Il12 mRNA lipid nanoparticle, preparation method therefor, and use thereof

Abstract

Provided is an IL12 mRNA lipid nanoparticle, comprising IL12 mRNA and a vector for delivering the IL12 mRNA, wherein the vector comprises one or more amino acid tocopheroloxyalkyl esters or pharmaceutically acceptable salts thereof. The present invention also relates to a preparation method for the lipid nanoparticle and use thereof in the preparation of an anti-bladder cancer drug. The lipid nanoparticle uses the amino acid tocopheroloxyalkyl ester as an ionizable lipid molecule vector for the delivery of the IL12 mRNA, thereby effectively delivering the IL12 mRNA into a tumor cell, avoiding the risk of mRNA being degraded by enzymes, exerting an anti-tumor effect in a mouse bladder cancer treatment group, inhibiting the growth of cancer cells, and exhibiting good biosafety.

Description

An IL-12 mRNA lipid nanoparticle, its preparation method, and its application. Technical Field

[0001] This invention belongs to the field of anti-bladder cancer drug technology, and relates to an IL 12 mRNA lipid nanoparticle, specifically to a lipid nanoparticle comprising an amino acid tocopheryl oxyalkylene ester as a carrier for delivering IL 12 mRNA, a method for preparing the lipid nanoparticle, and its application in the preparation of drugs for treating bladder cancer. Background Technology

[0002] Bladder cancer is a common malignant tumor of the urinary system, with approximately 500,000 new cases and 200,000 deaths worldwide each year. In China alone, there were approximately 100,000 new cases and 40,000 deaths in 2019. Based on whether it invades the muscle layer, bladder cancer can be divided into two main categories: muscle-invasive bladder cancer and non-muscle-invasive bladder cancer. Non-muscle-invasive bladder cancer accounts for 70% of cases, and transurethral resection of bladder tumors (TURP) and intravesical instillation of drugs are the mainstream treatment methods. Currently, traditional chemotherapy drugs such as gemcitabine and paclitaxel, as well as BCG, are mainly used clinically for intravesical instillation. BCG intravesical instillation is a classic method for treating bladder cancer, but some patients experience intolerance or treatment failure. Direct exposure to traditional chemotherapy drugs can irritate the bladder mucosa, easily leading to chemical cystitis, and its efficacy is limited. There is an urgent clinical need for safe and effective new drugs for intravesical instillation therapy of bladder cancer.

[0003] Interleukin-12 (IL-12) is a cytokine secreted by antigen-presenting cells such as monocytes / macrophages and B cells. Dendritic cells and helper T cells (Th cells) can also produce it. IL-12 exerts its anti-tumor effects by regulating innate and adaptive immunity and inhibiting tumor angiogenesis. Even with high IL-12 exposure, the infiltration level of IL-12 in the tumor microenvironment is very low, and high exposure has severe toxic effects on the body. High-dose infusion of IL-12 can lead to serious adverse reactions and is not suitable for systemic administration. Local administration of IL-12 may offer the possibility of enhancing anti-tumor effects and reducing toxic side effects. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional hematopoietic growth factor and an important immunomodulatory factor. GM-CSF can induce anti-tumor immune responses in the body. Combining GM-CSF with chemotherapy, radiotherapy, and immunotherapy can effectively kill tumors, prolong patient survival, and reduce toxic side effects. While GM-CSF is not very effective as a monotherapy for tumors, it can achieve good results as an adjuvant therapy. Combining GM-CSF with other drugs is a better option.

[0004] Currently, most clinical studies on IL-12's anti-tumor effects are in Phase I or II development stages, focusing on combining IL-12 with radiotherapy, chemotherapy, and immunotherapy to exert their combined anti-tumor effects. While IL-12 monotherapy has shown promising therapeutic effects in non-clinical studies, its clinical efficacy is not significant. This may be because the efficacy of IL-12 requires the pre-existing tumor immune response in the body. Non-clinical trials typically use newly inoculated tumor mice as test animals, which inherently possess some tumor immunity; whereas the tumor immunity of clinical patients has declined or been destroyed by previous treatments due to long-term illness. Furthermore, the very low dosage of IL-12 at the tumor site after systemic administration, coupled with its significant toxicity to patients, is also a factor that cannot be ignored.

[0005] IL-12 or GM-CSF, as biological macromolecular protein drugs, are difficult to be absorbed through biological membranes and are easily biodegraded, making them unsuitable for direct bladder instillation for the treatment of bladder cancer, and no relevant reports have been found.

[0006] mRNA drugs can be classified into three categories: preventative vaccines, therapeutic vaccines, and therapeutic drugs. mRNA can be translated and expressed within cells without entering the cell nucleus, thus avoiding integration into the host's genome and the risk of gene mutation. mRNA has low immunogenicity, making it less likely to trigger an immune response; it has a short half-life, and its metabolites are naturally occurring, eliminating the risk of persistent toxicity accumulation. Furthermore, mRNA can be synthesized through in vitro transcription, making it relatively inexpensive. mRNA is a highly promising drug that can meet the needs of gene therapy, cancer treatment, and vaccine development. However, because mRNA is easily degraded by enzymes and not easily taken up by target cells, the challenge lies in efficiently and stably delivering it intracellularly. In August 2021, the FDA approved the world's first mRNA COVID-19 vaccine (trade name Comirnay), jointly developed by Pfizer and BioNTech, which was also the first mRNA drug. In January 2022, the FDA approved Moderna's mRNA COVID-19 vaccine (trade name Spikevax). Using lipid nanoparticles as a carrier, it achieves efficient loading and delivery of mRNA. Summary of the Invention

[0007] To address the side effects of systemic IL-12 administration, the difficulty of protein drugs penetrating biological membranes, and the susceptibility of mRNA antitumor drugs to enzymatic degradation in existing technologies, the primary objective of this invention is to provide an IL-12 mRNA lipid nanoparticle. This lipid nanoparticle uses the amino acid tocopheryl oxyalkylene ester as an ionizable lipid molecular carrier for delivering IL-12 mRNA, effectively delivering IL-12 mRNA into tumor cells while avoiding the risk of mRNA degradation by enzymes. Furthermore, it exhibits antitumor effects in a mouse bladder cancer treatment group, inhibiting cancer cell growth and demonstrating good biocompatibility.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned IL 12 mRNA lipid nanoparticles, and the application of IL 12 mRNA lipid nanoparticles in the preparation of drugs for treating bladder cancer.

[0009] The present invention provides an IL 12 mRNA lipid nanoparticle comprising IL 12 mRNA and a carrier for delivering IL 12 mRNA, wherein the carrier comprises one or more ionizable lipid molecules or pharmaceutically acceptable salts thereof, having the general formula shown in (I):

[0010] Where B is R is n is 2-10;

[0011] T is

[0012] In one embodiment of the present invention, the ionizable lipid molecule is preferably histidine tocopheryl oxyethyl ester, lysine tocopheryl oxyethyl ester, arginine tocopheryl oxyethyl ester, histidine tocopheryl oxypropyl ester, lysine tocopheryl oxypropyl ester, or arginine tocopheryl oxypropyl ester; more preferably histidine tocopheryl oxyethyl ester.

[0013] In one embodiment of the present invention, preferably, the carrier further includes auxiliary lipid molecules, cholesterol, and PEGylated lipid molecules.

[0014] In one embodiment of the present invention, preferably, the auxiliary lipid molecule is distearylphosphatidylcholine (DSPC) or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE); the PEGylated lipid molecule is distearylphosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dimyristoylglycerol-polyethylene glycol 2000 (DMG2000), polyethylene glycol 1000 vitamin E succinate (TPGS1000), or polyethylene glycol 2000 vitamin E succinate (TPGS2000).

[0015] In one embodiment of the present invention, preferably, the molar ratio of the ionizable lipid molecule, the auxiliary lipid molecule, cholesterol and the PEGylated lipid molecule is 50:(5-20):(10-50):(1-5).

[0016] The present invention also provides a method for preparing the above-mentioned IL 12 mRNA lipid nanoparticles, characterized by comprising the following steps:

[0017] (1) Prepare acetate buffer (pH=3-4) for IL-12 mRNA and ethanol solution containing ionizable lipid molecules, auxiliary lipid molecules, cholesterol and PEGylated lipid molecules respectively.

[0018] (2) The above-prepared ethanol solution was mixed with IL-12 mRNA acetate buffer using a microfluidic mixer. The flow rate ratio was controlled to prepare a nanoparticle solution. The nanoparticle solution was diluted with 4-hydroxyethylpiperazine ethanesulfonic acid and concentrated by tangential flow ultrafiltration to obtain an IL-12 mRNA nanoparticle solution.

[0019] In one embodiment of the present invention, preferably, the particle size of the IL 12 mRNA lipid nanoparticles is 50-800 nm, more preferably 60-500 nm; and the zeta potential is -40-40 mV, more preferably -20-20 mV.

[0020] The present invention also provides a pharmaceutical composition comprising the above-described IL 12 mRNA lipid nanoparticles.

[0021] In one embodiment of the present invention, preferably, the pharmaceutical composition further includes GM-CSF mRNA or GM-CSF mRNA lipid nanoparticles.

[0022] In one embodiment of the present invention, preferably, the GM-CSF mRNA lipid nanoparticles include GM-CSF mRNA and a carrier for delivering GM-CSF mRNA, wherein the carrier includes one or more of the above-described ionizable lipid molecules or pharmaceutically acceptable salts thereof.

[0023] In one embodiment of the present invention, preferably, the particle size of the GM-CSF mRNA lipid nanoparticles is 50-800 nm, more preferably 60-500 nm; and the zeta potential is -40-40 mV, more preferably -20-20 mV.

[0024] In one embodiment of the present invention, the ionizable lipid molecule is preferably histidine tocopheryl oxyethyl ester, lysine tocopheryl oxyethyl ester, arginine tocopheryl oxyethyl ester, histidine tocopheryl oxypropyl ester, lysine tocopheryl oxypropyl ester, or arginine tocopheryl oxypropyl ester; more preferably histidine tocopheryl oxyethyl ester.

[0025] In one embodiment of the present invention, preferably, the carrier further includes an auxiliary lipid molecule, cholesterol, and a PEGylated lipid molecule. The auxiliary lipid molecule is distearylphosphatidylcholine (DSPC) or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE); the PEGylated lipid molecule is distearylphosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dimyristoylglycerol-polyethylene glycol 2000 (DMG2000), polyethylene glycol 1000 vitamin E succinate (TPGS1000), or polyethylene glycol 2000 vitamin E succinate (TPGS2000).

[0026] The present invention also provides a pharmaceutical formulation, which is a lyophilized powder injection made from the above-mentioned IL 12 mRNA lipid nanoparticles, or a lyophilized powder injection made from the above-mentioned pharmaceutical composition.

[0027] In one embodiment of the present invention, preferably, the freeze-drying protectant or additive used in the freeze-dried powder injection is one or more of lactose, sucrose, trehalose, mannose, etc., preferably sucrose or trehalose; the concentration of the freeze-drying protectant or additive used is 3%-20% (g / ml), preferably 5-10%.

[0028] The present invention also provides the use of the above-mentioned IL 12 mRNA lipid nanoparticles, the above-mentioned pharmaceutical composition, or the above-mentioned pharmaceutical preparation in the preparation of drugs for treating bladder cancer.

[0029] The IL 12 mRNA lipid nanoparticles of the present invention, the above-described pharmaceutical composition, or the above-described pharmaceutical formulation are preferably administered via bladder instillation.

[0030] In one administration method, an IL-12 mRNA lipid nanoparticle solution is instilled into bladder cancer animals or humans via bladder instillation for anti-bladder cancer treatment. Optional dosages include, for example, approximately 100 μL of IL-12 mRNA lipid nanoparticle solution instilled into the bladder of bladder cancer mice, with an IL-12 mRNA dose of 1-100 μg, preferably 2-20 μg, instilled once every 1-15 days, preferably once every 7 days.

[0031] In another administration method, IL-12 mRNA lipid nanoparticle solution and GM-CSF mRNA lipid nanoparticle solution are instilled together into the bladder of bladder cancer animals or humans for anti-bladder cancer treatment. An optional dosage ratio of IL-12 mRNA lipid nanoparticles to GM-CSF mRNA lipid nanoparticles is 30:1–1:30, preferably 10:1–1:10. For example, in bladder cancer mice, the total volume of the IL-12 mRNA lipid nanoparticle solution and GM-CSF mRNA lipid nanoparticle solution instilled into the bladder is approximately 100 μL, the mRNA dose is 1–100 μg, preferably 2–20 μg; the dosage ratio of IL-12 mRNA to GM-CSF mRNA is 30:1–1:30, preferably 10:1–1:10, and instilled once every 1–15 days, preferably once every 7 days.

[0032] In another administration method, IL-12 mRNA nanoparticle solutions and GM-CSF mRNA nanoparticle solutions are alternately instilled into the bladder of animals or humans with bladder cancer for anti-bladder cancer treatment. Optional dosages include a ratio of IL-12 mRNA lipid nanoparticles to GM-CSF mRNA lipid nanoparticles of 30:1–1:30, preferably 10:1–1:10. For example, in bladder cancer mice, the total volume of the nanoparticle solution instilled into the bladder is approximately 100 μL, the mRNA dose is 1–100 μg, preferably 2–20 μg, and the IL-12 mRNA to GM-CSF mRNA dosage ratio is 30:1–1:30, preferably 10:1–1:10. Instillation is performed every 1–15 days, preferably every 7 days. The first instillation uses one type of mRNA nanoparticle solution, the second uses another type, and so on, alternating between the two instillations for anti-bladder cancer treatment.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention discloses an IL-12 mRNA lipid nanoparticle, its preparation method, and its application. The IL-12 mRNA lipid nanoparticle uses an amino acid tocopheryl oxyalkylene ester as an ionizable lipid molecular carrier for delivering IL-12 mRNA, effectively delivering IL-12 mRNA into tumor cells, avoiding the risk of mRNA degradation by enzymes, and allowing for uptake and transfection by tumor cells. In vivo imaging showed that it exerted an anti-tumor effect in the mouse bladder cancer treatment group. Compared with the blank solution group, cancer cells did not grow and gradually underwent apoptosis. No cancer cells were observed after 21 days of treatment. Furthermore, it exhibited good biocompatibility, with no obvious adverse symptoms observed in mice; the mice showed increased appetite, were active, and did not experience weight loss. Combining GM-CSF mRNA lipid nanoparticles with IL-12 mRNA treatment in mice with bladder cancer showed even better anti-tumor effects. Attached Figure Description

[0035] Figure 1 shows the histidine tocopherol oxyethyl ester (HTE) of the present invention. 1 H NMR spectrum;

[0036] Figure 2 shows the MS spectrum of histidine tocopherol oxyethyl ester (HTE) of the present invention;

[0037] Figure 3 is a particle size distribution curve of the IL-12 mRNA lipid nanoparticles of Example 1 of the present invention;

[0038] Figure 4 is a particle size distribution curve of GM-CSF mRNA lipid nanoparticles in Example 2 of the present invention;

[0039] Figure 5 is a schematic diagram of the lyophilized powder injection of IL-12 mRNA lipid nanoparticles in Example 3 of the present invention, wherein (a) is the lyophilized powder state and (b) is the water-soluble state.

[0040] Figure 6 is a schematic diagram of the lyophilized powder injection of GM-CSF mRNA lipid nanoparticles in Example 3 of the present invention, wherein (a) is the lyophilized powder state and (b) is the water-soluble state.

[0041] Figure 7 is a particle size distribution curve of the IL-12 mRNA lipid nanoparticles of Example 4 of the present invention;

[0042] Figure 8 is an in vivo imaging image of mouse bladder cancer treated by bladder instillation of IL-12 mRNA lipid nanoparticles in Example 4 of the present invention, wherein (a) is the blank solution group and (b) is the lipid nanoparticle solution group.

[0043] Figure 9 shows the particle size distribution curve of the GM-CSF mRNA lipid nanoparticles in Example 5 of the present invention.

[0044] Figure 10 is an in vivo imaging image of mouse bladder cancer treated by combined bladder instillation of IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles in Example 5 of the present invention, wherein (a) is the blank solution group and (b) is the lipid nanoparticle solution group. Detailed Implementation

[0045] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0046] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0047] The ionizable lipid molecule of the present invention is prepared by the following steps:

[0048] (i) Tocopherol is prepared from a haloalkanol via a substitution reaction, wherein the general formula for the chemical reaction between tocopherol and the haloalkanol is as follows:

[0049] X: Halogen atom, such as bromine, chlorine or iodine; n = 2 to 10;

[0050] (ii) Tocopheryl oxyalkanols are prepared by esterification with base-protected amino acids (such as lysine, histidine, or arginine). The general chemical reaction formula is as follows:

[0051] RCOOH: Base-protected amino acids (such as lysine, histidine, or arginine); n = 2–10;

[0052] (iii) Preparation of amino acid tocopherol oxyalkyl esters by deprotection reaction, the general chemical reaction formula is as follows:

[0053] n = 2 to 10;

[0054] B is a residue of an amino acid or an amino acid derivative, preferably at least one of the following three:

[0055] In addition to the three amino acids mentioned above, B can also be a residue of other amino acids or derivatives of the aforementioned amino acids.

[0056] The following uses histidine tocopherol oxyethyl ester as an example to introduce the preparation method of ionizable lipid molecules. Other ionizable lipid molecules can be prepared by referring to this method.

[0057] (1) Preparation of tocopheryloxyethanol (TEOH)

[0058] 10.00 g of D-α-tocopherol and 1.39 g of sodium hydroxide were added to a flask containing 30 mL of DMF (N,N-dimethylformamide). Under magnetic stirring in a 90 °C oil bath, 10 mL of DMF solution containing 4.03 g of 2-bromoethanol was slowly added. After reacting for 90 h, the reaction solution was added to 200 mL of water and extracted three times with 50 mL of methyl tert-butyl ether. The organic phases were combined, and methyl tert-butyl ether was removed by rotary evaporation. A small amount of dichloromethane was added to dissolve the crude product, yielding a dichloromethane solution for column chromatography.

[0059] The dichloromethane solution containing the crude product was purified by separation using a silica gel column (300g 200-300 mesh silica gel, 70mm diameter). The column was eluted sequentially with a mixed solvent of petroleum ether / ethyl acetate at a volume ratio of 8:1 and 5:1, and the eluents were collected. The eluents containing only TEOH were collected and combined, and the solvent was removed by rotary evaporation to obtain a deep yellow viscous liquid, TEOH, with a yield of 75.0%.

[0060] (2) Preparation of histidine tocopheryl oxyethyl ester (HTE)

[0061] 2.13 g TEOH, 1.92 g N,N'-di-tert-butoxycarbonyl-L-histidine, 1.29 g 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 0.22 g 4-dimethylaminopyridine (DMAP) were added to a 50 mL dry flask containing 10.5 mL anhydrous dichloromethane. The flask was sealed and protected from light, and the reaction was carried out at room temperature with magnetic stirring. After 70 h of reaction, the solution was rotary evaporated to approximately 3 mL. The 3 mL dichloromethane solution containing the crude product was purified by silica gel column chromatography (90 g 200-300 mesh silica gel, 40 mm diameter), eluted with a 40:1 dichloromethane / methanol mixture, and the eluent was collected. The eluent containing only the product (N,N'-di-tert-butoxycarbonyl-L-histidine tocopherol oxyethyl ester, BHTE) was collected and combined. The solvent was removed by rotary evaporation at room temperature, and the residue was dried under vacuum at room temperature to constant weight to obtain a pale yellow semi-solid, which is BHTE, with a yield of 86.5%.

[0062] 3.15 g of BHTE was added to a flask containing 31.5 mL of dry dichloromethane. 10.5 mL of trifluoroacetic acid was slowly added dropwise. The flask was sealed and protected from light. The mixture was magnetically stirred and reacted at room temperature for 3 h. The reaction mixture was then removed by rotary evaporation at 40 °C to remove the solvent and trifluoroacetic acid, yielding the crude product. The crude product was dissolved in 5 mL of dichloromethane to prepare a dichloromethane solution for column chromatography purification. The dichloromethane solution containing the crude product was purified using a silica gel column (90 g 200-300 mesh silica gel, 40 mm diameter). The column was eluted sequentially with dichloromethane / methanol mixed solvents at volume ratios of 40:1, 20:2, and 20:3, and the eluates were collected. The eluates containing only HTE trifluoroacetate were collected and combined. The solvent was removed by rotary evaporation at room temperature. The residue was dried under vacuum at room temperature to constant weight, yielding a pale yellow semi-solid, HTE trifluoroacetate, with a yield of 87.8%.

[0063] 1.20 g of HTE trifluoroacetate was added to 12 mL of dichloromethane and extracted with 6 mL of saturated sodium bicarbonate solution. The organic phase was dried with anhydrous sodium sulfate, centrifuged, filtered, and the organic solvent was removed by rotary evaporation. The filtrate was then dried under vacuum to obtain a pale yellow semi-solid, namely HTE, with a yield of 82.9%. Its structural formula is shown in formula (1).1 The H NMR spectrum is shown in Figure 1, and the MS spectrum is shown in Figure 2.

[0064] 1 HNMR (400MHz, DMSO-d6): 0.83 (CH3 at 12H, 20, 25, 29, 30), 1.22 (CH3 at 3H, 31), 1.83-2.14 (CH3 at 9H, 7, 9, 10), 2.83-3.06 (CH2 at 2H, 37), 3.80 (CH2 at 2H, 32), 4.36 (CH2 at 2H, 33), 6.89 (CH at 1H, 44), 7.66 (CH at 1H, 42).

[0065] MS(ESI+):HTE(C37 H63 N3 O4)[M+H] + The theoretical m / z value is 612.4740, and the measured value is 612.4747. The errors are both within 5 ppm, and it is the main component.

[0066] Example 1: Preparation of IL-12 mRNA lipid nanoparticles

[0067] Prepare 30 mL of acetate buffer (pH = 3.6) containing 1000 μg IL-12 mRNA and 10 mL of ethanol solution containing 15 mg histidine tocopheryl oxypropyl ester and corresponding amounts of DSPC, cholesterol, and DMG2000 (histidine tocopheryl oxypropyl ester:DSPC:cholesterol:DMG2000 = 66.6:10:39.2:1.54 (molar ratio)). Use a microfluidic mixer (ring chip) to mix the 10 mL ethanol solution with the 30 mL IL-12 mRNA acetate buffer at a total flow rate of 28 mL / min and a flow rate ratio of acetate buffer to ethanol of 21:7. Prepare and collect the prepared nanoparticle solution. Dilute the prepared nanoparticle solution with 280 mL of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (pH = 7.4). Concentrate the nanoparticle solution by tangential flow ultrafiltration to prepare 60 mL of IL-12 mRNA nanoparticle solution. The particle size was determined using a dynamic laser light scattering (DLS) instrument, and the measured particle size was 160.7 nm with a polydispersity index (PDI) of 0.130. The particle size distribution curve is shown in Figure 3. Quant-iT... TM An RNA kit was used to quantitatively analyze mRNA using an ELISA reader, and the encapsulation efficiency was determined. The encapsulation efficiency of IL-12 mRNA was found to be 97%.

[0068] Example 2: Preparation of GM-CSF mRNA lipid nanoparticles

[0069] Prepare 18 mL of acetate buffer (pH = 3.6) containing 300 μg GM-CSF mRNA and 6 mL of ethanol solution containing 3 mg histidine tocopheryloxyethyl ester and corresponding amounts of DSPC, cholesterol, and DMG2000 (histidine tocopheryloxyethyl ester:DSPC:cholesterol:DMG2000 = 50:10:38.5:1.5 (molar ratio)). Use a microfluidic mixer (ring chip) to mix the 6 mL ethanol solution with the 18 mL GM-CSF mRNA acetate buffer at a total flow rate of 28 mL / min, with an acetate buffer to ethanol flow rate ratio of 21:7. Prepare and collect the prepared nanoparticle solution. Dilute the prepared nanoparticle solution with 100 mL of HEPES (pH = 7.4). The nanosolution was transferred to an ultrafiltration tube (molecular weight cutoff of 100 kJ), and subjected to low-temperature centrifugation at 4000 rpm for approximately 1 min to prepare a 7 mL concentrated solution of GM-CSF mRNA nanoparticles. The particle size was determined using a dynamic laser light scattering (DLS) instrument, yielding a nanoparticle size of 232.7 nm and a polydispersity index (PDI) of 0.093. The particle size distribution curve is shown in Figure 4. Quant-iT... TM The RNA kit was used to quantitatively analyze mRNA using an ELISA reader, and the encapsulation efficiency was measured. The encapsulation efficiency of GM-CSF mRNA was found to be 98%.

[0070] Example 3: Lyophilized powder injections of IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles were prepared respectively.

[0071] 5 w / v% lactose and 5 w / v% sucrose were added as lyophilization protectants to the IL 12 mRNA lipid nanoparticles or GM-CSF mRNA lipid nanoparticle solutions prepared in Examples 1 and 2, respectively, and then the solutions were placed in 7 ml vials for freeze-drying. The freeze-drying process was as follows: rapid freezing at -45℃ for 3 h, followed by sublimation at -35℃. A pressure rise test was performed to observe the sublimation inside the sample. When the pressure was <1 Pa / min, sublimation was stopped after 1-2 h. The sample was then heated to 20℃ for desorption, and desorption was stopped when the pressure rise was <1 Pa / min. The prepared freeze-dried powder injections were all intact, with full bottoms and no shrinkage. Figure 5(a) shows the freeze-dried powder injection of IL 12 mRNA lipid nanoparticles, and Figure 6(a) shows the freeze-dried powder injection of GM-CSF mRNA lipid nanoparticles. After redissolving in water, a clear nanoparticle solution was obtained. Figure 5(b) shows the aqueous solution of IL 12 mRNA lipid nanoparticles, and Figure 6(b) shows the aqueous solution of GM-CSF mRNA lipid nanoparticles.

[0072] Example 4: Bladder instillation of IL-12 mRNA lipid nanoparticles for the treatment of bladder cancer in mice

[0073] (I) Constructing a mouse orthotopic model of bladder cancer

[0074] The Balb / c mouse orthotopic bladder tumor model was constructed by introducing Luc-MB49 cells (luciferase-labeled mouse bladder cancer cells) into a bladder pretreated with polylysine (PLL). The specific steps are as follows:

[0075] ① Each mouse was anesthetized by intraperitoneal injection of approximately 130 μL of 1% (w / v) sodium pentobarbital anesthetic;

[0076] ② Clean the mouse's urethral opening with sterile cotton pads;

[0077] ③ Using sterile forceps, lift the upper side of the urethral opening and insert the pre-lubricated paraffin oil-lubricated catheter into the urethral opening at approximately 60°-90° to the mouse's tail. Then gently rotate the catheter to allow it to slowly slide into the urethra. After sliding into the urethra, gently push the catheter horizontally about 1 cm or to the base of the needle to reach the bladder.

[0078] ④ After flushing the bladder with 100 μL of phosphate buffer, inject 100 μL of 0.1 mg / mL PLL into the bladder through the catheter and retain for 20 min.

[0079] ⑤ Empty the mouse bladder and reinsert the catheter, then infuse 50 μL of tumor cell suspension (containing 1*10 Luc-MB49 cells) into the bladder of the PLL-pretreated mouse. 6 indivual).

[0080] ⑥ After bladder instillation, the mice were placed supine on a constant temperature electric heating pad (37℃) until they naturally woke up and urinated (this should take 2 hours or more).

[0081] (II) Bladder instillation of IL-12 mRNA lipid nanoparticle solution into bladder cancer mice

[0082] Except for the following differences, the preparation method of the IL-12 mRNA lipid nanoparticle solution used in Example 4 is the same as that in Example 1, except that the amount of IL-12 mRNA is 100 μg, and the prepared nanoparticle solution is diluted with 30 mL of HEPES. The diluted nanoparticle solution is transferred to an ultrafiltration tube (molecular weight cutoff of 100 kJ), and ultrafiltered by low-temperature centrifugation at 4000 rpm to prepare a concentrated nanoparticle solution. The particle size of the IL-12 mRNA lipid nanoparticles was determined to be 136.0 nm using dynamic laser light scattering, with a particle size distribution index of 0.104. The particle size distribution curve is shown in Figure 7, and the zeta potential is 0.93 mV. Quant-iT TM RNA kit, microplate reader for quantitative analysis of mRNA, mRNA encapsulation rate was measured to be 97.0%.

[0083] On day 3 post-modeling, IL-12 mRNA nanoparticle solution (5 μg IL-12 mRNA, 100 μL) was instilled into the bladder, followed by a second and third instillation on days 7 and 14. A blank solution (100 μL) without IL-12 mRNA nanoparticles was used as a control.

[0084] While the mouse is awake, first, lift its tail to induce a physiological reflex and urinate. For mice that have not urinated, gently and continuously squeeze the bladder area of ​​its lower abdomen on the cage rack to expel urine. If no urine is expelled, anuria should be considered. Then proceed with the following steps: Inject approximately 130 μL of 1% (w / v) sodium pentobarbital anesthetic into the mouse's peritoneum; clean the mouse's urethral opening with sterile cotton swabs; lift the upper side of the urethral opening with sterile forceps, and insert a pre-lubricated paraffin-coated catheter into the urethral opening at approximately 60°-90° to the mouse's tail. Then gently rotate the catheter to allow it to slowly slide into the urethra. After sliding into the urethra, gently push the catheter horizontally about 1 cm or to the base of the needle until it reaches the bladder. Infuse the bladder with physiological saline through the catheter, and slowly flush the bladder three times, about 100 μL each time. After flushing, slowly inject IL-12 mRNA nanoparticle solution, a total of 100 μL. Slowly remove the catheter, put the mouse back in the cage, and keep it lying flat.

[0085] Following bladder instillation of IL-12 mRNA lipid nanoparticle solution, fluorescence expression in the bladder and tumor tissues was detected and photographed at regular intervals using an in vivo imaging system (IVIS spectrum, PerkinElmer). On days 7, 14, and 21 after bladder instillation, the fluorescence intensity of mouse luciferase-labeled bladder cancer was measured using the in vivo imaging system and IVIS spectrum software. Increased fluorescence intensity indicated bladder cancer growth, while decreased fluorescence intensity indicated inhibited bladder cancer growth. Mouse photographs taken using the in vivo imaging system are shown in Figure 8. The average fluorescence intensity (unit: p / sec / cm² / sr) at the mouse bladder was quantitatively analyzed using IVIS spectrum software, and the results are shown in Table 1.

[0086] Table 1 Note: The fluorescence intensity in the bladder of a healthy mouse was 3.982*10. 4 p / sec / cm 2 / sr.

[0087] As shown in the table above, the fluorescence intensity in the bladder region of mice in the blank solution perfusion group was 10. 7 -10 9 p / sec / cm 2 / sr, significantly higher than the fluorescence intensity of 3.982*10 in the bladder of healthy mice. 4 p / sec / cm 2 / sr, this is due to luciferase labeling of mouse bladder tumors, and the fluorescence intensity increases over time, indicating that the tumor is growing; the fluorescence intensity in the bladder of mice in the IL-12 mRNA nanoparticle instillation group was 10. 4 -10 5 p / sec / cm 2 / sr indicates that perfusion of IL-12 mRNA lipid nanoparticle solution has a good anti-tumor effect.

[0088] Mice in the bladder instillation group of IL-12 mRNA lipid nanoparticle solution ate more, were more active, and did not lose weight; mice in the bladder instillation group of blank solution gradually reduced their food intake, became less active, and lost weight, indicating that instillation of IL-12 mRNA lipid nanoparticle solution did not cause obvious adverse symptoms in mice and had good biocompatibility. Due to the inhibition of tumor growth, the mice were in good condition; bladder instillation of blank solution could not inhibit tumor growth, and as the tumor continued to grow, the mice's condition deteriorated.

[0089] Example 5: Combined instillation of IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles for the treatment of bladder cancer in mice.

[0090] (I) The construction of the mouse bladder cancer orthotopic model and the bladder instillation procedure are the same as in Example 4. The difference from Example 4 is that the bladder instillation is performed using a solution of 100 μL of IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles (containing 2.5 μg IL-12 mRNA and 2.5 μg GM-CSF mRNA).

[0091] (II) Bladder instillation of IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles into bladder cancer mice:

[0092] The IL-12 mRNA lipid nanoparticle solution used in Example 5 is the same as that in Example 4;

[0093] Except for the following differences, the preparation method of the GM-CSF mRNA lipid nanoparticle solution used in Example 5 is the same as that in Example 2, except that the amount of GM-CSF mRNA used is 100 μg. The particle size of the GM-CSF mRNA lipid nanoparticles was determined to be 142.0 nm using dynamic laser light scattering, with a particle size distribution index of 0.126. The particle size distribution curve is shown in Figure 9. The zeta potential was 1.22 mV; the Quant-iT... TM An RNA kit was used to quantitatively analyze mRNA using an ELISA reader, and the mRNA encapsulation rate was determined to be 99.3%.

[0094] On day 3 post-modeling, a solution of IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles (containing 2.5 μg IL-12 mRNA and 2.5 μg GM-CSF mRNA, 100 μL) was instilled into the bladder. A second and third instillation of the same lipid nanoparticle solution was performed on days 7 and 14. A blank solution (100 μL) without lipid nanoparticles was used as a control.

[0095] Following bladder instillation, fluorescence expression in the bladder and tumor tissues was detected and photographed at regular intervals using an in vivo imaging system (IVIS spectrum, PerkinElmer). On days 7, 14, and 21 after bladder instillation, the fluorescence intensity of mouse luciferase-labeled bladder cancer was measured using the in vivo imaging system and IVIS spectrum software. Increased fluorescence intensity indicated bladder cancer enlargement, while decreased fluorescence intensity indicated inhibited bladder cancer growth. Mouse photographs taken using the in vivo imaging system are shown in Figure 10. The average fluorescence intensity (unit: p / sec / cm² / sr) at the mouse bladder was quantitatively analyzed using IVIS spectrum software, and the results are shown in Table 2.

[0096] Table 2 Note: The fluorescence intensity in the bladder of a healthy mouse was 3.982*10. 4 p / sec / cm 2 / sr.

[0097] As shown in the table above, the fluorescence intensity in the bladder region of mice in the blank solution group was 10. 7 -10 9 p / sec / cm 2 / sr, significantly higher than the fluorescence intensity of 3.982*10 in the bladder of healthy mice. 4 p / sec / cm 2 The fluorescence intensity increased over time, indicating tumor growth; the fluorescence intensity in the mouse bladder after perfusion with IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles was 10. 4 -10 5 p / sec / cm 2 / sr indicates that the combined infusion of IL-12 mRNA nanoparticles and GM-CSF mRNA nanoparticles has a good anti-tumor effect.

[0098] Mice in the bladder instillation groups of IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles showed increased appetite, increased activity, and no weight loss. Mice in the bladder instillation group of blank solution showed a gradual decrease in appetite, decreased activity, and weight loss, indicating that instillation of IL-12 mRNA lipid nanoparticles and GM-CSF mRNA lipid nanoparticles did not cause significant discomfort in mice and had good biocompatibility. The mice were in good condition due to the inhibition of tumor growth. Bladder instillation of blank solution did not inhibit tumor growth, and the mice's condition deteriorated as the tumor continued to grow.

[0099] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. An IL 12 mRNA lipid nanoparticle, characterized in that, This includes IL 12 mRNA and a vector for delivering IL 12 mRNA, said vector comprising one or more ionizable lipid molecules or pharmaceutically acceptable salts thereof, having the general formula shown in (I): Where B is R is n is 2-10; T is 2. The IL 12 mRNA lipid nanoparticles according to claim 1, characterized in that, The ionizable lipid molecules are histidine tocopheryl oxyethyl ester, lysine tocopheryl oxyethyl ester, arginine tocopheryl oxyethyl ester, histidine tocopheryl oxypropyl ester, lysine tocopheryl oxypropyl ester, or arginine tocopheryl oxypropyl ester.

3. The IL 12 mRNA lipid nanoparticles according to claim 1 or 2, characterized in that, The carrier also includes assist lipid molecules, cholesterol, and PEGylated lipid molecules.

4. The IL 12 mRNA lipid nanoparticles according to claim 3, characterized in that, The auxiliary lipid molecule is distearylphosphatidylcholine or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine; the PEGylated lipid molecule is distearylphosphatidylethanolamine-polyethylene glycol, dimyristoylglycerol-polyethylene glycol 2000, polyethylene glycol 1000 vitamin E succinate, or polyethylene glycol 2000 vitamin E succinate.

5. The IL 12 mRNA lipid nanoparticles according to claim 3, characterized in that, The molar ratio of the ionizable lipid molecules, auxiliary lipid molecules, cholesterol and PEGylated lipid molecules is 50:(5-20):(10-50):(1-5).

6. A method for preparing IL-12 mRNA lipid nanoparticles according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Prepare acetate buffer (pH=3-4) for IL-12 mRNA and ethanol solution containing ionizable lipid molecules, auxiliary lipid molecules, cholesterol and PEGylated lipid molecules respectively. (2) The above-prepared ethanol solution was mixed with IL-12 mRNA acetate buffer using a microfluidic mixer. The flow rate ratio was controlled to prepare a nanoparticle solution. The nanoparticle solution was diluted with 4-hydroxyethylpiperazine ethanesulfonic acid and concentrated by tangential flow ultrafiltration to obtain an IL-12 mRNA nanoparticle solution.

7. A pharmaceutical composition, characterized in that, Includes the IL 12 mRNA lipid nanoparticles according to any one of claims 1-5.

8. The pharmaceutical composition according to claim 7, characterized in that, It also includes GM-CSF mRNA or GM-CSF mRNA lipid nanoparticles.

9. The pharmaceutical composition according to claim 8, characterized in that, The GM-CSF mRNA lipid nanoparticles comprise GM-CSF mRNA and a carrier for delivering GM-CSF mRNA, wherein the carrier comprises one or more ionizable lipid molecules of claim 1 or pharmaceutically acceptable salts thereof.

10. A pharmaceutical preparation, characterized in that, The lyophilized powder injection of IL 12 mRNA lipid nanoparticles according to any one of claims 1-5, or the lyophilized powder injection of the pharmaceutical composition according to any one of claims 7-9.

11. The use of the IL 12 mRNA lipid nanoparticles according to any one of claims 1-5, the pharmaceutical composition according to any one of claims 7-9, or the pharmaceutical formulation according to claim 10 in the preparation of a drug for treating bladder cancer.

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