add GalNAc-PEG 2000 -DSPE, cholesterol, DOTAP, and DSPC were dissolved in anhydrous ethanol to obtain a carrier solution. siMKK4 was dissolved in DEPC water. The siMKK4 solution was added dropwise to the carrier solution under ultrasonic water bath to obtain solution A. Solution A was collected in a round-bottom flask, and ethanol was removed by rotary evaporation under reduced pressure. The evaporated solution was stabilized at room temperature to obtain the hepatocyte-targeted gene delivery system GalNAc-LNP-siMKK4 loaded with siMKK4.

3. The method for preparing the liver parenchymal cell-targeted gene delivery system according to claim 2, characterized in that, In step (1), the molar ratio of GalNAc, succinic anhydride and 4-dimethylaminopyridine is 1:0.5:0.1 to 1:5:2, the reaction temperature is 0 ℃ to 100 ℃, and the reaction time is 0.1 to 24 h.

4. The method for preparing the liver parenchymal cell-targeted gene delivery system according to claim 2, characterized in that, In step (2), DSPE-PEG 2000 The molar ratio of -NH2, GalNAc-COOH, PyBOP to triethylamine is 1:0.5:0.5:0.5 to 1:7.5:7.5:12.5, the reaction temperature is 0 ℃ to 100 ℃, and the reaction time is 0.1 to 24 h.

5. The method for preparing the hepatocyte-targeted gene delivery system according to claim 2, characterized in that, In step (3), GalNAc-PEG 2000 - The molar ratio of DSPE, cholesterol, DOTAP, and DSPC is 0.5:40:50:10~10: The ratio of ultrasonic time to 40:50:0.5 was 0.1 h to 3 h, and the ultrasonic temperature was 0 ℃ to 60 ℃. The rotary evaporation time was 0.1 h to 12 h, and the rotary evaporation temperature was 25 ℃ to 60 ℃. The stabilization time was 0.1 h to 6 h.

6. The use of the liver parenchymal cell-targeted gene delivery system prepared by any one of claims 1-5 in the preparation of liver regeneration drugs for treating liver failure.

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