Nucleic acid-small molecule drug co-delivery system as well as preparation method and application thereof

Abstract

The invention relates to the field of genetic engineering medicine and vaccine manufacturing, in particular to a nucleic acid-small molecule medicine co-delivery system and a preparation method and application thereof. According to the nucleic acid-small molecule drug co-delivery system, lipid components serve as a carrier, the carrier encapsulates drug components, and the lipid components comprise cationic lipid, neutral phospholipid, cholesterol and PEG-lipid; the medicine component comprises a nucleic acid medicine and a small molecule medicine; the particle size of the nucleic acid-small molecule drug co-delivery system is 50-300 nm. Experiments prove that the nucleic acid-small molecule drug co-delivery system disclosed by the invention has a wide application prospect in the fields of anti-inflammation, immunoregulation and anti-tumor.

Description

Technical Field

[0001] This invention relates to the field of genetically engineered drugs and vaccine manufacturing, and in particular to a nucleic acid-small molecule drug co-delivery system, its preparation method, and its application. Background Technology

[0002] Nucleic acid drugs can specifically target specific molecules, blocking or increasing the expression of corresponding proteins, and have significant application potential in anti-inflammatory, anti-tumor, and immunomodulatory fields. However, the cellular environment in the human body is complex, and single-method treatment with nucleic acid drugs often fails to achieve good results. Therapies that combine drugs with different mechanisms of action to achieve a "1+1>2" effect are gradually becoming an important research direction.

[0003] The combined delivery of small molecule drugs and small interfering RNA (siRNA) is a promising therapeutic strategy. 1) The combination of the two can achieve synergistic treatment through different mechanisms, thereby enhancing the overall efficacy; 2) silencing drug resistance genes using siRNA can overcome tumor drug resistance; 3) chemotherapy drugs can achieve better therapeutic effects at lower doses through the synergistic effect of siRNA, thereby reducing the toxic side effects of the drugs; 4) regulating the tumor microenvironment through siRNA can assist small molecule drugs in exerting their effects.

[0004] Autophagy plays a variety of roles in many diseases, particularly cancer, inflammatory diseases, neurodegenerative diseases, and immune diseases. In some cases, autophagy can provide nutrients to tumors by recycling protein deposits and damaged organelles, promoting the survival and adaptation of cancer cells in harsh microenvironments such as hypoxia and nutrient deprivation. Chloroquine and its analogues have received widespread attention in the industry as autophagy inhibitors due to their safety and excellent clinical performance.

[0005] Lipid nanoparticles (LNPs) offer high nucleic acid encapsulation efficiency and excellent physicochemical stability, making them a widely used non-viral delivery system with significant potential in gene-engineered drugs and vaccine manufacturing, particularly in clinical applications of gene therapies. Currently marketed nanoparticles consist of four components: cholesterol, phospholipids, PEG lipids, and ionizable lipids. These ionizable lipids can bind negatively charged siRNA, forming neutral lipid nanoparticles with high encapsulation efficiency. This effectively protects siRNA from degradation while enhancing its cellular uptake efficiency.

[0006] There have been attempts to enhance the efficacy of small molecule drugs by co-delivering them with nucleic acid drugs using lipid carriers. For example, Jose A. et al., AAPS PharmSciTech 19:166–175 (2018) used cationic lipids to co-deliver curcumin and STAT3 siRNA for the treatment of skin cancer. Oh, HR et al., Nanomaterials 6:141 (2016) co-loaded doxorubicin and siRNA for the treatment of liver cancer. Lin, C. et al., J. Drug Target. 27:797–805 (2019) developed a GE-11 peptide-coupled liposome co-delivery system loaded with gemcitabine and siRNA for the targeted treatment of pancreatic cancer.

[0007] Kulkarni JA et al., Langmuir 37(24):7312-7319 (2021) developed an LNP system for co-encapsulating the hydrophobic small molecule drug amphotericin B (AmpB) and siRNA using a rapid mixing technique; Slaughter KV et al., Adv Mate 15:e2403701 (2024) designed an ionizable analog of the selective estrogen receptor degrader fulvestrant to replace the ionizable lipids in traditional lipid nanoparticles, autonomously encapsulating siRNA to form colloidal nanoparticles, thus achieving the co-delivery of ionizable small molecule drugs and siRNA.

[0008] Given the above background, it is essential to study nucleic acid-small molecule drug co-delivery systems. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a nucleic acid-small molecule drug co-delivery system, its preparation method and application.

[0010] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0011] Technical Topic 1

[0012] A nucleic acid-small molecule drug co-delivery system, wherein the co-delivery system uses a lipid component as a carrier, and the carrier encapsulates the drug component;

[0013] The lipid components include cationic lipids, neutral phospholipids, cholesterol, and PEG-lipids;

[0014] The drug components include nucleic acid drugs and small molecule drugs.

[0015] As a further improvement of the present invention, the particle size of the co-delivery system is 50-300 nm;

[0016] As a further improvement of the present invention, the particle size of the co-delivery system is 100-250 nm; as a further improvement of the present invention, the particle size of the co-delivery system is 150-220 nm.

[0017] As a further improvement of the present invention, the cationic lipid is at least one of 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) (SM-102), and ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315);

[0018] The neutral phospholipids are selected from the following compounds: distearylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), soybean lecithin (SL), egg yolk lecithin (EPC), hydrogenated soybean lecithin (HSPC), hydrogenated egg yolk lecithin (HEPC), phosphatidic acid, and sphingomyelin;

[0019] The PEG lipid is selected from one or more of the following compounds: PEG lipid is selected from PEG-modified distearate phosphatidylethanolamine (DSPE-PEG), PEG-modified dimyristoyl glycerol (DMG-PEG), PEG-modified dipalmitoyl phosphatidylethanolamine (DPPE-PEG), PEG-modified dimyristoyl phosphatidylethanolamine (DMPE-PEG), PEG-modified dilauroyl phosphatidylethanolamine (DLPE-PEG), PEG-modified ceramide (Ceramide-PEG), and mPEG-modified bis(tetradecyl)acetamide (mPEG-DTA).

[0020] As a further improvement of the present invention, the cationic lipid is methyl 4-(N,N-dimethylamino)butyrate (dilinyl) ester;

[0021] As a further improvement of the present invention, the molecular weight of the PEG or mPEG is 1000-5000.

[0022] As a further improvement of the present invention, the nucleic acid drug is selected from one or more of mRNA, siRNA, dsRNA, snRNA, tRNA, rRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, lncRNA, shRNA, ssDNA, aiRNA, nucleic acid aptamers, and ribozymes.

[0023] The small molecule drug is selected from chloroquine and its analogues, including 4-[(7-chloroquinoline-4-yl)amino]-2-[(diethylamino)methyl]phenol (amodiquine), 2-[[4-[(7-chloroquinoline-4-yl)amino]pentyl](ethyl)amino]ethanol (hydroxychloroquine), or quinine.

[0024] As a further improvement of the present invention, the chloroquine and its analogues are 2-[[4-[(7-chloroquinoline-4-yl)amino]pentyl](ethyl)amino]ethanol.

[0025] As a further improvement of the present invention, the chloroquine and its analogues are quinine.

[0026] As a further improvement of the present invention, the nucleic acid drug is siRNA.

[0027] Technical Theme Two

[0028] A method for preparing a nucleic acid-small molecule drug co-delivery system as described in Technical Subject 1, comprising the following steps:

[0029] S1: Dissolve cationic lipids, neutral phospholipids, cholesterol, and PEG-lipids in solvent I respectively; dissolve nucleic acid drugs and small molecule drugs in solvent II to form aqueous solution B;

[0030] S2: Mix the cationic lipids, neutral phospholipids, cholesterol, and PEG-lipid solutions obtained in S1 in a certain proportion to form organic phase solution A.

[0031] S3: Using a microfluidic system, solution A and solution B are mixed at a flow rate ratio of 1:1-5 to obtain mixture D. Mixture D is then subjected to ultrafiltration and centrifugation to collect the resulting sample E.

[0032] S4: Incubate the collected sample E with the small molecule drug solution C at 50-60℃ for 15-45 min, and then ultrafilter and centrifuge to obtain the nucleic acid-small molecule drug co-delivery system.

[0033] As a further improvement of the present invention, solvent I is ethanol and solvent II is citrate-sodium citrate buffer.

[0034] As a further improvement of the present invention, the molar ratio of cationic lipids, cholesterol, neutral phospholipids and PEG-lipids in S2 is 20-50:28.5-48.5:10-40:1.5.

[0035] The mass ratio of nucleic acid drugs to small molecule drugs in S1 is 1:1 to 1:25.5.

[0036] As a further improvement of the present invention, the molar ratio of cationic lipids, cholesterol, neutral phospholipids, and PEG-lipids in S2 is 30:28.5:40:1.5;

[0037] As a further improvement of the present invention, the mass ratio of nucleic acid drug to small molecule drug in S1 is 1:1.

[0038] As a further improvement of the present invention, the nitrogen / phosphorus ratio of the cationic lipid to the nucleic acid drug is 1-10:1;

[0039] As a further improvement of the present invention, the nitrogen / phosphorus ratio of the cationic lipid to the nucleic acid drug is 10:1;

[0040] As a further improvement of the present invention, the mass ratio of the small molecule drug to the nucleic acid drug in S1 and S4 is 100-5:1.

[0041] As a further improvement of the present invention, in the entire reaction system, the mass ratio of small molecule drug to nucleic acid drug in S1 and S4 is 11:1.

[0042] Technical Theme 3

[0043] A pharmaceutical composition comprising the co-delivery system described in Technical Subject 1 and a pharmaceutically acceptable carrier.

[0044] Technical Theme 4

[0045] Application of the co-delivery system described in Technical Topic 1 in the preparation of anti-inflammatory, immunomodulatory and antitumor drugs.

[0046] The beneficial effects of adopting the above technical solution are as follows:

[0047] This invention provides a nucleic acid-small molecule drug co-delivery system. Experimental verification shows that this system achieves co-encapsulation of nucleic acids and small molecule drugs with good stability. Experimental verification also shows that chloroquine and its analogues in this nucleic acid-small molecule drug co-delivery system can inhibit autophagy. Under acidic conditions, chloroquine carries a positive charge and, under these conditions, has the ability to bind to nucleic acid molecules (which carry a negative charge), promoting the encapsulation of both drugs, improving the delivery efficiency of the nucleic acid drug, and enhancing the therapeutic effect.

[0048] The present invention also provides a method for preparing a nucleic acid-small molecule drug co-delivery system, which has controllable process, high reproducibility, and can be scaled up for production, and has good prospects for industrialization.

[0049] The nucleic acid-small molecule drug co-delivery system disclosed in this invention has broad application prospects in the fields of anti-inflammatory, immunomodulatory, and anti-tumor applications. Attached Figure Description

[0050] Figure 1 The images show the cryo-electron microscopy and transmission electron microscopy results of lipid nanoparticles prepared by the method described in Example 4 with siRNA of type siN.C., where A and B are cryo-electron microscopy images; and C and D are transmission electron microscopy images.

[0051] Figure 2 This is a graph showing the placement stability results of the LNP in Example 2.

[0052] Figure 3 The graph shows the serum stability results of LNP in Example 2 of the present invention.

[0053] Figure 4 This is a laser confocal image of cells taken up in Example 3 of the present invention, in which, from left to right, are the cytoskeleton, Cy5-siRNA, cell nucleus, and overall image.

[0054] Figure 5 The effect of different formulations on the proliferation capacity of 4T1 cells is shown in Example 4 of this invention.

[0055] Figure 6 The effect of different formulations on MCF-7 cell colony formation is shown in Example 4 of the present invention.

[0056] Figure 7 The effect of different formulations on 4T1 cell colony formation is shown in Example 4 of the present invention.

[0057] Figure 8 The image shows the qRT-PCR results in Example 5 of this invention.

[0058] Figure 9 This is a Western Blot result graph from Example 5 of the present invention.

[0059] Figure 10 This is a Western Blot result graph from Example 6 of the present invention.

[0060] Figure 11 The blood compatibility results in Example 7 of the present invention are shown in the figure.

[0061] Figure 12 The biodistribution results of the 4T1 subcutaneous tumor model in Example 8 of the present invention are shown in the figure. A is the animal distribution result and B is the organ distribution result. The fluorescence intensity increases from red-black to yellow.

[0062] Figure 13 The biodistribution results of the 4T1-Luc in situ tumor model in Example 8 of the present invention are shown in Figure A, where A is the animal distribution result and B is the organ distribution result. In the 4T1-Luc group, the fluorescence intensity increases from blue to red, and in the DIR and Cy5 groups, the fluorescence intensity increases from red-black to yellow.

[0063] Figure 14 The tumor volume change curve of the 4T1-Luc in situ tumor model in Example 9 of the present invention.

[0064] Figure 15 In vivo bioluminescence image of the 4T1-Luc in situ tumor model in Example 9 of the present invention.

[0065] Figure 16 The graph shows the expression level of CDK4 / 6 mRNA in tumor tissue in Example 9 of the present invention.

[0066] Figure 17 Immunohistochemical and H&E staining results of tumor sections in Example 9 of the invention.

[0067] Figure 18 The curve showing the change in mouse body weight in Example 10 of the present invention.

[0068] Figure 19 The H&E staining results of the main organs in Example 10 of the present invention are shown in the figure.

[0069] Figure 20 The result of serum biochemical analysis in Example 10 of the invention.

[0070] Figure 21 Figure 10 shows the results of the differential analysis of red blood cells and platelets in Example 10 of the invention. Detailed Implementation

[0071] The present invention will be described in more detail through specific embodiments. The following embodiments are for illustrative purposes only and do not limit the invention in any way.

[0072] It should be noted that the terms "first" and "second" are used only to distinguish different purposes or features, and do not imply their relative technical importance, nor do they suggest the number of technical features indicated.

[0073] The siRNA sequences involved in the embodiments of this invention are shown in Table 1:

[0074] siN.C. is the negative control siRNA.

[0075] Cy5-siRNA is a fluorescently labeled siRNA that is labeled with the 5-carboxycyclopentadiene (Cy5) group.

[0076] siCDK4 / 6 is a mixture of siCDK4 and siCDK6 in a mass ratio of 1:1.

[0077] Table 1. List of siRNA sequences used in this invention

[0078]

[0079] The siRNAs mentioned in Table 1 were all purchased from Shanghai Gemma Gene Co., Ltd.

[0080] Example 1: Preparation of lipid nanoparticles

[0081] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0082] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 50:38.5:10:1.5 to prepare a mixed lipid solution.

[0083] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0084] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0085] Example 2 Preparation of lipid nanoparticles

[0086] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0087] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 40:28.5:30:1.5 to prepare a mixed lipid solution.

[0088] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0089] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0090] Example 3 Preparation of lipid nanoparticles

[0091] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0092] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 30:38.5:30:1.5 to prepare a mixed lipid solution.

[0093] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0094] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0095] Example 4 Preparation of lipid nanoparticles

[0096] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0097] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 30:28.5:40:1.5 to prepare a mixed lipid solution.

[0098] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0099] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0100] Example 5 Preparation of lipid nanoparticles

[0101] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0102] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 20:48.5:30:1.5 to prepare a mixed lipid solution.

[0103] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0104] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0105] Example 6 Preparation of lipid nanoparticles

[0106] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0107] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 20:38.5:40:1.5 to prepare a mixed lipid solution.

[0108] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0109] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0110] Example 7 Preparation of DiR fluorescently labeled lipid nanoparticles

[0111] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0112] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)] (DSPE-PEG 2000) and 1,1'-dioctyl-3,3,3',3'-tetramethylindole diiodide (DiR fluorescent dye) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52 μmol / mL, 1.44 μmol / mL, 2.02 μmol / mL, 0.08 μmol / mL and 0.5 mg / mL, respectively. Mix the components thoroughly according to the molar ratio of DLin-MC3-DMA, Chol, DSPC, DSPE-PEG 2000 and DiR fluorescent dye of 30:28.5:40:1.5:1.5 to prepare a mixed lipid solution.

[0113] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0114] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0115] Example 8: Preparation of a nucleic acid-small molecule drug co-delivery system

[0116] S1: Dissolve quinine and siRNA in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with a quinine concentration of 16.38 μg / mL and an siRNA concentration of 16.67 μg / mL.

[0117] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 20:38.5:40:1.5 to prepare a mixed lipid solution.

[0118] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0119] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with quinine solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of quinine to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0120] Example 9: Preparation of a nucleic acid-small molecule drug co-delivery system

[0121] S1: Dissolve amodiaquine and siRNA in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with amodiaquine concentration of 17.97 μg / mL and siRNA concentration of 16.67 μg / mL.

[0122] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 20:38.5:40:1.5 to prepare a mixed lipid solution.

[0123] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0124] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with amodiaquine solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of amodiaquine to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0125] Example 10: Preparation of a nucleic acid-small molecule drug co-delivery system

[0126] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0127] S2: Heptadecano-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) (SM-102), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL, respectively. The components were then mixed thoroughly in a molar ratio of SM-102, Chol, DSPC, and DSPE-PEG2000 of 20:38.5:40:1.5 to prepare a mixed lipid solution.

[0128] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0129] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0130] Example 11 Preparation of a nucleic acid-small molecule drug co-delivery system

[0131] S1: Hydroxychloroquine (HCQ) and siRNA were dissolved in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with HCQ concentration of 16.96 μg / mL and siRNA concentration of 16.67 μg / mL.

[0132] S2: A stock solution was prepared by dissolving ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)]] (DSPE-PEG 2000) in anhydrous ethanol, with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL for each component. The components were then mixed thoroughly in a molar ratio of 20:38.5:40:1.5 to prepare a mixed lipid solution.

[0133] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0134] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube. The medium was replaced with 1×PBS (pH=7.4). The mixture was then incubated with HCQ solution (solvent: PBS pH=7.4, 1 mg / mL) at 55°C for 30 min (the mass ratio of HCQ to siRNA was 11:1 throughout the reaction). After incubation on ice for 5 min, the mixture was ultrafiltered and centrifuged. The sample was then collected and sterilized by filtration through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0135] Comparative Example 1

[0136] S1: Dissolve siRNA in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with a siRNA concentration of 16.67 μg / mL.

[0137] S2: A stock solution was prepared by dissolving methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), and 1,2-distearate-sn-glycerol-3-phosphate ethylene glycol ester-PEG 2000 (DSPE-PEG 2000) in anhydrous ethanol, with concentrations of 1.52, 1.44, 2.02, and 0.08 μmol / mL for each component. The components were then thoroughly mixed at a molar ratio of DLin-MC3-DMA, Chol, DSPC, and DSPE-PEG 2000 of 30:28.5:40:1.5 to prepare a mixed lipid solution.

[0138] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0139] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube, with the medium replaced by 1×PBS (pH=7.4). The collected sample was then sterilized and filtered through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0140] Comparative Example 2

[0141] S1: Dissolve siRNA in citrate-sodium citrate buffer (pH=4) to prepare an aqueous solution with a siRNA concentration of 16.67 μg / mL.

[0142] S2: 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester (DLin-MC3-DMA), cholesterol (Chol), distearate phosphatidylcholine (DSPC), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[polyethylene glycol (2000)] (DSPE-PEG 2000) and 1,1'-dioctyl-3,3,3',3'-tetramethylindole diiodide (DiR fluorescent dye) were dissolved in anhydrous ethanol to prepare a mother liquor with concentrations of 1.52 μmol / mL, 1.44 μmol / mL, 2.02 μmol / mL, 0.08 μmol / mL and 0.5 mg / mL, respectively. Mix the components thoroughly according to the molar ratio of DLin-MC3-DMA, Chol, DSPC, DSPE-PEG 2000 and DiR fluorescent dye of 30:28.5:40:1.5:1.5 to prepare a mixed lipid solution.

[0143] S3: Using a microfluidic device, the aqueous solution prepared in step S1 and the organic solution prepared in step S2 are mixed in a microfluidic chip. The aqueous and organic solutions are injected from the flow paths on both sides of the chip and merge through the middle structure of the chip. The flow rate of the organic phase is set to 1 mL / min, the flow rate of the aqueous phase is set to 3 mL / min, and the volume ratio of organic phase to aqueous phase is 1:3, resulting in a mixture containing lipid nanoparticles.

[0144] S4: The mixture containing lipid nanoparticles obtained in S3 was ultrafiltered using an ultrafiltration centrifuge tube, with the medium replaced by 1×PBS (pH=7.4). The collected sample was then sterilized and filtered through a 0.22 μm filter membrane to obtain a lipid nanoparticle suspension.

[0145] Example of effect 1

[0146] The nucleic acid-small molecule drug co-delivery system used in this example is a nucleic acid-small molecule drug co-delivery system prepared by the method described in Examples 1-6, using siRNA as siN.C.

[0147] 1. The particle size and PDI were tested, and the results are shown in Table 2.

[0148] Table 2 Effect of different embodiments on particle size and PDI

[0149]

[0150] The results showed that the nucleic acid-small molecule drug co-delivery system of the present invention has a particle size of 150-220 nm and a uniform particle size distribution.

[0151] 2. Utilizing Quant-iT TM Ribogreen TM The encapsulation efficiency of siRNA was determined using the RNA Assay Kit (Thermo). LNP was diluted with 1×TE solution, mixed with Ribogreen, and allowed to stand for 3 min. Fluorescence values ​​were measured using a microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Samples were divided into two groups: those without Triton X-100 and those with 5% Triton X-100. These groups were used as samples before and after LNP degradation, respectively, for fluorescence value measurement. The encapsulation efficiency (EE%) was calculated using the following formula. The results are shown in Table 3.

[0152] EE% = (Concentration after destruction - Concentration before destruction) / Concentration after destruction × 100%

[0153] 3. After the nucleic acid-small molecule drug co-delivery system was destroyed with 5% Triton-X100, it was passed through a chromatographic column, and the HCQ concentration was determined by high performance liquid chromatography. The results are shown in Table 3.

[0154] Chromatographic column: Kromasil 100-5 C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-water (50:50, v / v, containing 0.05% trifluoroacetic acid); flow rate: 1.0 mL / min; column temperature: 40 °C; detection wavelength: 331 nm; injection volume: 5 μL.

[0155] Table 3. Effect of different embodiments on drug encapsulation effect

[0156]

[0157] The nucleic acid-small molecule drug co-delivery systems prepared with different formulations all showed good encapsulation effects with an encapsulation rate of >80% for siRNA, and also showed a certain degree of encapsulation for HCQ, realizing the co-encapsulation of small molecule drugs and nucleic acid drugs by lipid nanoparticles.

[0158] 4. Morphological characterization of lipid nanoparticles

[0159] The nucleic acid-small molecule drug co-delivery system prepared according to the method described in Example 4, with siRNA of type siN.C., was characterized morphologically. The results are shown in the appendix. Figure 1 As shown, the nucleic acid-small molecule drug co-delivery system is approximately spherical, with an outer layer coated by a lipid bilayer; cryo-electron microscopy images show that it has a typical electron-dense core structure.

[0160] Example 2: Stability Assessment

[0161] The nucleic acid-small molecule drug co-delivery system prepared using the method described in Example 4 with siRNA as siN.C., and the lipid nanoparticles prepared using the method described in Comparative Example 1 with siRNA as siN.C., were placed at 4°C for 7 days. The particle size and PDI changes were monitored daily at the same time using a Malvern laser particle size analyzer to assess their stability. The results are attached. Figure 2 As shown, the results indicate that neither the single-loaded nor the co-loaded formulation showed significant changes in particle size and PDI within 7 days, demonstrating good stability.

[0162] Lipid nanoparticles prepared using the method described in Comparative Example 1 (siN.C.-LNP) with siRNA as siN.C., and a nucleic acid-small molecule drug co-delivery system prepared using the method described in Example 4 (siN.C.-HCQ-LNP) with siRNA as siN.C. were mixed with fetal bovine serum (FBS) at a 1:1 volume ratio and incubated at 37 °C. Samples were taken at 0, 2, 4, 6, 8, and 24 h. 1 μL of 0.5 mol / L EDTA and 4 μL of 2% Triton X-100 were added to the samples, and the samples were frozen (-80 °C). Analysis was performed by agarose gel electrophoresis.

[0163] As attached Figure 3 As shown, naked siN.C. degrades in serum; LNPs encapsulating siRNA can effectively avoid nuclease degradation and have good stability.

[0164] Example 3: Examination of Cellular Uptake Levels

[0165] This example uses laser scanning confocal microscopy (CLSM) to observe the uptake of Cy5-siRNA.

[0166] Rhodamine phalloidin staining shows the cytoskeleton in red, DAPI staining shows the nucleus in blue, and Cy5-siRNA staining shows the nucleus in green.

[0167] 4T1 cells were cultured in RPMI-1640 complete medium at a concentration of 1×10⁻⁶. 5 Cells were seeded in confocal microscopy dishes with glass bottoms and incubated at 37 °C for 24 h. The original culture medium was discarded, and the cells were washed twice with PBS. RPMI-1640 complete culture medium containing the drug (drug concentration based on siRNA, 200 nM) was added to the corresponding wells (1 mL / well), and the cell culture plates were incubated at 37 °C for 2 h and 5 h, respectively. After incubation, the drug-containing culture medium was discarded, and the cells were fixed with 4% paraformaldehyde and stained. The fixative was discarded, and the cells were washed three times with pre-cooled PBS and incubated with 0.1% Triton X-100 for 10 min. After washing twice with PBS, the cells were incubated with rhodamine phalloidin in the dark for 30 min. After washing twice with PBS, the cells were incubated with DAPI for 15 min. After washing three times with PBS, the cells were observed under a laser confocal microscope.

[0168] The RPMI-1640 complete culture medium containing the drug solution was divided into four groups: RPMI-1640 complete culture medium, RPMI-1640 complete culture medium containing Cy5-siRNA, RPMI-1640 complete culture medium containing lipid nanoparticles prepared by the method described in Comparative Example 1 (wherein the siRNA is Cy5-siRNA), and RPMI-1640 complete culture medium containing the nucleic acid-small molecule drug co-delivery system prepared by the method described in Example 4 (wherein the siRNA is Cy5-siRNA).

[0169] The results are as follows Figure 4 As shown, the LNPs treatment group significantly enhanced cellular uptake of siRNA, and the cellular uptake level of the formulation increased over time, exhibiting a time-dependent characteristic, indicating that the prepared nucleic acid-small molecule drug co-delivery system showed good cellular uptake capability.

[0170] Example 4: Evaluation of the effect of cell proliferation inhibition

[0171] To verify whether the formulation could inhibit cancer cell proliferation in vitro, this efficacy example included a CCK-8 assay and a colony formation assay.

[0172] The free siRNA of siCDK4 / 6 used in the 4T1 cell experimental group is shown in the Mus group in Table 1.

[0173] The free siRNA of siCDK4 / 6 used in the MCF-7 cell experimental group is shown in the Homo group in Table 1.

[0174] CCK-8 assay: 4T1 cells were cultured in RPMI-1640 complete medium and seeded into 96-well plates at a density of 5000 cells / well (100 μL / well). The cells were incubated at 37 °C for 24 h; the original medium was then discarded. Lipid nanoparticles (siCDK4 / 6 siRNA) were prepared according to the method described in Example 4. Lipid nanoparticles (siCDK4 / 6 siRNA) were prepared according to the method described in Comparative Example 1. Culture media containing siRNA concentrations of 1, 10, 25, 50, 100, 200, and 400 nM were prepared (equal siRNA concentration, PBS (pH=7.4) was used as the preparation medium). Cells were incubated with the drug-containing medium for 48 h. After incubation, 100 μL of CCK-8 solution (CCK-8:complete medium = 1:9, v / v) was added to each well, and the cells were incubated for 1 h. The absorbance was measured at a wavelength of 450 nm using a multi-functional microplate reader (BMG Labtech, Germany).

[0175] Colony formation assay: 4T1 cells were cultured in RPMI-1640 complete medium and seeded into 6-well plates at a density of 5000 cells / well with 2 mL of medium. The plates were incubated at 37 °C for 24 h.

[0176] Each group of drugs was prepared into a culture medium containing the corresponding drug. The groups included: 1. Blank group (no drug treatment group; control); 2. Free HCQ; 3. Free negative control siRNA (siN.C.); 4. Lipid nanoparticles prepared according to the method described in Comparative Example 1, wherein the siRNA was siN.C.; 5. Lipid nanoparticles prepared according to the method described in Example 4, wherein the siRNA was siN.C.; 6. Free siCDK4 / 6 targeting CDK4 / 6; 7. Lipid nanoparticles prepared according to the method described in Comparative Example 1, wherein the siRNA was siCDK4 / 6; 8. Lipid nanoparticles prepared according to the method described in Example 4, wherein the siRNA was siCDK4 / 6. The drug medium was PBS, and the drugs were administered at equal drug concentrations (siRNA: 400 nM; HCQ: 9.8 ug / mL in the culture medium).

[0177] Discard the original culture medium and replace it with 2 mL of drug-containing culture medium. Incubate at 37°C for 24 h, then replace with fresh culture medium and continue culturing for 5 days. Wash the well plate twice with PBS, fix with 4% paraformaldehyde for 5 min, add 0.5 mL of crystal violet staining solution, stain at room temperature for 20 min, discard, wash repeatedly with distilled water, and take photos for record.

[0178] The CCK-8 assay and colony formation assay for MCF-7 cells are performed using the same methods as those for 4T1 cells. The difference is that MCF-7 cells are used instead of 4T1 cells, and the free siCDK4 / 6 siRNAs used are shown in the Homo group in Table 1.

[0179] The results are as follows Figure 5-7 As shown, siCDK4 / 6-HCQ-LNP exhibits the best cell proliferation inhibition effect.

[0180] Example 5: Evaluation of gene silencing effect

[0181] The gene silencing effect of LNP was evaluated in this study using qRT-PCR and Western blot assays, respectively.

[0182] qRT-PCR: RPMI-1640 complete medium was used to culture 4T1 cells at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 / 2 well in 6-well plates and incubated at 37 °C for 24 h. The original culture medium was discarded, and 2 mL of RPMI-1640 complete medium containing the drug (drug dosage: siRNA: 400 nM; HCQ: 9.8 ug / mL) was added to each well. After incubation for 6 h, the medium was replaced with RPMI-1640 complete medium and cultured for another 24 h. Cells were digested and collected for RNA extraction, and CDK4 / 6 gene levels were determined using qRT-PCR.

[0183] The drug-containing RPMI-1640 complete culture medium was divided into four groups: RPMI-1640 complete culture medium containing siCDK4 / 6, RPMI-1640 complete culture medium containing HCQ, RPMI-1640 complete culture medium containing lipid nanoparticles prepared by the method described in Comparative Example 1 (where siRNA is siCDK4 / 6), and RPMI-1640 complete culture medium containing a nucleic acid-small molecule drug co-delivery system prepared by the method described in Example 4 (where siRNA is siCDK4 / 6).

[0184] Western blot: RPMI-1640 complete medium was used to inoculate 4T1 cells at a concentration of 1×10⁻⁶. 5Cells were seeded at a density of 1 / 2 well in 6-well plates and incubated at 37 °C for 24 h. The original culture medium was discarded, and 2 mL of RPMI-1640 complete culture medium containing the drug (drug dosage: siRNA: 400 nM; HCQ: 9.8 ug / mL) was added to each well. After incubation for 6 h, the medium was replaced with RPMI-1640 complete culture medium and cultured for another 48 h. Cells were then digested and collected to extract total protein for Western blot analysis.

[0185] The drug-containing RPMI-1640 complete culture medium was divided into four groups: RPMI-1640 complete culture medium containing siCDK4 / 6, RPMI-1640 complete culture medium containing HCQ, RPMI-1640 complete culture medium containing lipid nanoparticles prepared by the method described in Comparative Example 1 (wherein siRNA is siCDK4 / 6), and RPMI-1640 complete culture medium containing the nucleic acid-small molecule drug co-delivery system prepared by the method described in Example 4 (wherein siRNA is siCDK4 / 6).

[0186] like Figure 8 As shown, siCDK4 / 6-HCQ-LNP effectively silences the CDK4 / 6 gene. Figure 9 As shown, siCDK4 / 6-HCQ-LNP downregulated the expression level of CDK4 / 6 protein, which further led to a decrease in the level of its downstream pRb protein.

[0187] Example 6: Evaluation of Autophagy Level

[0188] This example evaluates the autophagy-inhibiting ability of LNPs.

[0189] The expression levels of autophagy-related proteins were assessed using Western blot assays.

[0190] The Western blot experiment was performed in the same manner as in Example 5, detecting the expression levels of marker proteins (LC3B-II and p62) of the autophagy pathway.

[0191] Figure 10 The levels of autophagy pathway marker proteins (LC3B-II and p62) in the siCDK4 / 6-HCQ-LNP treatment group were significantly higher than those in the control group, demonstrating the effective autophagy blocking effect of the co-delivery formulation.

[0192] Example 7: Blood compatibility test

[0193] This example demonstrates the blood compatibility of the formulation by co-incubating LNP with a red blood cell suspension.

[0194] Blood was collected from the abdominal aorta of SD rats, centrifuged, washed, and diluted with physiological saline to a 2% red blood cell suspension. PBS was used as a negative control, and a 1% Triton X-100 treatment group was used as a positive control.

[0195] Experimental group 1 was a PBS solution of lipid nanoparticles prepared by the method described in Comparative Example 1, wherein the siRNA was selected as siN.C.;

[0196] Experimental group 2 is a PBS solution of a nucleic acid-small molecule drug co-delivery system prepared by the method described in Example 4, wherein the siRNA is siN.C.;

[0197] Each group was mixed with red blood cell suspension at a 1:1 volume ratio (siRNA concentration in the experimental group mixture was 12.5 ug / mL) and incubated at 37 ℃. Samples were taken at 1, 3, and 6 h, centrifuged (4 ℃, 3500 rpm, 10 min), observed, photographed, and the supernatant was transferred to a 96-well plate. The absorbance was measured at 545 nm using a microplate reader, and the hemolysis rate was calculated. Simultaneously, the morphology of red blood cells was observed under a microscope for any changes. Figure 11 As shown, the formulation exhibits no obvious hemolysis and has good blood compatibility.

[0198] Example 8: Evaluation of biodistribution in vivo

[0199] The mice used in this study were female BALB / c mice, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0200] In the experimental group, Cy5-siRNA-DiR-LNP was prepared using the method described in Comparative Example 1, wherein Cy5-siRNA was selected as the siRNA.

[0201] In the experimental group, Cy5-siRNA-DiR-HCQ-LNP was prepared using the method described in Example 4, wherein Cy5-siRNA was selected as the siRNA.

[0202] This embodiment examines the biodistribution of LNP in vivo after intravenous injection.

[0203] Subcutaneous and in situ breast cancer tumor models were constructed to validate the in vivo biodistribution of the drug. 1×10 6 1 × 10⁴ T1 cells were subcutaneously injected into the right axillary region of mice to establish a subcutaneous breast cancer tumor model. 6 4T1-Luc cells were orally inoculated into the mammary fat pads of mice to establish an in situ breast cancer tumor model. The model mice were randomly divided into 3 groups, with 3 mice in each group.

[0204] Groups and dosing regimens: Dosage: Cy5-siRNA (0.3 mg / kg), 1,1'-dioctyl-3,3,3',3'-tetramethylindole diiodide (DiR) (0.05 mg / kg) (different groups were administered the same amount).

[0205] The formulation was prepared according to Formulation 4 of Example 1.

[0206] Control group: Free Cy5-siRNA and free DiR mixed solution (code: Free Cy5-siRNA-DiR), intravenously injected, volume 0.2 mL, solvent PBS (pH=7.4).

[0207] Experimental group: 1. Cy5-siRNA-DiR-LNP;

[0208] 2. Cy5-siRNA-DiR-HCQ-LNP;

[0209] The volume was 0.2 mL, and the solvent was PBS (pH=7.4).

[0210] At 1, 4, 6, 8, 10, 12, 24, and 48 hours after administration, whole-body images of small animals were acquired using a small animal in vivo imaging system (Cy5: Ex / Em=640 / 680 nm, DiR: Ex / Em=745 / 800 nm) to observe the distribution of the formulation in the animals. After imaging, the mice were euthanized, and major organs (heart, liver, spleen, lung, and kidney) and tumor tissue were collected for ex vivo tissue imaging at the same wavelength. For bioluminescence imaging of the in situ tumor model, D-Luciferin (150 mg / kg) was injected intraperitoneally before imaging, and other procedures were the same as for the subcutaneous tumor model.

[0211] In both the 4T1 subcutaneous tumor model and the orthotopic tumor model, significant DiR fluorescence signals were observed at the tumor site in the experimental group, indicating that the vector could effectively reach the tumor site; and compared with the free siRNA administration group, LNP protected siRNA from degradation and prolonged its circulation time in vivo. Figure 12 , 13 ).

[0212] Example 9: In vivo pharmacodynamic evaluation

[0213] This example demonstrates the use of LNP for antitumor treatment of orthotopic breast cancer mice.

[0214] A 4T1-Luc orthotopic breast cancer mouse model was constructed, and the modeling method was the same as in Example 8.

[0215] The mice used were female BALB / c mice, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0216] The model mice were randomly divided into 7 groups, with 5 mice in each group.

[0217] Groups and dosing regimens: Dosage of siRNA: 0.6 mg / kg; HCQ: 1.3 mg / kg (different groups were administered at the same dose).

[0218] Experimental group 3 was prepared using the method described in Example 1, wherein the siRNA used was siN.C.;

[0219] Experimental group 4 was prepared using the method described in Example 4, wherein the siRNA used was siN.C.;

[0220] Experimental group 5 was prepared using the method described in Comparative Example 1, wherein the siRNA used was siCDK4 / 6;

[0221] Experimental group 6 was prepared using the method described in Example 4, wherein the siRNA used was siCDK4 / 6;

[0222] Control group: normal saline, intravenous injection, once every other day, volume of 0.2 mL, for seven consecutive times.

[0223] The experimental group specifically includes 6 groups: 1. Free HCQ group;

[0224] 2. Free siCDK4 / 6 siRNA group;

[0225] 3. Lipid nanoparticles (siN.C.-LNP) loaded with negative control siRNA;

[0226] 4. Nucleic acid-small molecule drug co-delivery system (siN.C.-HCQ-LNP) co-loaded with negative control siRNA and HCQ;

[0227] 5. Lipid nanoparticles carrying CDK4 / 6 siRNA (siCDK4 / 6-LNP group)

[0228] 6. Nucleic acid-small molecule drug co-delivery system (siCDK4 / 6-HCQ-LNP) co-loaded with CDK4 / 6 siRNA and HCQ.

[0229] Administer the injection every other day, using PBS (pH=7.4) as the solvent, in a volume of 0.2 mL, for seven consecutive administrations.

[0230] The results are as follows Figure 14-17As shown, the siCDK4 / 6-HCQ-LNP administration group exhibited the best anti-tumor efficacy. Specifically, this formulation inhibited the expression of the CDK4 / 6 gene and related proteins, and had a significant inhibitory effect on tumor growth in mice.

[0231] Example 10: In vivo safety evaluation

[0232] Record the changes in animal weight during the treatment period in Example 9. After treatment, blood was collected for complete blood count and blood biochemistry tests. Major organs (heart, liver, spleen, lung, kidney) and tumors of mice were collected for H&E staining. The specific procedure was as follows: tissues were fixed with 4% paraformaldehyde, sectioned, dewaxed and placed in water, stained with hematoxylin, rinsed with tap water, differentiated with hydrochloric acid ethanol, and then blued with running water; then stained with eosin, followed by graded ethanol dehydration, xylene clearing, and finally mounted with neutral resin for observation.

[0233] The results are as follows Figure 18-21 As shown, the nucleic acid-small molecule drug co-delivery system disclosed in this invention has no obvious toxic side effects. Specifically, during the treatment period, the mice showed no significant change in body weight; no obvious lesions in major organs; and no significant abnormalities in blood biochemistry and routine blood tests.

[0234] Finally, it should be noted that the above embodiments are merely several specific implementations of the present invention, intended to facilitate a detailed understanding of the technical solution of the present invention. However, the patent protection scope of the present invention is not limited thereto. It should be pointed out that those skilled in the art can still make various modifications or improvements, and all such modifications or improvements should be included within the protection scope of the present invention.

Claims

1. A nucleic acid-small molecule drug co-delivery system, characterized in that, The co-delivery system uses a lipid component as a carrier, which encapsulates the drug component. The lipid components include cationic lipids, neutral phospholipids, cholesterol, and PEG-lipids; The drug components include nucleic acid drugs and small molecule drugs.

2. The nucleic acid-small molecule drug co-delivery system according to claim 1, characterized in that, The particle size of the co-delivery system is 50-300 nm, preferably 100-250 nm, and most preferably 150-220 nm.

3. The nucleic acid-small molecule drug co-delivery system according to claim 1, characterized in that, The cationic lipid is at least one of 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate, and ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); The neutral phospholipids are selected from the following compounds: distearylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, phosphatidic acid, and sphingomyelin; The PEG lipid is selected from one or more of the following compounds: PEG lipid is selected from PEG-modified distearylphosphatidylethanolamine, PEG-modified dimyristoylglycerol, PEG-modified dipalmitoylphosphatidylethanolamine, PEG-modified dimyristoylphosphatidylethanolamine, PEG-modified dilauroylphosphatidylethanolamine, PEG-modified ceramide, and mPEG-modified bis(tetradecylacetamide); more preferably, the molecular weight of the PEG or mPEG is 1000-5000.

4. The nucleic acid-small molecule drug co-delivery system according to claim 1, characterized in that, The nucleic acid drug is selected from one or more of mRNA, siRNA, dsRNA, snRNA, tRNA, rRNA, circRNA, saRNA, antisense oligonucleotide, miRNA, lncRNA, shRNA, ssDNA, aiRNA, nucleic acid aptamers, and ribozymes, with siRNA being preferred; The small molecule drug is selected from chloroquine and its analogues, including quinine, 4-[(7-chloroquinoline-4-yl)amino]-2-[(diethylamino)methyl]phenol, 2-[[4-[(7-chloroquinoline-4-yl)amino]pentyl](ethyl)amino]ethanol, and most preferably 2-[[4-[(7-chloroquinoline-4-yl)amino]pentyl](ethyl)amino]ethanol or quinine.

5. A method for preparing a nucleic acid-small molecule drug co-delivery system as described in any one of claims 1-4, characterized in that, The preparation steps are as follows: S1: Dissolve cationic lipids, neutral phospholipids, cholesterol, and PEG-lipids in solvent I respectively; dissolve nucleic acid drugs and small molecule drugs in solvent II to form aqueous solution B; S2: Mix the cationic lipids, neutral phospholipids, cholesterol, and PEG-lipid solutions obtained in S1 in a certain proportion to form organic phase solution A; S3: Using a microfluidic system, solution A and solution B are mixed at a flow rate ratio of 1:1-5 to obtain mixture D. Mixture D is then subjected to ultrafiltration and centrifugation to collect the resulting sample E. S4: Incubate the collected sample E with the small molecule drug solution C at 50-60℃ for 15-45 min, and then ultrafilter and centrifuge to obtain the nucleic acid-small molecule drug co-delivery system.

6. The preparation method according to claim 5, wherein solvent I is ethanol and solvent II is citrate-sodium citrate buffer.

7. The preparation method according to claim 5, characterized in that, In S2, the molar ratio of cationic lipids, cholesterol, neutral phospholipids, and PEG-lipids is 20-50:28.5-48.5:10-40:1.5, preferably 30:28.5:40:1.5; in S1, the mass ratio of nucleic acid drugs to small molecule drugs is 1:1-1:25.5, preferably 1:

1.

8. The preparation method according to claim 5, characterized in that, The nitrogen / phosphorus ratio of cationic lipids to nucleic acid drugs is 1-10:1, preferably 10:1; The mass ratio of the sum of the small molecule drugs in S1 and S4 to the mass of the nucleic acid drug is 100-5:1, preferably 11:

1.

9. A pharmaceutical composition comprising the co-delivery system of any one of claims 1-4 and a pharmaceutically acceptable carrier.

10. The use of the co-delivery system according to any one of claims 1-4 in the preparation of anti-inflammatory, immunomodulatory and antitumor drugs.

Images