Lipid nanoparticle for targeted delivery of nucleic acid to peritoneum and peritoneal tumor

Abstract

The invention discloses lipid nanoparticles for targeted delivery of nucleic acids to peritoneum and peritoneal tumors. The invention provides a lipid nanoparticle. The lipid nanoparticle comprises an ionizable lipid, a cationic auxiliary lipid containing a trimethylamine head, sterol and a PEG (polyethylene glycol) lipid. According to the lipid nanoparticles, nucleic acid can be efficiently delivered to peritoneal tissues, meanwhile, distribution of the nucleic acid and a delivery carrier in other tissues and organs of the whole body is effectively reduced, and the side effect risk caused by nonspecific accumulation of the nanoparticles is remarkably reduced.

Description

Technical Field

[0001] This invention relates to lipid nanoparticles for targeted delivery of nucleic acids to the peritoneum and peritoneal tumors. Background Technology

[0002] Intraperitoneal administration is a routine method of delivering drugs locally by direct intraperitoneal injection. Compared to systemic administration (such as intravenous injection), it has the advantages of increasing drug concentration at the target site and reducing systemic toxicity, demonstrating irreplaceable advantages in the treatment of peritonitis, peritoneal tumors, and other abdominal diseases.

[0003] In recent years, lipid nanoparticles (LNPs), as an advanced drug delivery system, have made significant breakthroughs in the field of nucleic acid drug delivery due to their excellent biocompatibility and biosafety.

[0004] However, existing clinically approved lipid nanoparticle systems exhibit serious limitations when used to treat peritoneal diseases: significant non-specific accumulation of lipid nanoparticles in the liver and spleen occurs after intraperitoneal injection, resulting in low local targeting efficiency and accompanying systemic toxicity risks, severely limiting their clinical application value. Therefore, there is an urgent need to develop a novel lipid nanoparticle system to reduce the accumulation of nanoparticles in non-target organs (tissues), promote efficient retention of lipid nanoparticles in peritoneal tissues, and achieve efficient delivery of nucleic acid drugs to the peritoneum and peritoneal-related lesions. Summary of the Invention

[0005] To address the problem of significant non-specific accumulation of lipid nanoparticles in the liver and spleen after intraperitoneal injection, leading to low local targeting efficiency in the peritoneum, this invention provides lipid nanoparticles for targeted delivery of nucleic acids to the peritoneum and peritoneal tumors. The lipid nanoparticles of this invention can achieve efficient delivery of nucleic acids to peritoneal tissue while effectively reducing the distribution of nucleic acids and delivery carriers in other tissues and organs throughout the body, significantly reducing the risk of side effects caused by non-specific accumulation of nanoparticles.

[0006] The present invention provides a lipid nanoparticle comprising: ionizable lipid, cationic auxiliary lipid containing a trimethylammonium head, sterol and PEG lipid.

[0007] In this invention, "cationic auxiliary lipid containing a trimethylammonium head" means that the cationic auxiliary lipid contains .

[0008] In this invention, the structure of the cationic auxiliary lipid containing a trimethylammonium head is a compound as shown in Formula I or its stereoisomers or tautomers:

[0009] ;

[0010] Where L is independently -O-, -CH2O-, -OC(=O)-, -CH2OC(=O)-; R is independently C1-C 20 Alkyl, C2-C 20 alkenyl or C2-C 20 Alkyne group.

[0011] In this invention, the C1-C 20 Alkyl groups are straight-chain alkanes, such as C10 and C20. 10 -C 20 Alkyl groups, for example, C 11 -C 15 alkyl.

[0012] In this invention, the C2-C 20 The alkenyl group is a straight-chain alkenyl group, such as C. 15 -C 20 Alkenyl, for example C 17 -C 18 Alkenyl group.

[0013] In this invention, the C2-C 20 Alkenyl groups contain a double bond, for example, between the 8th and 9th or the 9th and 10th carbons.

[0014] In this invention, preferably, the cationic auxiliary lipid containing a trimethylammonium head is selected from DOTAP (… ), DOTMA ( ) and DMTAP ( One or more of them.

[0015] In this invention, the sterol can be a conventional sterol. The sterol includes animal, plant, or fungal sterols; preferably, the sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, and α-tocopherol, such as cholesterol.

[0016] In this invention, the lipid nanoparticles have a particle size of 100–200 nm.

[0017] In this invention, the surface potential of the lipid nanoparticles is +1 to +5 mV.

[0018] In this invention, the ionizable lipid is selected from SM-102 ( ), DLin-MC3-DMA ( ) and ALC-0315 ( One or more of them.

[0019] In this invention, the PEG lipid is a lipid molecule modified with a polyethylene glycol hydrophilic end. The PEG lipid is preferably selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified dimethylglycine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0020] Preferably, the PEG lipid is selected from one or more of DMG-PEG, DSPE-PEG, and DOPE-PEG;

[0021] Preferably, the PEG lipid is DMG-PEG or DSPE-PEG.

[0022] Preferably, the average molecular weight of the polyethylene glycol in the PEG lipid is 2000-5000. In this invention, the PEG lipid is preferably DMG-PEG 2K.

[0023] In this invention, preferably, the nitrogen-to-phosphorus ratio (N / P) of the lipid nanoparticles is 8 to 13 (the nitrogen-to-phosphorus ratio refers to the ratio of the number of moles of ionizable nitrogen atoms in one or more ionizable lipid compounds to the number of moles of phosphate ester groups in RNA).

[0024] In this invention, the molar ratio of the ionizable lipid to the cationic auxiliary lipid containing a trimethylammonium head is (10~50):(10~25); preferably (30~40):(10~15); more preferably (1-2.3):1; even more preferably (1.8-2.1):1, and most preferably 2:1.

[0025] In this invention, the molar ratio of the ionizable lipid to the sterol is (10~50):(25~80); more preferably (30~40):(45~60); even more preferably 1:(0.8-2.0); even more preferably 1:(1.8-1.9); and most preferably 30:54.5.

[0026] In this invention, the molar ratio of the ionizable lipid to the PEG lipid is (10~50):(0.5~3.5); preferably, (30~40):(45~60); more preferably, (25-65):1; even more preferably, (58-62):1; and most preferably, 60:1.

[0027] In this invention, the proportions of the various components described refer to the proportions of the raw materials.

[0028] The lipid nanoparticles of the present invention can be prepared using conventional methods known in the art.

[0029] The present invention also provides a pharmaceutical composition comprising a therapeutic agent and / or a preventive agent and the aforementioned lipid nanoparticles.

[0030] Preferably, the pharmaceutical composition is an injectable preparation.

[0031] In this invention, the therapeutic agent and / or preventive agent is one or more nucleic acids. The nucleic acid may be a conventional nucleic acid in the art. The therapeutic agent and / or preventive agent may be primary or metastatic peritoneal cancer.

[0032] The present invention also provides the use of the aforementioned lipid nanoparticles or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention or treatment of peritoneal tumors.

[0033] Preferably, in the application, the drug is administered via intraperitoneal injection.

[0034] The positive and progressive effects of this invention are as follows: the lipid nanoparticles of this invention can efficiently deliver nucleic acids to peritoneal tissue, while effectively reducing the distribution of nucleic acids and delivery carriers in other tissues and organs throughout the body, and significantly reducing the risk of side effects caused by non-specific accumulation of nanoparticles. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the preparation process of the peritoneal-targeting lipid nanoparticles described in Example 1;

[0036] Figure 2 The delivery efficiency of lipid nanoparticles with different lipid compositions in mouse colon cancer cells, human peritoneal mesothelial cells and macrophages;

[0037] Figure 3 This is a diagram showing the organ expression distribution of luciferase mRNA delivered intraperitoneally by the peritoneal-targeting lipid nanoparticles (Peritoneum-LNP-SM102) in normal mice.

[0038] Figure 4 Organ expression distribution of luciferase mRNA delivered intraperitoneally by peritoneal-targeting lipid nanoparticles (Peritoneum-LNP-DSPE-PEG) in normal mice;

[0039] Figure 5 Organ expression distribution of peritoneal-targeted lipid nanoparticles (Peritoneum-LNP-DOTMA or Peritoneum-LNP-DMTAP) delivering luciferase mRNA intraperitoneally in normal mice;

[0040] Figure 6The image shows the organ expression distribution of control lipid nanoparticles (Control-LNP, DLin-MC3-DMA:DSPC:cholesterol:PEG2000-DMG molar ratio of 50%:10%:38.5%:1.5%) and peritoneal targeted lipid nanoparticles (Peritoneum-LNP) described in Example 1 in a mouse model of colon cancer peritoneal metastasis.

[0041] Figure 7 This is a diagram showing the organ expression distribution of luciferase mRNA delivered intraperitoneally by the control lipid nanoparticles (Control-LNP) and the peritoneal-targeting lipid nanoparticles (Peritoneum-LNP) described in Example 1 in normal mice.

[0042] Figure 8 This is an experimental protocol for treating a colon cancer peritoneal metastasis model by intraperitoneal injection of peritoneal-targeting lipid nanoparticles loaded with IL-12 mRNA obtained in Example 1.

[0043] Figure 9 The efficacy of intraperitoneal injection of peritoneal-targeting lipid nanoparticles loaded with IL-12 mRNA obtained in Example 1 was evaluated in a colon cancer peritoneal metastasis model. Detailed Implementation

[0044] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0045] Example 1 (Microfluidic Method)

[0046] like Figure 1 The schematic diagram of the preparation process shows the preparation of the lipid mixture: Ionizable lipids (DLin-MC3-DMA), auxiliary lipids (DOTAP, 1,2-dioleoyl-3-trimethylammoniumpropane), cholesterol (Cholesterol), and polyethylene glycol-modified lipids (PEG lipids, DMG-PEG 2K) are mixed in a molar ratio of 30:15:54.5:0.5. After adding anhydrous ethanol, the mixture is stirred or sonicated using a magnetic stirrer or ultrasonic oscillator until completely transparent and free of precipitate, resulting in a lipid mixture with an ionizable lipid concentration of 2 mg / mL.

[0047] Nucleic acid (Luciferase mRNA) was dissolved in citrate buffer at pH 4.0 to obtain a solution with a nucleic acid concentration of 1 mg / mL;

[0048] Using a microfluidic device, the lipid mixture and nucleic acid solution were rapidly mixed in a cross-flow manner at a volume ratio of 1:3 (mixing rate ratio of lipid mixture to nucleic acid solution: 1:3, total flow rate: 800 μL / min), using 1× PBS buffer and M... w Dialysis with a dialysis tube of 5-10 KD for 4-6 hours removes ethanol and unencapsulated components to obtain the peritoneal targeted lipid nanoparticles (Peritoneum-LNP); the obtained peritoneal targeted lipid nanoparticles can be stored for a short time at 4°C.

[0049] Using a Malvern Zetasizer dynamic light scattering device with a zeta potential measurement module, the measurement parameters were set, including a temperature of 25°C, the refractive index of the medium, and the dielectric constant, to test the particle size of the peritoneal targeted lipid nanoparticles. The average particle size of the peritoneal targeted lipid nanoparticles was 122 nm, the distribution coefficient PDI was 0.134, and the zeta potential was 1.94 mV.

[0050] The encapsulation efficiency of nucleic acids in the peritoneal-targeting lipid nanoparticles was determined: the mRNA encapsulation efficiency of the lipid nanoparticles was calculated using a Quant-iT-RiboGreen assay (Thermo Fisher Scientific, Waltham, MA). Each lipid nanoparticle sample was diluted to approximately 2 ng / μL in two microcentrifuge tubes containing either 1× TE buffer or 0.1% (v / v) Triton X-100. The lipid nanoparticles in Triton X-100 were lysed for 20 min. After incubation, the lipid nanoparticles in TE buffer, Triton X-100, and mRNA standards were added in triplicate to black 96-well plates, and fluorescent RiboGreen reagent was added according to the manufacturer's instructions. Fluorescence intensity was read at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using a SpectraMax iD5 Molecular Devices. RNA content was quantified by comparison with a standard curve estimated using least-squares linear regression (LSLR). Encapsulation efficiency is calculated as follows: where A is the RNA content in the TE buffer, and B is the RNA content in the Triton X-100 buffer. Finally, the encapsulation ratio is calculated using the following formula: ;

[0051] In Example 1, the encapsulation rate was 97.3%.

[0052] Example 2

[0053] Lipid nanoparticles were prepared according to the method of Example 1 and according to the molar contents of Table 1 below.

[0054] Table 1

[0055]

[0056] The delivery efficiency of four different lipid nanoparticles prepared in Example 2 was tested in cells: Four different lipid nanoparticles loaded with luciferase mRNA were transfected into mouse colon cancer cells, macrophages and human peritoneal mesothelial cells.

[0057] Mouse colon cancer cells (TCM37, Chinese Academy of Sciences Cell Bank), macrophages (TCM13, Chinese Academy of Sciences Cell Bank), and human peritoneal mesothelial cells (ELISA, ml096343) were seeded into 48-well plates and allowed to adhere overnight.

[0058] The lipid nanoparticles were administered at a dose of 50 ng mRNA per well via direct drop-up.

[0059] Cells were collected 24 hours after drug administration, and the cells were lysed using cell lysis buffer, with the supernatant collected.

[0060] Add luciferase substrate to the supernatant and measure absorbance using a spectrophotometer. Figure 2 Table 2 shows the delivery efficiency of luciferase mRNA in different cells by lipid nanoparticles prepared according to the Patisiran formulation (Molar ratio of Control-LNP, DLin-MC3-DMA:DSPC:cholesterol:PEG2000-DMG 50%:10%:38:5%:1:5%) and lipid nanoparticles obtained in Example 2. Figure 2 As shown in Table 2, D1 and D2 had similar or higher transfection efficiencies than Control-LNP in mouse colon cancer cells, macrophages, and human peritoneal mesothelial cells, while D1 had the best delivery efficiency in the three cell lines, and this was designated as Peritoneum-LNP.

[0061] Table 2

[0062]

[0063] Following the method of Example 1, the molar ratio of lipid nanoparticles was fixed, and lipid nanoparticles Peritoneum-LNP-SM102 were prepared by replacing DLin-MC3-DMA with SM102.

[0064] Three healthy Balb / c mice (male or female, aged 6-8 weeks, weighing 18-22 g) from Beijing Vital River Laboratory Animal Technology Co., Ltd. were selected and injected intraperitoneally with lipid nanoparticles (Peritoneum-LNP-SM102) loaded with luciferase mRNA.

[0065] The injection dose of lipid nanoparticles is 0.5 mg / kg body weight (calculated based on nucleic acid content, 100-200 μL / mouse, the specific volume is calculated according to the weight of the mouse), and the injection method is intraperitoneal injection (IP injection). Aseptic operation is required during the injection process to avoid damage to the animal's internal organs.

[0066] Six hours after injection, the mice were anesthetized, euthanized by dislocation of the neck, and the heart, liver, spleen, lungs, kidneys, and mesentery were removed.

[0067] The removed organs were immersed in a D-fluorescein potassium solution (concentration of 10 mg / ml) for five minutes.

[0068] In vivo imaging of dissected organs was performed using an in vivo imaging system (IVIS), and the fluorescence values ​​of each organ were quantified using Living Image. Figure 3 Table 3 shows the organ expression distribution of luciferase mRNA delivered intraperitoneally by the peritoneal-targeting lipid nanoparticles (Peritoneum-LNP-SM102) in normal mice. Figure 3 As shown in Table 3, Peritoneum-LNP-SM102 can efficiently deliver mRNA to the mesentery while reducing non-specific delivery to organs such as the liver and spleen, demonstrating significant peritoneal targeting.

[0069] Table 3

[0070]

[0071] Following the method of Example 1, the molar ratio of lipid nanoparticles was fixed, and lipid nanoparticles Peritoneum-LNP-DSPE-PEG were prepared by replacing DMG-PEG 2K with DSPE-PEG2K.

[0072] Four healthy Balb / c mice (male or female, aged 6-8 weeks and weighing 18-22 g) from Beijing Vital River Laboratory Animal Technology Co., Ltd. were selected and injected intraperitoneally with lipid nanoparticles (Peritoneum-LNP-DSPE-PEG) loaded with luciferase mRNA.

[0073] The injection dose of lipid nanoparticles is 0.5 mg / kg body weight (calculated based on nucleic acid content, 100-200 μL / mouse, the specific volume is calculated according to the weight of the mouse), and the injection method is intraperitoneal injection (IP injection). Aseptic operation is required during the injection process to avoid damage to the animal's internal organs.

[0074] Six hours after injection, the mice were anesthetized, euthanized by dislocation of the neck, and the heart, liver, spleen, lungs, kidneys, and mesentery were removed.

[0075] The removed organs were immersed in a D-fluorescein potassium solution (concentration of 10 mg / ml) for five minutes.

[0076] In vivo imaging of dissected organs was performed using an in vivo imaging system (IVIS), and the fluorescence values ​​of each organ were quantified using Living Image. Figure 4 Table 4 shows the organ expression distribution of luciferase mRNA delivered intraperitoneally by the peritoneal-targeting lipid nanoparticles (Peritoneum-LNP-DSPE-PEG) in normal mice. Figure 4 As shown in Table 4, Peritoneum-LNP-DSPE-PEG can efficiently deliver mRNA to the mesentery while reducing non-specific delivery to organs such as the liver and spleen, demonstrating significant peritoneal targeting.

[0077] Table 4

[0078]

[0079] Following the method of Example 1, the molar ratio of lipid nanoparticles was fixed, and lipid nanoparticles Peritoneum-LNP-DOTMA and Peritoneum-LNP-DMTAP were prepared by replacing DOTAP with DOTMA or DMTAP.

[0080] Five healthy Balb / c mice (male or female, aged 6-8 weeks and weighing 18-22 g) from Beijing Vital River Laboratory Animal Technology Co., Ltd. were selected and injected intraperitoneally with lipid nanoparticles loaded with luciferase mRNA (Peritoneum-LNP-DOTMA or Peritoneum-LNP-DMTAP).

[0081] The injection dose of lipid nanoparticles is 0.5 mg / kg body weight (calculated based on nucleic acid content, 100-200 μL / mouse, the specific volume is calculated according to the weight of the mouse), and the injection method is intraperitoneal injection (IP injection). Aseptic operation is required during the injection process to avoid damage to the animal's internal organs.

[0082] Six hours after injection, the mice were anesthetized, euthanized by dislocation of the neck, and the heart, liver, spleen, lungs, kidneys, and mesentery were removed.

[0083] The removed organs were immersed in a D-fluorescein potassium solution (concentration of 10 mg / ml) for five minutes.

[0084] In vivo imaging of dissected organs was performed using an in vivo imaging system (IVIS), and the fluorescence values ​​of each organ were quantified using Living Image. Figure 5Table 5 shows the organ expression distribution of luciferase mRNA delivered intraperitoneally by the peritoneal-targeting lipid nanoparticles (Peritoneum-LNP-DOTMA or Peritoneum-LNP-DMTAP) in normal mice. Figure 5 As shown in Table 5, both Peritoneum-LNP-DOTMA and Peritoneum-LNP-DMTAP can efficiently deliver mRNA to the mesentery while reducing non-specific delivery to organs such as the liver and spleen, demonstrating significant peritoneal targeting.

[0085] Table 5

[0086]

[0087] Application Example 1

[0088] The targeted delivery efficiency of the peritoneal-targeting lipid nanoparticles described in Example 1 in a mouse model of peritoneal tumors was tested.

[0089] Healthy Balb / c mice, of any sex, aged 6–8 weeks and weighing 18–22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. A colon cancer peritoneal metastasis model was established by intraperitoneal injection of colon cancer cells into the mice. After tumor formation on days 7–10, the mice were randomly divided into experimental and control groups, with four mice in each group: Peritoneum-targeted lipid nanoparticle group: intraperitoneal injection of lipid nanoparticles loaded with luciferase mRNA (Peritoneum-LNP); Control group: intraperitoneal injection of lipid nanoparticles loaded with luciferase mRNA (Control-LNP).

[0090] The injection dose of lipid nanoparticles is 0.5 mg / kg body weight (calculated based on nucleic acid content, 100-200 μL / mouse, the specific volume is calculated according to the weight of the mouse), and the injection method is intraperitoneal injection (IP injection). Aseptic operation is required during the injection process to avoid damage to the animal's internal organs.

[0091] Six hours after injection, the mice were anesthetized, euthanized by dislocation, and the heart, liver, spleen, lungs, kidneys, and abdominal tumors were removed.

[0092] The removed organs were immersed in a D-fluorescein potassium solution (concentration of 10 mg / ml) for five minutes.

[0093] In vivo imaging of dissected organs was performed using an in vivo imaging system (IVIS), and the fluorescence values ​​of each organ were quantified using Living Image. Figure 6Table 6 shows the organ expression distribution of lipid nanoparticles Control-LNP prepared according to the Patisiran formulation (composed of DLin-MC3-DMA:DSPC:cholesterol:PEG2000-DMG in a molar ratio of 50%:10%:38.5%:1.5%, prepared using the same method as in Example 1) and peritoneal-targeting lipid nanoparticles Peritoneum-LNP obtained in Example 1, which deliver luciferase mRNA intraperitoneally, in a mouse model of colon cancer peritoneal metastasis. Figure 6 As shown in Table 6, compared with Control-LNP, the Peritoneum-LNP group showed high expression of luciferase in peritoneal tumors, while no significant expression was observed in extra-abdominal organs such as the liver and spleen; conversely, the Control-LNP group showed high expression of luciferase in liver and spleen tissues. This indicates that Peritoneum-LNP can achieve selective nucleic acid delivery to peritoneal tumors, demonstrating significant peritoneal tumor targeting.

[0094] Table 6

[0095]

[0096] Application Example 2

[0097] Testing the peritoneal-targeting lipid nanoparticles described in Example 1 to assess their peritoneal-targeting delivery capability in normal mice:

[0098] Healthy Balb / c mice from Beijing Vital River Laboratory Animal Technology Co., Ltd., of any sex, aged 6–8 weeks and weighing 18–22 g, were used. The mice were randomly divided into experimental and control groups, with 4 mice in each group: Peritoneum-targeted lipid nanoparticle group: lipid nanoparticles loaded with luciferase mRNA were injected intraperitoneally (Peritoneum-LNP); Control group: lipid nanoparticles loaded with luciferase mRNA were injected intraperitoneally (Control-LNP).

[0099] The injection dose of lipid nanoparticles is 0.5 mg / kg body weight (calculated based on nucleic acid content, 100-200 μL / mouse, the specific volume is calculated according to the weight of the mouse), and the injection method is intraperitoneal injection (IP injection). Aseptic operation is required during the injection process to avoid damage to the animal's internal organs.

[0100] Six hours after injection, the mice were anesthetized, euthanized by dislocation of the neck, and the heart, liver, spleen, lungs, kidneys, and mesentery were removed.

[0101] The removed organs were immersed in a D-fluorescein potassium solution (concentration of 10 mg / ml) for five minutes.

[0102] In vivo imaging of dissected organs was performed using an in vivo imaging system (IVIS), and the fluorescence values ​​of each organ were quantified using Living Image. Figure 7 Table 7 shows the organ expression distribution of luciferase mRNA delivered intraperitoneally by the control lipid nanoparticles (Control-LNP) and the peritoneal-targeting lipid nanoparticles (Peritoneum-LNP) described in Example 1 in normal mice. Figure 7 As shown in Table 7, compared with the Control-LNP group, Peritoneum-LNP can efficiently deliver mRNA to the mesentery, while reducing non-specific delivery to organs such as the liver and spleen, demonstrating significant peritoneal targeting.

[0103] Table 7

[0104]

[0105] Application Example 3

[0106] Balb / c mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., were used to establish a colon cancer peritoneal metastasis model. Tumor cell suspension (CT26-Luc cells, constructed by stably transfecting CT26 cells with Luciferase from the Cell Bank of the Chinese Academy of Sciences, 5 × 10⁶ m²) was used. 5 ~10×10 5 (1 cell / mouse) was injected into mice via intraperitoneal injection. After tumor formation, mice were randomly divided into three groups: the peritoneal-targeting lipid nanoparticle Peritoneum-LNP loaded with IL-12 mRNA (experimental group), the IL-2 mRNA-loaded Control-LNP (control group), and PBS (blank group). Each group of mice was treated with intraperitoneal injection at a predetermined dose and frequency (experimental design as described in Example 1). Figure 8 (As shown); after treatment, the therapeutic effect of lipid nanoparticles was reflected by recording continuous changes in the fluorescence intensity of tumors in the peritoneal cavity of mice. Figure 9 Table 8 shows the therapeutic effect of the peritoneal-targeted lipid nanoparticles described in Example 1 on inhibiting peritoneal metastasis of colon cancer. Figure 9 It was found that the fluorescence intensity of peritoneal metastases in the experimental group mice was significantly lower than that in the control group on day 14 (P<0.05), indicating that the peritoneal-targeting lipid nanoparticles loaded with IL-12 mRNA have a significant inhibitory effect on the formation of peritoneal metastases.

[0107] Table 8

[0108]

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lipid nanoparticle, characterized in that, It contains: ionizable lipids, cationic auxiliary lipids with a trimethylammonium head, sterols, and PEG lipids.

2. The lipid nanoparticles as described in claim 1, characterized in that, The structure of the cationic auxiliary lipid containing the trimethylammonium head is a compound as shown in Formula I or its stereoisomers or tautomers: ； Where L is independently -O-, -CH2O-, -OC(=O)-, -CH2OC(=O)-; R is independently C1-C 20 Alkyl, C2-C 20 alkenyl or C2-C 20 Alkyne group.

3. The lipid nanoparticles as described in claim 2, characterized in that, The structure of the cationic auxiliary lipid containing the trimethylammonium head satisfies one or more of the following conditions: (1) The C1-C 20 Alkyl groups are straight-chain alkanes; (2) The C2-C 20 The alkenyl group is a straight-chain alkenyl group; (3) The C2-C 20 The alkenyl group contains a double bond.

4. The lipid nanoparticles as described in claim 3, characterized in that, The structure of the cationic auxiliary lipid containing the trimethylammonium head satisfies one or more of the following conditions: (1) The C1-C 20 Alkyl groups are straight-chain alkanes; (2) The C2-C 20 The alkenyl group is C 15 -C 20 alkenyl; (3) The C2-C 20 The alkenyl group contains a double bond, which is located between the 8th and 9th or 9th and 10th carbons.

5. The lipid nanoparticles as described in claim 4, characterized in that, The structure of the cationic auxiliary lipid containing the trimethylammonium head satisfies one or two of the following conditions: (1) The C1-C 20 Alkyl group is C 11 -C 15 alkyl; (2) The C2-C 20 The alkenyl group is C 17 -C 18 Alkenyl group.

6. The lipid nanoparticles according to any one of claims 1-5, characterized in that, Lipid nanoparticles satisfy one or more of the following conditions: (1) The ionized lipids are selected from one or more lipid nanoparticles selected from SM-102, DLin-MC3-DMA and ALC-0315; (2) The PEG lipid is a lipid molecule modified with a polyethylene glycol hydrophilic end; (3) The molar ratio of the ionizable lipid to the cationic auxiliary lipid containing the trimethylammonium head is (10~50): (10~25); (4) The molar ratio of the ionizable lipids to sterols is (10~50):(25~80); (5) The molar ratio of the ionizable lipid to the PEG lipid is (10~50): (0.5~3.5).

7. The lipid nanoparticles as described in claim 6, characterized in that, Lipid nanoparticles satisfy one or more of the following conditions: (1) The PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified dimethylglycine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol; (2) The molar ratio of the ionizable lipid to the cationic auxiliary lipid containing a trimethylammonium head is (1.8-2.1):1; (3) The molar ratio of the ionizable lipids to sterols is 1:(1.8-1.9). (4) The molar ratio of the ionizable lipid to the PEG lipid is (58-62):1; (5) The average molecular weight of polyethylene glycol in the PEG lipid is 2000-5000.

8. The lipid nanoparticles as described in claim 7, characterized in that, Lipid nanoparticles satisfy one or more of the following conditions: (1) The PEG lipid is selected from one or more of DMG-PEG, DSPE-PEG and DOPE-PEG; (2) The molar ratio of the ionizable lipid to the cationic auxiliary lipid containing the trimethylammonium head is 2:1; (3) The molar ratio of the ionizable lipids and sterols is 30:54.5; (4) The molar ratio of the ionizable lipid to the PEG lipid is 60:

1.

9. A pharmaceutical composition comprising a therapeutic agent and / or a preventive agent, and lipid nanoparticles as described in any one of claims 1-8.

10. The use of a lipid nanoparticle as described in any one of claims 1-8 or a pharmaceutical composition as described in claim 9 in the preparation of a medicament for the prevention or treatment of peritoneal tumors.

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