Oligonucleotide lung targeting delivery system and application thereof in treatment of lung diseases

Abstract

The invention relates to an oligonucleotide lung targeting delivery system and application thereof in treatment of lung diseases, and relates to the technical field of biological medicines and nano-medicines. The invention designs a high-efficiency lung targeting oligonucleotide delivery drug, which comprises lipid nanoparticles, the lipid nanoparticles contain oligonucleotide and lipid components, and the lipid components are composed of 4-(N, N-dimethylamino) butyric acid (dilinoleyl) methyl ester, (2, 4-dimethylamino) butyric acid (dilinoleyl) methyl ester, (2, 4-dimethylamino) butyric acid (2, 4-dimethylamino) butyric acid (2, 4-dimethylamino) butyric acid (2, 4-dimethylamino) butyric acid (2, 4-dimethylamino) butyric acid The composition is composed of 1, 3-dioleoyl-propyl)-trimethylamine, dipalmitoyl phosphatidylcholine and pegylated lipid. A breakthrough of a nucleic acid medicine lung delivery technology is realized, in-vivo experiment verification results show that the lung aggregation efficiency is improved by about 2-4 times compared with that of traditional lipid nanoparticles, the lipid nanoparticles have remarkable lung metastatic tumor resisting activity, in-vivo experiments show that the lipid nanoparticles can inhibit growth of colorectal cancer lung metastatic tumors and prolong the survival time of mice, and the lipid nanoparticles have good application prospects. And the effect is better than that of 5-fluorouracil and regorafenib.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomedicine, and in particular to an oligonucleotide lung-targeted delivery system and its application in the treatment of lung diseases.

[0002] Background technology particles.

[0003] Colorectal cancer is the third most common cancer worldwide. The latest epidemiological data from 2025 shows over 1.9 million new cases and over 930,000 deaths annually. Nearly 20% of these cases are diagnosed in patients under 50 years old, and the annual growth rate of new cases in developed countries in Europe and America reaches 3.2%. The lungs are the most common extra-abdominal metastasis organ for colorectal cancer, affecting approximately 15%-20% of patients, and the 5-year survival rate for these patients is less than 10%.

[0004] Among current treatment options, surgical resection combined with radiotherapy and chemotherapy (such as fluorouracil and oxaliplatin) can improve the five-year survival rate of early-stage colorectal cancer to 70%-80%, but the efficacy drops significantly in patients with advanced metastases. Targeted therapies such as anti-EGFR (epidermal growth factor receptor) monoclonal antibodies (cetuximab) and anti-VEGF (vascular endothelial growth factor) monoclonal antibodies (bevacizumab) are only effective in some wild-type patients and are prone to developing resistance; PD-1 inhibitors (such as pembrolizumab) are only suitable for microsatellite instability-high (MSI-H) patients (accounting for <15%). For patients with colorectal cancer lung metastases, the lung lesion response rate of traditional chemotherapy drugs is less than 30%, and they have serious systemic toxicity, urgently requiring new targeted therapy strategies.

[0005] Oligonucleotide drugs (such as small interfering RNA, microRNA, and antisense oligonucleotides) demonstrate the potential for precision medicine by specifically regulating the expression of target genes. In 2018, the first small interfering RNA drug, Patisiran (targeting the thyroxine transporter TTR gene), was launched for the treatment of hereditary amyloidosis polyneuropathy. In 2021, Inclisiran (targeting the proprotein convertase subtilisin type 9 PCSK9) was approved for hypercholesterolemia, confirming the clinical value of this class of drugs. However, traditional delivery systems (such as liposomes and polymer nanoparticles) lack organ specificity, making precise delivery to the lungs difficult.

[0006] Lipid nanoparticles, as the most mature nucleic acid delivery platform currently available, are typically formulated with ionizable lipids (such as methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester), cofactor phospholipids (such as distearylphosphatidylcholine), cholesterol, and polyethylene glycol-modified lipids. They encapsulate nucleic acids and mediate endosome escape through positive and negative charge interactions. The 2018 launch of Patisiran validated the druggability of lipid nanoparticles. However, due to surface charge and polyethylene glycol modification, approximately 70%-80% of traditional lipid nanoparticles accumulate in the liver and spleen, with only 5%-10% reaching distant organs such as the lungs and brain. This organ distribution characteristic limits their wider application. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the problem that most lipid nanoparticles in the prior art aggregate in the liver and spleen, and to provide a lung-targeting lipid nanoparticle that can efficiently deliver gene drugs (such as oligonucleotides), which has significant efficacy in the treatment of lung metastases, especially in the treatment of lung metastases of colorectal cancer.

[0008] This invention provides a lipid nanoparticle for lung targeting, comprising oligonucleotides and lipid components, and including the following components in molar percentages based on the total molar mass of the lipid components as 100%:

[0009] 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 0-66%;

[0010] (2,3-Dioleoyl-propyl)-trimethylamine, 0-66%;

[0011] Dipalmitoylphosphatidylcholine, 0-66%;

[0012] Polyethylene glycol-modified lipids, 1%-5% (more preferably 1%-3%);

[0013] The molar percentages of 4-(N,N-dimethylamino)butyric acid (dioleoyl) methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, and dipalmitoylphosphatidylcholine are not all 0.

[0014] Furthermore, of 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, and dipalmitoylphosphatidylcholine, at least two have a molar percentage that is not 0; preferably:

[0015] When the molar percentage of 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester is 0, the molar percentages of (2,3-dioleoyl-propyl)-trimethylamine and dipalmitoylphosphatidylcholine are 30%-66% and 30%-66%, respectively.

[0016] Alternatively, when the molar percentage of dipalmitoylphosphatidylcholine is 0, the molar percentages of 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester and (2,3-dioleoyl-propyl)-trimethylamine are 30%-66% and 30%-66%, respectively, with the most preferred percentages being 30%-55% and 45%-66%.

[0017] Alternatively, when the molar percentages of 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, and dipalmitoylphosphatidylcholine are all not 0, their molar percentages are 15%-66%, 15%-66%, and 15%-66%, respectively. Most preferably, the molar percentages of the three are 15%-20%, 45%-66%, and 15%-35%, or 30%-35%, 15%-20%, and 45%-50%, or 60%-66%, 15%-20%, and 15%-20%, respectively.

[0018] Furthermore, in the polyethylene glycol-modified lipid, the average molecular weight of the polyethylene glycol segments is 1000-5000 Da (more preferably 2000-2700 Da).

[0019] Furthermore, the oligonucleotide is a gene silencing agent, including but not limited to microRNA, small interfering RNA, antisense oligonucleotide, short hairpin RNA, etc.

[0020] Furthermore, the mass ratio of the oligonucleotide to the lipid component is 1:(10-20).

[0021] A second objective of this invention is to provide a method for preparing the lipid nanoparticles, comprising the following steps:

[0022] S1. Dissolve the oligonucleotides to prepare an aqueous phase;

[0023] Dissolve the lipid components to prepare an oil phase;

[0024] S2. The lipid nanoparticles are prepared by mixing the aqueous phase and the oil phase (using microfluidic methods, thin film hydration methods, etc.).

[0025] A third objective of this invention is to provide an oligonucleotide lung-targeted delivery system comprising the lipid nanoparticles.

[0026] A fourth object of the present invention is to provide a pharmaceutical composition for preparing an oligonucleotide lung-targeted delivery system for lung targeting, comprising: a gene drug and a lipid component, wherein the gene drug is an oligonucleotide, and the lipid component comprises methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester, (2,3-dioleoylpropyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipids. The proportions of each component are as described above.

[0027] A fifth object of the present invention is to provide the use of the lipid nanoparticles, oligonucleotide lung-targeted delivery system or pharmaceutical composition in the preparation of medicaments for the treatment of lung diseases.

[0028] Furthermore, the lung diseases mentioned include lung cancer, lung metastases, pulmonary fibrosis, and lung infections.

[0029] Furthermore, the lung metastatic tumor can be a lung metastasis of any solid tumor, such as lung metastasis of liver cancer, lung metastasis of breast cancer, lung metastasis of gastric cancer, lung metastasis of colorectal cancer, lung metastasis of pancreatic cancer, lung metastasis of kidney cancer, lung metastasis of ovarian cancer, lung metastasis of sarcoma, lung metastasis of melanoma, lung metastasis of head and neck squamous cell carcinoma, lung metastasis of prostate cancer, lung metastasis of bladder cancer, lung metastasis of cervical cancer, etc.

[0030] Furthermore, the oligonucleotide can be a gene silencing agent for any drug target gene of the disease to be treated. For example, when the lung metastasis is colorectal cancer lung metastasis, the oligonucleotide can be a sequence that targets and silences the poly(ADP-ribose) polymerase gene PARP1 or the ETS variant transcription factor 4 gene ETV4.

[0031] Furthermore, when the target gene is the poly(ADP-ribose) polymerase gene PARP1, the oligonucleotide is the sequence shown in SEQ ID NO.10 and SEQ ID NO.18.

[0032] Furthermore, when the target gene is the ETS variant transcription factor 4 gene ETV4, the oligonucleotides are any of the following groups:

[0033] (1) The sequences shown in SEQ ID NO.3 and SEQ ID NO.11;

[0034] (2) The sequences shown in SEQ ID NO.4 and SEQ ID NO.12;

[0035] (3) The sequences shown in SEQ ID NO.5 and SEQ ID NO.13 (preferred);

[0036] (4) The sequences shown in SEQ ID NO.6 and SEQ ID NO.14 (preferred);

[0037] (5) The sequences shown in SEQ ID NO.7 and SEQ ID NO.15;

[0038] (6) The sequences shown in SEQ ID NO.8 and SEQ ID NO.16.

[0039] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0040] This invention significantly enhances the lung affinity of lipid nanoparticles through the synergistic effect of different lipid components, achieving high lung targeting. In vivo experiments also show that its lung aggregation efficiency is approximately 2-4 times higher than that of traditional lipid nanoparticles. Furthermore, the lipid nanoparticles prepared by this invention exhibit good stability and low cytotoxicity: the particle size shows no significant change after 14 days of storage at 4°C, exhibits strong resistance to nucleases, protects oligonucleotide molecules from degradation, has a hemolysis rate of <5%, and shows low toxicity to normal cells (such as HEK-293T) and tumor cells (such as T84 and CT26), demonstrating good biocompatibility. It shows excellent efficacy in the treatment of lung diseases, especially significant anti-tumor activity. In vivo experiments show that the lipid nanoparticles of this invention can inhibit the growth of colorectal cancer lung metastases and prolong the survival of mice, with effects superior to 5-fluorouracil and regorafenib. Attached Figure Description

[0041] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0042] Figure 1 The results show the particle size, potential, and encapsulation efficiency of the lipid nanoparticles prepared in this invention; (A, B) particle size diagrams of LuT-LNP and AM22@LuT-LNP; (C) Zeta potentials of LuT-LNP, AM22@LuT-LNP, and AM22; and (D) encapsulation efficiency of LuT-LNP.

[0043] Figure 2 The data are for the safety and stability of lipid nanoparticles; (A) particle size stability of LuT-LNP in PBS at 4°C; (B, C, D) survival rates of HEK-293T, T84 and CT26 cells treated with different concentrations of LuT-LNP; (E) hemolysis assay to examine the biocompatibility of LuT-LNP; (F) nuclease resistance assay of LuT-LNP.

[0044] Figure 3 The distribution of lipid nanoparticles in the heart, lungs, liver, spleen, and kidneys of mice.

[0045] Figure 4 The inhibitory effect of AM22 on PARP1 gene expression in T84 cells.

[0046] Figure 5 The inhibitory effect of AM22@LuT-LNP on lung metastases of colorectal cancer in mice is shown in the following figures: (A) in vivo imaging of mice before administration and (B) fluorescence intensity statistics; (C) in vivo imaging of mice after administration and (D) fluorescence intensity statistics; (E) mouse body weight change curve; and (F) mouse survival curve.

[0047] Figure 6This study aimed to investigate the inhibitory effect of small interfering RNA on ETV4 gene expression in human colorectal cancer lung metastasis cells T84 and mouse colorectal cancer cells CT26.

[0048] Figure 7 To demonstrate the inhibitory effect of ETV4 siRNA@LuT-LNP on lung metastases from colorectal cancer in mice. (A) In vivo imaging of mice before administration and (B) Statistical results of fluorescence intensity; (C) In vivo imaging of mice after administration and (D) Statistical results of fluorescence intensity; (E) Curve of mouse body weight change; (F) Curve of mouse survival. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0050] The solution involved in this invention is as follows:

[0051] To address the current lack of highly efficient lipid nanoparticles for targeting the lungs, this invention constructs a highly stable and lung-affinity delivery system by optimizing the component ratios and targeting strategies of lipid nanoparticles, thus filling a technological gap in targeted therapy for lung diseases. Specifically:

[0052] This invention provides an oligonucleotide lung-targeted delivery system, wherein the delivery system is lipid nanoparticles, the lipid nanoparticles comprising: methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester, (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipids; the polyethylene glycol-modified lipids have a molecular weight of 2000-2700.

[0053] Specifically, in the lipid nanoparticles, the molar ratio of 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipids is (0-66):(0-66):(0-66):1.5.

[0054] Preferably, in the lipid nanoparticles, the molar ratio of 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipid is (0-50):(0-66):(0-50):1.5.

[0055] More preferably, in the lipid nanoparticles, the molar ratio of (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipid is (0-66):(0-50):1.5.

[0056] More preferably, the molar ratio of (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipids in the lipid nanoparticles is 65.67:32.83:1.5.

[0057] The lipid nanoparticles also include oligonucleotide molecules, such as small interfering RNA, microRNA, and antisense oligonucleotides. Preferably, the oligonucleotide molecules are microRNAs targeting the PARP1 gene or small interfering RNAs targeting the ETV4 gene.

[0058] The small interfering RNA targeting the ETV4 gene has the active strand RNA sequence shown in SEQ ID NO. 3-8 and the complementary strand RNA sequence shown in SEQ ID NO. 11-16. Alternatively, it may have any one of the following nucleotide sequences:

[0059] I. Nucleotide sequences obtained by modification, substitution, deletion, or addition of one or more bases to the nucleotide sequences shown in Table 1; or

[0060] II. Nucleotide sequences obtained by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 ribonucleotides having the nucleotide sequences shown in Table 1; or

[0061] III. Sequences that have at least 80% homology with the nucleotide sequences shown in Table 1.

[0062] The method for preparing the oligonucleotide delivery system includes the following steps:

[0063] S1. Preparation of lipid-ethanol solution: Dissolve 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipids in ethanol to prepare a lipid-ethanol solution.

[0064] S2. Prepare nucleic acid-buffer solution: Dissolve oligonucleotides in acidic buffer solution;

[0065] S3. Mixing and Collection: Thoroughly mix the lipid-ethanol solution from step S1 with the oligonucleotide solution from step S2 to form lipid nanoparticles, and collect the effluent lipid nanoparticles.

[0066] in:

[0067] The acidic buffer solution mentioned in step S2 is sodium citrate buffer solution.

[0068] In step S3, the lipid-ethanol solution has a mass-to-volume ratio of lipid to ethanol of (1-4) mg / mL.

[0069] The concentration of the oligonucleotide in step S3 is 200-300 μg / ml.

[0070] The mixing described in step S3 employs microfluidic mixing technology or thin-film hydration.

[0071] Applications:

[0072] The oligonucleotide lung-targeted delivery system of the present invention can be used to prepare drugs for treating lung diseases, including colorectal cancer lung metastases, lung cancer, pulmonary fibrosis, and lung infections.

[0073] The oligonucleotide lung-targeted delivery system of the present invention can be used to prepare gene silencing agents, the genes including ETV4, PARP1 and other genes related to lung diseases.

[0074] The oligonucleotide lung-targeted delivery system of the present invention can be used to prepare inhibitors.

[0075] The oligonucleotide lung-targeted delivery system of the present invention can be used to prepare pharmaceutical compositions for treating lung metastases of colorectal cancer, comprising the oligonucleotide lung-targeted delivery system and pharmaceutically acceptable excipients.

[0076] The materials and methods involved in this invention are as follows:

[0077] (1) Polyethylene glycol-modified lipids: 1,2-dimyristic-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG) 2000 ).

[0078] (2) The method for preparing lipid nanoparticles using microfluidic methods is as follows:

[0079] The liposome nanoparticle synthesis system requires a flow control system and a microfluidic chip, specifically including a 2-channel microinjection pump, two flow sensors, two reservoirs, and the microfluidic chip. The workflow of the liposome nanoparticle synthesis system is as follows: a pressure controller connected to a pressure source provides a stable pressure value, applying a fixed pressure to the sealed reservoir through a pipeline, thereby stably outputting the liquid from the reservoir. The liquid then passes through a flow sensor, which measures the flow rate and feeds it back to the pressure controller, achieving precise flow control. Subsequently, the fluid is stably and accurately delivered to the microfluidic chip, with specific parameters controlled and set via computer software. The 2-channel microinjection pump separately controls the ethanol and aqueous phase solutions containing lipids. The flow rates of the two phases can be adjusted independently. After passing through the flow sensors and dampers, they are mixed in the microfluidic chip, thus synthesizing liposome nanoparticles. Specifically, by adjusting the flow rates of the ethanol and aqueous phases, the flow rate ratio, and the total flow rate, nanoliposomes of a fixed particle size can be obtained.

[0080] For oligonucleotide encapsulation, the lipid components are first dissolved in anhydrous ethanol according to the above lipid nanoparticle formulation and ratio to prepare a lipid-ethanol solution. Simultaneously, the oligonucleotides are dissolved in an acidic buffer solution to ensure they are in a suitable form for lipid binding. The lipid-ethanol solution and oligonucleotide solution are then thoroughly mixed in a micromixer using a microfluidic chip to form lipid nanoparticles. During this process, the lipids are protonated in ethanol to form cations, which bind to the negatively charged oligonucleotides, self-assembling into lipid nanoparticles through electrostatic interactions.

[0081] The mixed lipid nanoparticle solution contains a high concentration of ethanol, which needs to be removed by dialysis or ultrafiltration, and the solution system should be replaced with a neutral buffer for subsequent biological experiments and long-term preservation.

[0082] Quality testing of lipid nanoparticles: The particle size and monodispersity of lipid nanoparticles are tested using equipment such as dynamic light scattering instruments to ensure that the quality of lipid nanoparticles meets the experimental requirements.

[0083] Storage of lipid nanoparticles: Lipid nanoparticles that have passed quality testing should be stored at -80℃ for a long period of time.

[0084] (3) Genes and sequences involved

[0085] PARP1, poly(ADP-ribose) polymerase 1, human Gene ID:142.

[0086] ETV4, ETS variant transcription factor 4, human sequence as shown in SEQ ID NO.1, mouse sequence as shown in SEQ ID NO.2.

[0087] SEQ ID NO.1:

[0088]

[0089] SEQ ID NO.2:

[0090]

[0091] The microRNA and small interfering RNA sequences targeting each gene are shown in Table 1.

[0092] Table 1. MicroRNA and Small Interfering RNA Sequences

[0093]

[0094] Example 1: Preparation of lipid nanoparticles using microfluidic mixing technology

[0095] The composition of the lipid nanoparticles in this embodiment is shown in Table 2:

[0096] Table 2. Component ratio of lipid nanoparticles

[0097]

[0098] A method for preparing an oligonucleotide lung-targeted delivery system includes the following steps:

[0099] (1) Preparation of lipid solution (oil phase): 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipids are dissolved in anhydrous ethanol to prepare a lipid-ethanol solution, wherein the mass-volume ratio of the lipids to ethanol is 4 mg / mL;

[0100] (2) Preparation of nucleic acid solution (aqueous phase): Dissolve AM22 in sodium citrate buffer at pH 4.0 to prepare nucleic acid-buffer solution, wherein the concentration of the oligonucleotide molecule is 146.5 μg / mL;

[0101] (3) Mixing and shaping: The aqueous phase and oil phase were loaded into a microfluidic mixing device and mixed through a 200 μm × 200 μm chip. The mixing conditions were set as follows: the volume ratio of aqueous phase to oil phase was 3:1 (the mass ratio of oligonucleotide and lipid components was 1:17.9), and the total flow rate was 20 mL / min to form lipid nanoparticles.

[0102] (4) Post-processing: Remove residual ethanol by dialysis or ultrafiltration and replace it with neutral buffer;

[0103] (5) Quality testing: The particle size, potential, encapsulation efficiency and stability of the lipid nanoparticles are tested.

[0104] Example 2: Preparation of lipid nanoparticles by thin-film hydration method

[0105] (1) Weigh out 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, dipalmitoylphosphatidylcholine, and polyethylene glycol-modified lipids according to each group in Table 2, dissolve them in 5 mL of chloroform, and transfer them to a 50 mL round-bottom flask.

[0106] (2) The trichloromethane was removed by rotary evaporation at 60℃ to form a uniform lipid film;

[0107] (3) Add PBS buffer or UP water at 60℃ and hydrate for more than 1 hour;

[0108] (4) Use a handheld ultrasound processor to sonicate for 10 minutes to form colostrum;

[0109] (5) After standing overnight, add buffer containing microRNA AM22 and repeatedly extrude through 0.4μm and 0.2μm polycarbonate membranes 24 times to obtain lipid nanoparticles, and store at 4℃.

[0110] The lipid nanoparticles prepared in this embodiment were characterized and their safety was verified. The results are shown in the figure. Figure 1-2 .

[0111] Example 3: In vivo lung-targeting experiment of lipid nanoparticles

[0112] (1) Establishment of CT26-luc mouse model of colorectal cancer lung metastasis: 4×10 4 CT26-luc cells (mouse colon cancer cells - luciferase-labeled);

[0113] (2) Five days after modeling, the group was injected with 100 μg of DiR (1,1'-dioctyl-3,3,3',3'-tetramethylindolecarbonylcyanine perchlorate) labeled lung-targeting lipid nanoparticles (LuT), MC3 lipid nanoparticles and SORT lipid nanoparticles via the tail vein.

[0114] in:

[0115] Lung-targeting lipid nanoparticles (LuT) are group I-XIX from Example 2;

[0116] The preparation of MC3 lipid nanoparticles was the same as in Example 2, except that the lipid composition was different. The molar ratio of 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, distearate phosphatidylcholine, polyethylene glycol-modified lipids and cholesterol was 50:10:1.5:38.5.

[0117] The preparation of SORT lipid nanoparticles was the same as in Example 2, except that the lipid composition was different. The molar ratio of 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, distearate phosphatidylcholine, polyethylene glycol-modified lipids, and cholesterol was 25:50:5:0.8:19.2.

[0118] (3) Six hours later, the mice were sacrificed and the heart, liver, spleen, lungs and kidneys were removed. The fluorescence distribution was detected by IVIS-Lumina in vivo imaging system.

[0119] (4) Organ distribution and lung fluorescence intensity of lipid nanoparticles are shown in Table 3 and Figure 3 .

[0120] Table 3 Organ distribution and lung fluorescence intensity of LuT lipid nanoparticles

[0121]

[0122] The fluorescence intensity in the lungs of the MC3 lipid nanoparticle group was only 19% of that of the LuT lipid nanoparticle IX group, and significant aggregation was observed in the liver and spleen.

[0123] The fluorescence intensity in the lungs of the SORT lipid nanoparticle group was only 49% of that of the LuT lipid nanoparticle IX group, and significant aggregation was observed in the liver and spleen.

[0124] As can be seen, the proportion of fluorescence intensity in the lungs of the LuT lipid nanoparticle group was 9.55-39.83%, which was significantly higher than that of MC3 lipid nanoparticles and SORT lipid nanoparticles (P<0.01), while the fluorescence intensity in the liver and spleen was significantly reduced.

[0125] Example 4: Anti-colorectal cancer lung metastases activity of microRNA lipid nanoparticles

[0126] Human colorectal cancer lung metastasis cells T84 were treated with the negative control RNAs SEQ ID NO.9&17 and SEQ ID NO.10&18, respectively, and the microRNA AM22 was found to inhibit the expression of the PARP1 gene in T84 cells (using the glyceraldehyde-3-phosphate dehydrogenase GAPDH gene as an internal control). The results are shown in the figure below. Figure 4 Therefore, lipid nanoparticles were prepared using this sequence, following the same steps as group III in Example 2, and named AM22@LuT-LNP.

[0127] (1) Grouping of colorectal cancer lung metastasis model mice (n=6): negative control group (negative control RNA SEQ ID NO.9&17 lipid nanoparticles, 1OD), positive control group (5-fluorouracil 5-FU, 20mg / kg), AM22 lipid nanoparticle group (AM22@LuT-LNP, SEQ ID NO.10&18, 1OD);

[0128] (2) The negative control group and the AM22 lipid nanoparticle group were administered the drug via the tail vein once every 4 days for a total of 4 times; the positive control group was administered the drug via the peritoneal cavity once every 2 days for a total of 7 times.

[0129] (3) Weigh yourself every 4 days, monitor tumor progression with IVIS-Lumina live imaging system, and record survival time;

[0130] (4) Figure 5 The results showed that tumor growth was significantly inhibited in the AM22 lipid nanoparticle group, the survival time of mice was extended by 55% compared with the negative control group (P<0.05), and there was no significant decrease in body weight.

[0131] Example 5: Anti-colorectal cancer lung metastases activity of small interfering RNA lipid nanoparticles

[0132] Human colorectal cancer lung metastasis cells T84 and mouse colorectal cancer cells CT26 were treated with negative control RNAs SEQ ID NO. 9 & 17 and SEQ ID NO. 3-16, respectively. The results showed that both small interfering RNAs inhibited ETV4 gene expression in T84 cells, with SEQ ID NO. 5 & 13 showing the best performance. (See attached figures). Figure 6 Therefore, lipid nanoparticles were prepared using this sequence, following the same steps as group III in Example 2, and named ETV4@LuT-LNP.

[0133] (1) Grouping of colorectal cancer lung metastasis model mice (n=6): negative control group (negative control RNA SEQ ID NO.9&17 lipid nanoparticles, 1OD), positive control group (regorafenib, 10mg / kg), ETV4 gene small interfering RNA lipid nanoparticle group (ETV4@LuT-LNP, SEQ ID NO.5&13, 1OD).

[0134] (2) The negative control group and the ETV4 gene small interfering RNA lipid nanoparticle group were administered the drug via tail vein once every 4 days for a total of 4 times; the positive control group was administered the drug via intraperitoneal injection once every 2 days for a total of 7 times.

[0135] (3) Weigh yourself every 4 days, monitor tumor progression with IVIS-Lumina live imaging system, and record survival time;

[0136] (4) Figure 7The results showed that tumor growth was significantly inhibited in the ETV4 gene small interfering RNA lipid nanoparticle group, the survival time of mice was extended by 31% compared with the control group (P<0.005), and there was no significant decrease in body weight.

[0137] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A lipid nanoparticle, characterized in that, The lipid nanoparticles are for lung targeting and contain oligonucleotides and lipid components. Based on the total molar mass of the lipid components (100%), they include the following components in the following molar percentages: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 0-66%; (2,3-Dioleoyl-propyl)-trimethylamine, 0-66%; Dipalmitoylphosphatidylcholine, 0-66%; Polyethylene glycol-modified lipids, 1%-5%; Among them, at least two of the following have a molar percentage that is not 0: methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, and dipalmitoylphosphatidylcholine.

2. The lipid nanoparticles according to claim 1, characterized in that, When the molar percentage of 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester is 0, the molar percentages of (2,3-dioleoyl-propyl)-trimethylamine and dipalmitoylphosphatidylcholine are 30%-66% and 30%-66%, respectively. Alternatively, when the molar percentage of dipalmitoylphosphatidylcholine is 0, the molar percentages of 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester and (2,3-dioleoyl-propyl)trimethylamine are 30%-66% and 30%-66%, respectively; Alternatively, when the molar percentages of 4-(N,N-dimethylamino)butyrate (dioleoyl)methyl ester, (2,3-dioleoyl-propyl)-trimethylamine and dipalmitoylphosphatidylcholine are all not 0, the molar percentages are 15%-66%, 15%-66% and 15%-66%, respectively.

3. The lipid nanoparticles according to claim 2, characterized in that, When the molar percentage of dipalmitoylphosphatidylcholine is 0, the molar percentages of 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester and (2,3-dioleoyl-propyl)trimethylamine are 30%-55% and 45%-66%, respectively. Alternatively, when the molar percentages of 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, and dipalmitoylphosphatidylcholine are all not 0, the molar percentages are 15%-20%, 45%-66%, and 15%-35%, or 30%-35%, 15%-20%, and 45%-50%, or 60%-66%, 15%-20%, and 15%-20%, respectively.

4. The lipid nanoparticles according to any one of claims 1-3, characterized in that, In the PEGylated lipids, the average molecular weight of the polyethylene glycol segments is 1000-5000 Da; And / or, the oligonucleotide is a gene silencing agent, which includes one or more of microRNA, small interfering RNA, antisense oligonucleotide, and short hairpin RNA; And / or, the mass ratio of the oligonucleotide to the lipid component is 1:(10-20).

5. The method for preparing lipid nanoparticles according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Dissolve the oligonucleotide to prepare an aqueous phase; Dissolve the lipid components to prepare an oil phase; S2. Mix the aqueous phase and the oil phase to prepare the lipid nanoparticles.

6. An oligonucleotide lung-targeted delivery system, characterized in that, Includes the lipid nanoparticles according to any one of claims 1-4.

7. A pharmaceutical composition for preparing an oligonucleotide lung-targeted delivery system, characterized in that, The pharmaceutical composition comprises a gene-producing drug and a lipid component, wherein the gene-producing drug is an oligonucleotide, and the lipid component comprises the following components in molar percentages, based on a total molar mass of 100%: 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 0-66%; (2,3-Dioleoyl-propyl)-trimethylamine, 0-66%; Dipalmitoylphosphatidylcholine, 0-66%; Polyethylene glycol-modified lipids, 1%-5%; Among them, at least two of the following have a molar percentage that is not 0: methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester, (2,3-dioleoyl-propyl)-trimethylamine, and dipalmitoylphosphatidylcholine.

8. The use of the lipid nanoparticles of any one of claims 1-4, the oligonucleotide lung-targeted delivery system of claim 6, or the pharmaceutical composition of claim 7 in the preparation of a medicament for treating lung diseases.

9. The application according to claim 8, characterized in that, The lung diseases mentioned include lung cancer, lung metastases, pulmonary fibrosis, or lung infections; And / or, the oligonucleotide is a gene silencing sequence of the drug target gene; And / or, when the lung metastasis is a colorectal cancer lung metastasis, the oligonucleotide is a sequence targeting the silent poly(ADP-ribose) polymerase gene PARP1 or the ETS variant transcription factor 4 gene ETV4.

10. The application according to claim 9, characterized in that, When the target gene is the poly(ADP-ribose) polymerase gene PARP1, the oligonucleotides are the sequences shown in SEQ ID NO.10 and SEQ ID NO.18; And / or, when the target gene is the ETS variant transcription factor 4 gene ETV4, the oligonucleotides are any of the following groups: (1) The sequences shown in SEQ ID NO.3 and SEQ ID NO.11; (2) The sequences shown in SEQ ID NO.4 and SEQ ID NO.12; (3) The sequences shown in SEQ ID NO.5 and SEQ ID NO.13; (4) The sequences shown in SEQ ID NO.6 and SEQ ID NO.14; (5) The sequences shown in SEQ ID NO.7 and SEQ ID NO.15; (6) The sequences shown in SEQ ID NO.8 and SEQ ID NO.16.

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