MRNA (messenger ribonucleic acid) lipid nanoparticles and application thereof in treatment of triple-negative breast cancer

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to mRNA (messenger ribonucleic acid) lipid nanoparticles and application thereof in treatment of triple-negative breast cancer. The invention constructs an mRNA lipid nanoparticle, breaks through the dilemma of single drug curative effect of a glucose transporter (GLUT) inhibitor and a glutaminase (GLS) inhibitor for triple negative breast cancer, and realizes double-track treatment of mRNA metabolism reprogramming and small molecule energy deprivation for the first time. The mRNA lipid nanoparticles enhance the sensitivity of triple negative breast cancer to small molecule energy deprivation treatment, so that the clinical transformation feasibility of BAY-876 and CB-839 is improved to a new dimension. The delivery efficiency of the mRNA lipid nanoparticles is high, the biological safety is good, meanwhile, the production process of the nanoparticles is simpler and more standard compared with plasmids and viruses, and the drug production cost is lower.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and in particular to an mRNA lipid nanoparticle and its application in the treatment of triple-negative breast cancer. Background Technology

[0002] Breast cancer (BC) is prevalent worldwide. Early-stage patients have a five-year survival rate exceeding 98%, but 30% of patients are diagnosed at an advanced stage (with a five-year survival rate of only 20-27%). Current treatment relies on surgery combined with chemotherapy / targeted therapy. The triple-negative subtype has a survival rate as low as 65-70% due to high chemotherapy toxicity and strong immune resistance (response rate <50%).

[0003] Triple-negative breast cancer (TNBC) lacks estrogen receptors, progesterone receptors, and HER2 receptors, making traditional endocrine and targeted therapies less effective. Currently, first-line treatment options for TNBC are limited to chemotherapy and immunotherapy. Traditional chemotherapy drugs (such as paclitaxel) suffer from poor tumor selectivity due to their systemic distribution and high toxicity rates, including myelosuppression, resulting in a long-term five-year survival rate of less than 30% for TNBC patients. When using PD-1 inhibitors to treat TNBC, over 50% of patients develop resistance, and subsequent line-of-care treatment has an efficacy rate of only about 10%. Encouragingly, research has revealed a unique "reprogramming" phenomenon in the metabolic processes of TNBC cells, offering a new breakthrough for treatment. Currently, in cutting-edge research on TNBC treatment, inhibiting key glycolytic targets such as glucose transporters (GLUTs) or targeting glutamine metabolizing enzymes (GLS) holds promise as a metabolic therapy option for TNBC.

[0004] The Warburg effect refers to the tendency of cancer cells (including breast cancer cells) to preferentially break down glucose for energy via glycolysis, even in oxygen-rich conditions, rather than using the more efficient oxidative phosphorylation. Glycolytic intermediates (such as glucose-6-phosphate and fructose-6-phosphate) are the raw materials for synthesizing cellular "building blocks" such as nucleic acids, amino acids, and lipids. This is more important than simply producing energy, ensuring the continuous replication and division of cancer cells. Researchers have developed many drugs targeting various aspects of the Warburg effect. BAY-876, a glucose transporter (GLUT) inhibitor developed by Bayer AG, is currently the most active and selective GLUT1 inhibitor, effectively inhibiting glucose uptake by cancer cells. Glutamine is the most abundant amino acid in the human body and an important carbon and nitrogen source for cellular biosynthesis, participating in various processes such as energy metabolism, amino acid synthesis, nucleotide synthesis, and redox balance regulation. Glutamine can enter the tricarboxylic acid cycle (TCA cycle) as α-ketoglutarate through various reactions. The TCA cycle is the hub of intracellular energy and metabolism. Although tumor cells, due to the Warburg effect, tend to convert glucose into lactate via anaerobic glycolysis, thus obtaining intermediate metabolites necessary for their rapid growth, glycolysis is a relatively inefficient energy-producing mechanism. Tumor cells still require a large amount of energy to maintain their malignant phenotype. The tricarboxylic acid (TCA) cycle is not only one of the most efficient energy-producing reactions within cells but also provides the necessary molecules for biosynthesis. In recent years, increasing evidence suggests that tumorigenesis and development are highly dependent on TCA cycle reprogramming for energy production and macromolecule synthesis. Telaglenastat (CB-839), a GLS1 inhibitor, was initially developed by Calithera Biosciences, Inc. and is currently in Phase II clinical trials. Its mechanism of action is to inhibit glutaminase (GLS1) activity, hindering the TCA cycle and depriving tumor cells of sufficient energy to maintain their malignant phenotype. In combination with the GLUT1 inhibitor BAY-876, mRNA lipid nanoparticles induce disulfide death in tumor cells by inducing the uptake of large amounts of cysteine. This combination regimen increased the efficacy of BAY-876 but decreased its effective concentration. When mRNA lipid nanoparticles were used in combination with the GLS1 inhibitor CB-839, they induced tumor cells to eliminate large amounts of glutamate, hindering the tricarboxylic acid cycle and leading to impaired energy supply in tumor cells. This combination regimen increased the efficacy of CB-839 but decreased its effective concentration.

[0005] However, monotherapy trials using the GLUT inhibitor BAY-876 or the GLS inhibitor CB-839 face significant challenges. The difficulties with BAY-876 monotherapy include: ① Not all breast cancer patients are suitable for a monotherapy strategy with the GLUT1 inhibitor BAY-876. Based on published studies, BAY-876 only has a therapeutic effect on a subset of RB1-positive triple-negative breast cancer cells, limiting its indications; ② GLUT1 is highly expressed in key tissues such as erythrocytes, the blood-brain barrier, and the kidneys, and high doses of GLUT1 inhibitors may lead to serious adverse reactions; ③ In in vitro experiments, BAY-876 monotherapy showed a high half-maximal inhibitory concentration (IC50) (20 μM) against breast cancer cells, far exceeding clinically achievable doses; ④ By inhibiting glucose transporters in SLC7A11high cells (e.g., using the GLUT1 inhibitor BAY-876), disulfide death can be induced, thereby achieving an anti-tumor effect; however, not all breast cancer patients have high SLC7A11 expression. Challenges faced by CB-839 monotherapy: CB-839 did not achieve clinical benefit in a phase 2 clinical trial for advanced triple-negative breast cancer, which led to the termination of clinical research on the indication for triple-negative breast cancer (see NCT03057600 for details).

[0006] Activating Transcription Factor 4 (ATF4) regulates numerous metabolism-related genes, its function can be summarized as "resource generation and resource conservation," helping cells survive in nutrient-deprived environments. However, the metabolic "reprogramming" effect that tumor cells rely on becomes their Achilles' heel. Solute Carrier Family 7 Member 11 (SLC7A11; also known as xCT) is one of the main downstream targets of ATF4. SLC7A11 mediates cystine uptake and glutamate efflux. Cystine uptake leads to increased glycolysis dependence in tumor cells, while glutamate efflux leads to increased glutamine metabolism dependence. On the one hand, inhibiting glucose transporters in SLC7A11 high cells (e.g., using the GLUT1 inhibitor BAY-876) leads to a deficiency of raw materials for the pentose phosphate pathway in tumor cells, reducing NADPH production. Combined with the large uptake of cysteine, intracellular NADPH is rapidly depleted, resulting in insufficient NADPH to clear disulfide bonds and inducing disulfide death, thus achieving an anti-tumor effect. On the other hand, inhibiting glutamine metabolism in SLC7A11 high cells (e.g., using the GLS inhibitor CB-839) reduces glutamate production in tumor cells. Combined with the large excretion of glutamate, intracellular glutamate levels drop significantly, inhibiting the tricarboxylic acid cycle and drastically reducing ATP production, thus achieving an anti-tumor effect. However, not all breast cancer patients have high SLC7A11 expression. Therefore, overexpressing SLC7A11 can increase the sensitivity of tumors to BAY-876 and CB-839.

[0007] For overexpression of the SLC7A11 target, the commonly used techniques are plasmid transfection or viral infection, which face significant challenges in drug development: ① Plasmid drug systems: Plasmid drug development requires strict impurity control, resulting in low delivery efficiency, poor in vivo stability, and a low success rate. Naked plasmid DNA is difficult to cross cell membranes, leading to very low cell entry efficiency. Once inside the body, plasmid DNA is rapidly degraded by DNases in the blood, resulting in a short half-life. Even if it enters the cytoplasm, plasmid DNA still needs to enter the nucleus to exert its effects, a process that is also inefficient. Bacterial plasmid DNA may contain immunostimulatory sequences such as CpG motifs, potentially triggering unwanted immune responses. ② Viral drug systems: Viral drug development requires high viral titers and activity, and is difficult to control immunogenicity. High purity is required, and viral products need to remove a large number of process-related impurities (such as host cell DNA / protein, serum, endotoxins, etc.) and product-related impurities (such as empty virus particles, incompletely packaged viruses, etc.). Empty virus particles have no therapeutic effect but may trigger immune responses, but their isolation and removal are extremely difficult. While the ability of retroviruses and lentiviruses to integrate into the host genome facilitates long-term expression, it can also lead to insertional mutations, posing a potential risk of activating proto-oncogenes or inactivating tumor suppressor genes. Their safety faces significant challenges.

[0008] Therefore, the potential of monotherapy is limited, and overexpression technologies (such as plasmid transfection, viral infection, etc.) have problems such as high drug cost, high difficulty, high drug risk, and difficulty in controlling adverse events. Developing new effective treatments for breast cancer is urgent. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the existing technology and provide an mRNA lipid nanoparticle that, when used in combination with BAY-876 and CB839, can improve the therapeutic effect of triple-negative breast cancer.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an mRNA lipid nanoparticle, the mRNA lipid nanoparticle comprising lipid nanoparticles LNP and ATF4 mRNA; the lipid nanoparticles LNP encapsulate ATF4 mRNA; the nucleotide sequence of the ATF4 mRNA is shown in SEQ ID NO. 1.

[0011] Patients with triple-negative breast cancer (TNBC) experience a sharp decline in survival after first-line chemotherapy resistance due to the lack of ER / PR / HER2 targets. While BAY-876 and CB839 have shown some progress, their potential as monotherapy is limited. Overexpression of ATF4 can further increase tumor sensitivity to BAY-876 and CB-839. The specific mechanisms are as follows: ① When BAY-876 inhibits cellular glucose uptake, overexpression of SLC7A11 leads to increased cysteine ​​uptake. The large-scale reduction of cysteine ​​results in the depletion of reduced nicotinamide adenine dinucleotide phosphate (NADPH), leading to the accumulation of intracellular disulfide molecules and ultimately cell death. This novel cell death mechanism is named disulfide death. ② When CB-839 inhibits glutaminase, glutamate production decreases. Overexpression of SLC7A11 leads to the excretion of glutamate from the cell, further resulting in intracellular glutamate deficiency, exacerbating the obstruction of the tricarboxylic acid cycle, leading to a significant reduction in ATP production and cell death.

[0012] Due to the high cost, difficulty, high risk, and uncontrollable adverse events associated with current overexpression technologies (such as plasmid transfection and viral infection), this invention selects the LNP lipid nanoparticle nucleic acid delivery platform to achieve overexpression by delivering mRNA. This delivery platform has the following characteristics: it is a non-viral vector, avoiding the risk of insertional mutations; the process is relatively standardized, allowing for large-scale production and cost control. However, its delivery efficiency is affected by the size of the encapsulated mRNA; smaller mRNA molecules have higher delivery efficiency. Therefore, this invention further optimized the encapsulation system and screened targets through extensive research, ultimately selecting ATF4 as the target to construct ATF4 mRNA lipid nanomedicines (NPs). ATF4 (Activated Transcription Factor 4) can promote the transcription of SLC7A11, leading to metabolic reprogramming in breast cancer cells, promoting cystine uptake and glutamate efflux. The mRNA size of SLC7A11 is 9645 nt, which is relatively long, making it difficult to guarantee the delivery efficiency of LNPs. The length of ATF4 mRNA is 1419 nt, significantly shorter than that of SLC7A11. This invention utilizes the shorter ATF4 mRNA to construct ATF4 mRNA lipid nanoparticles (NPs) (ATF4). When NPs (ATF4) are used, the expression level of SLC7A11 in tumor cells significantly increases. At this point, glutamate levels in tumor cells are low, while cysteine ​​levels are high. Based on this, using BAY-876 to inhibit glucose uptake leads to disulfide death in tumor cells, and using CB-839 to inhibit the conversion of glutamine to glutamate inhibits the tricarboxylic acid cycle, leading to energy supply disorders. This drug overcomes the efficacy limitations of single-agent GLUT1 inhibitors and GLS inhibitors in breast cancer, achieving for the first time a dual-track therapy of mRNA metabolic reprogramming and small molecule energy deprivation, elevating the clinical translation feasibility of BAY-876 and CB-839 to a new level. Furthermore, the preparation cost of these mRNA lipid nanoparticles is low, and they exhibit high efficacy and safety.

[0013] Preferably, the lipid nanoparticles (LNPs) include at least one of ionizable lipids, cofactor lipids, cholesterol, neutral cofactor phospholipids, and PEGylated lipids.

[0014] Preferably, the average particle size of the mRNA lipid nanoparticles is 106-142 nm.

[0015] Secondly, the present invention provides a method for preparing the above-mentioned mRNA lipid nanoparticles, including the step of encapsulating ATF4 mRNA with lipid nanoparticles (LNP).

[0016] Specifically, the encapsulation step is as follows: LNP is mixed with P6-Cit-100, cholesterol, DSPC, and DMG-PEG2000 in a molar ratio of 4.5:31:10:0.4 (mass ratio 16:12:8:1), and dialyzed overnight. LNP is mixed with ATF4 mRNA at a mass ratio of 10:1 on ice for 30 minutes and stored in the dark (refer to the specification of invention patent CN120501756A—a pharmaceutical composition and its application, paragraphs

[0306] -

[0308] ). The preparation method of P6-Cit-100 can be referred to patent document with application number CN202310278694.4.

[0017] Thirdly, the present invention provides the application of the above-mentioned mRNA lipid nanoparticles in combination with GLUT1 inhibitors and / or GLS inhibitors in the preparation of drugs or formulations for treating triple-negative breast cancer.

[0018] Patients with triple-negative breast cancer (TNBC) experience a sharp decline in survival after first-line chemotherapy resistance due to the lack of ER / PR / HER2 targets. Although GLUT1 overexpression is >80% in TNBC, the existing GLUT1 inhibitor BAY-876 has failed to enter clinical trials due to its limited indications and toxicity. The GLS inhibitor CB839 failed in its phase 2 clinical trial, temporarily halting the development of GLS inhibitor treatment strategies for triple-negative breast cancer. This invention develops a novel combination strategy of mRNA lipid nanoparticles with BAY-876 and / or CB839, overcoming these limitations. In combination with the GLUT1 inhibitor BAY-876, the mRNA lipid nanoparticles induce tumor cells to take up large amounts of cysteine, inducing disulfide death. This combination increases the efficacy of BAY-876 while reducing its effective concentration. In combination with the GLS1 inhibitor CB-839, the mRNA lipid nanoparticles induce tumor cells to eliminate large amounts of glutamate, hindering the tricarboxylic acid cycle and leading to impaired energy supply in tumor cells. This combination regimen increased the efficacy of CB-839 while reducing its effective concentration. The mRNA lipid nanoparticles exhibited high delivery efficiency and effectively promoted SLC7A11 expression; furthermore, they demonstrated high biocompatibility. Additionally, the production process of LNPs is simpler and more standardized compared to plasmids and viruses, resulting in lower drug costs.

[0019] Preferably, the GLUT1 inhibitor is BAY-876; the GLS inhibitor is CB-839.

[0020] Preferably, the dosage of the mRNA lipid nanoparticles is not less than 150ug / kg, the dosage of BAY-876 is 3mg / kg, and the dosage of CB-839 is 100mg / kg.

[0021] Preferably, the mRNA lipid nanoparticles are administered via local injection, and BAY-876 / CB-839 is administered orally (by gavage).

[0022] Fourthly, the present invention provides the use of the above-mentioned mRNA lipid nanoparticles in the preparation of formulations that enhance the sensitivity of triple-negative breast cancer patients to GLUT1 inhibitors and / or GLS inhibitors.

[0023] Preferably, the mRNA lipid nanoparticles enhance the sensitivity of tumor cells to GLUT1 inhibitors and / or GLS inhibitors by increasing the expression level of SLC7A11.

[0024] Preferably, the GLUT1 inhibitor is BAY-876; the GLS inhibitor is CB-839.

[0025] Preferably, the effective content of NPs (ATF4) is not less than 150ug / kg, the dosage of BAY-876 is 3mg / kg, and the dosage of CB-839 is 100mg / kg.

[0026] Fifthly, the present invention provides a pharmaceutical composition for treating triple-negative breast cancer, comprising the following components: the above-mentioned mRNA lipid nanoparticles, BAY-876, and CB-839.

[0027] The beneficial effects of this invention are as follows: This invention develops a novel strategy for combining mRNA lipid nanoparticles (NPs) (ATF4) with BAY-876 and / or CB839. This strategy addresses the limitation of BAY-876 monotherapy, which is only effective in certain breast cancer cell lines, thus expanding its applicability; it also reduces the effective dose, increasing the sensitivity of breast cancer treatment; it addresses the efficacy issues of CB-839 monotherapy in breast cancer indications, increasing its sensitivity to breast cancer treatment; and it addresses the problems of high cost and high risk associated with SLC7A11 overexpression drugs.

[0028] The mRNA lipid nanoparticles encode the transcription factor ATF4 and effectively promote SLC7A11 expression. On one hand, the combination of these mRNA lipid nanoparticles and the glucose transporter inhibitor BAY-876 promotes disulfide death in triple-negative breast cancer cells; on the other hand, the combination of these mRNA lipid nanoparticles and the glutaminase inhibitor CB-839 further promotes glutamine metabolism disorder in triple-negative breast cancer cells. As a novel therapeutic agent, these mRNA lipid nanoparticles overcome the efficacy limitations of single-agent GLUT1 inhibitors and GLS inhibitors in breast cancer, achieving for the first time a dual-track treatment of mRNA metabolic reprogramming and small molecule energy deprivation, elevating the clinical translation feasibility of BAY-876 and CB-839 to a new level. Furthermore, these mRNA lipid nanoparticles have low preparation costs and exhibit high efficacy and safety.

[0029] When mRNA lipid nanoparticles are used in combination with the GLUT1 inhibitor BAY-876, they induce disulfide death in tumor cells by causing them to take up large amounts of cystine. This combination regimen increases the efficacy of BAY-876 but reduces its effective concentration.

[0030] When mRNA lipid nanoparticles are used in combination with the GLS1 inhibitor CB-839, they induce tumor cells to eliminate large amounts of glutamate, thereby hindering the tricarboxylic acid cycle and causing energy supply disorders in tumor cells. This combination regimen increases the efficacy of CB-839 while reducing its effective concentration.

[0031] mRNA lipid nanoparticles have high delivery efficiency and effectively promote SLC7A11 expression; moreover, they exhibit high biocompatibility. Furthermore, the production process of LNPs is simpler and more standardized compared to plasmids and viruses, resulting in lower drug costs. Attached Figure Description

[0032] Figure 1 Figure A shows the identification results of nanoparticles; Figure B shows the results of microscopic observation; Figure C and D show the results of effective particle size determination of nanoparticles using a nanoparticle size analyzer and a Zeta potential analyzer; and Figure D shows the results of Zeta potential testing.

[0033] Figure 2Figure 1 shows the effectiveness analysis results of ATF4 mRNA nanoparticles; Figure A shows the IC50 detection results of NPs(ATF4); Figure B shows the ATF4 mRNA expression level detection results (RT-qPCR) in each group; Figure C shows the SLC7A11 mRNA expression level detection results (RT-qPCR) in each group; Figure D shows the ATF4 protein expression level detection results (WB); Figure E shows the cysteine ​​detection results in each group; Figure F shows the glutamate detection results in each group; Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001; t test.

[0034] Figure 3 Figure A shows the results of biosafety analysis; Figure B shows the results of HE staining and the results of weight changes in mice in each group; data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001; ttest.

[0035] Figure 4 This diagram illustrates the mechanism of action of BAY-876 in combination with NPs (ATF4). The main action of BAY-876 is to inhibit the glucose transporter GLUT1 / 3, thereby suppressing the glucose uptake capacity of tumor cells. The glucose content in tumor cells decreases significantly, leading to a deficiency of glucose-6-phosphate, a raw material for the pentose phosphate pathway, resulting in a significant decrease in NADPH production. The main action of NPs (ATF4) is to deliver ATF4 mRNA into tumor cells via nanoliposomes, thereby promoting the transcription of SLC7A11, which in turn leads to a large uptake of cystine by tumor cells. When the two drugs are used in combination, cystine increases significantly while NADPH decreases significantly, inducing disulfide death.

[0036] Figure 5 Figure 1 shows the results of BAY-876 combined with NPs (ATF4) promoting disulfide death in triple-negative breast cancer cells. Figure A shows the changes in cysteine ​​levels in each group; Figure B shows the changes in glucose levels in each group; Figure C shows the changes in NADPH levels in each group; Figure D shows the changes in cell viability; Figure E shows the results of plate colony assay; Figure F shows the results of rhodamine-phalloidin staining. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001; ttest.

[0037] Figure 6Figure 1 shows the results of inhibiting tumor growth in a triple-negative breast cancer model mouse model using NPs (ATF4) in combination with BAY-876. Figure A shows the changes in tumor size in each group of mice; Figure B shows the statistical changes in tumor growth in each group of mice; Figure C shows the statistical changes in tumor weight in each group of mice; Figure D shows the glucose content in the tumors of each group of mice; Figure E shows the cysteine ​​content in the tumors of each group of mice; Figure F shows the NADPH content in each group of mice; Figure G shows the Ki67 positivity rate in the tumor tissues of each group of mice. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001; t test.

[0038] Figure 7 This diagram illustrates the mechanism of action of CB-839 in combination with NPs (ATF4). The main function of CB-839 is to inhibit the function of glutaminase GLS, preventing the metabolism of glutamine into glutamate within tumor cells. The reduced glutamate content in tumor cells, coupled with the fact that glutamate is the primary raw material for the tricarboxylic acid cycle in tumor cells, leads to a disruption of the tricarboxylic acid cycle and a significant decrease in ATP production. NPs (ATF4) promote the transcription of SLC7A11, resulting in a large efflux of glutamate from tumor cells. The combined use of these two drugs significantly reduces glutamate production while simultaneously causing the cells to excrete large amounts of glutamate. Under this dual effect, ATP production in tumor cells is greatly inhibited, leading to cell death.

[0039] Figure 8 Figure 1 shows the results of CB-839 combined with NPs (ATF4) inhibiting the tricarboxylic acid cycle in triple-negative breast cancer cells, leading to metabolic disorders. Figure A shows the changes in cell viability in each group (CCK8 assay), Figure B shows the glutamate content in tumor cells in each group, and Figure C shows the ATP content in tumor cells in each group. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001; ttest.

[0040] Figure 9 Figure 1 shows the results of inhibiting tumor growth in a triple-negative breast cancer model mouse model using NPs (ATF4) in combination with CB-839. Figure A shows the changes in tumor size in each group of mice; Figure B shows the statistical changes in tumor growth in each group of mice; Figure C shows the statistical changes in tumor weight in each group of mice; Figure D shows the changes in body weight in each group of mice; Figure E shows the results of detecting glutamate content in tumors in each group of mice; Figure F shows the results of detecting ATP content in tumors in each group of mice. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001; ttest. Detailed Implementation

[0041] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0042] Unless otherwise specified, the experimental methods used in this invention are conventional methods, and the materials and reagents used are commercially available products that can be obtained through commercial channels.

[0043] Example 1: Preparation of mRNA nanoparticles This invention uses a kit to synthesize mRNA encoding ATF4 (NM_182810.3) through in vitro transcription.

[0044] This invention uses mRNA encoding ATF4 for in vivo mouse evaluation. During mRNA synthesis, the invention modifies different sites on the mRNA, including adding a 5' cap and adding poly-A to the 3' end, thereby enhancing the stability of the in vitro transcribed mRNA. The designed coding region sequence can be replaced with epitopes of different target genes as needed, thus making it suitable for the design of various mRNA drugs.

[0045] The preparation method of lipid nanoparticles (LNP) and their encapsulation of mRNA to obtain nanoparticles is described in the invention patent CN120501756A - a pharmaceutical composition and its application, paragraphs

[0306] -

[0308] .

[0046] This invention uses electron microscopy to observe and image ATF4 mRNA nanoparticles. Figure 1 A, and its effective particle size was determined by a nanoparticle size analyzer and a Zeta potential analyzer. Figure 1 B and 1C), and Zeta potential ( Figure 1 D).

[0047] The above results demonstrate that the present invention successfully constructed ATF4 mRNA nanoparticles, hereinafter referred to as NPs(ATF4).

[0048] Example 2: NPs (ATF4) can effectively promote the expression of SLC7A11 in triple-negative breast cancer cells, leading to metabolic reprogramming. After successfully constructing ATF4 mRNA nanoparticles using LNP, this example validates its effectiveness.

[0049] The cell line used in this experiment was MDA-MB-231, derived from human cells; cultured in DMEM complete medium; and seeded at a density of 60%.

[0050] Test items: 1. IC50 detection: Set up drug concentration gradients of 0 ng, 250 ng / mL, 500 ng / mL, 750 ng / mL, 1000 ng / mL, 1250 ng / mL, and 1500 ng / mL, and use a CCK8 to detect IC50.

[0051] 2. ATF4 expression detection: The control groups were NPs (ctrl), i.e., LNPs without mRNA wrapping, and Naked mRNA, i.e., naked mRNA without LNP wrapping.

[0052] 3. The concentration of NPs (ATF4) was 1 μg / mL, and the time was 24 h. The mRNA expression of ATF4 was detected by RT-qPCR, and the protein expression of ATF4 was detected by WB.

[0053] 4. Cystine and glutamate detection: The control groups consisted of NPs (ctrl), i.e., LNPs without mRNA encapsulation, and Naked mRNA, i.e., naked mRNA without LNPs. NPs (ATF4) were administered at a concentration of 1 μg / mL over 24 hours, and detected using commercially available Cys and Glc detection kits.

[0054] The IC50 of this mRNA drug was first determined, and the result was 893.3 ng / mL ( Figure 2 A).

[0055] Compared with the control group, the expression level of ATF4 mRNA in triple-negative breast cancer cells in the NPs(ATF4) treatment group was significantly increased. Figure 2 B). Compared with the control group, the expression level of SLC7A11 mRNA in triple-negative breast cancer cells in the NPs(ATF4) treatment group was significantly increased ( Figure 2 C). Compared with the control group, the expression level of ATF4 protein in triple-negative breast cancer cells in the NPs(ATF4) treatment group was significantly increased ( Figure 2 D).

[0056] This indicates that the drug can effectively deliver ATF4 mRNA into tumor cells, and the delivered mRNA can effectively perform its function, successfully translating the ATF4 protein and promoting the expression of SLC7A11.

[0057] Due to the drug's promoting effect on SLC7A11 expression, the intracellular cystine content in triple-negative breast cancer cells in the NPs(ATF4) treatment group was significantly increased. Figure 2 E), NPs(ATF4) treatment group showed a significant decrease in intracellular glutamate in triple-negative breast cancer cells (E), Figure 2 F).

[0058] The above results indicate that NPs (ATF4) can be effectively delivered into tumor cells and promote the uptake of cystine and the excretion of glutamate by influencing SLC7A11.

[0059] Example 3: NPs (ATF4) have good biocompatibility. To verify the biosafety of this mRNA drug, in this embodiment, 1×10⁻⁶ mRNA molecules were injected orally into the mammary fat pads of Balb / c Nude mice. 6 A triple-negative breast cancer mouse model was constructed using MDA-MB-231 cells.

[0060] The patients were divided into a medication group and a control group for treatment. The drug group was administered NPs (ATF4) at a concentration of 150 ug / kg over 24 hours via intratumoral injection.

[0061] The control groups consisted of NPs (ctrl) (i.e., LNPs not wrapped with mRNA) and naked mRNA (i.e., naked mRNA not wrapped with LNPs).

[0062] The morphology of the heart, liver, spleen, lung, and kidney tissues of mice in the treatment group (NPs(ATF4)) and the control group (NPs(Ctrl) and Naked ATF4) was examined using HE staining. The mice were also weighed.

[0063] The results showed that HE staining revealed that the use of NPs (ATF4) did not cause significant morphological changes in the heart, liver, spleen, lungs, and kidneys of mice. Figure 3 A), meaning they were not harmed by the drug. At the same time, compared with the control group, the body weight of the mice in the drug-treated group was not significantly different ( Figure 3 B). This indicates that local injection of NPs (ATF4) into the tumor has good biocompatibility.

[0064] Example 4: Mechanism analysis of disulfide death induced in triple-negative breast cancer cells by NPs (ATF4) in combination with BAY-876 The primary function of BAY-876 is to inhibit the glucose transporter GLUT1 / 3, thereby suppressing the glucose uptake capacity of tumor cells. The significant decrease in intracellular glucose levels leads to a deficiency of glucose-6-phosphate, a precursor in the pentose phosphate pathway, resulting in a substantial decrease in NADPH production. The main function of NPs (ATF4) is to deliver ATF4 mRNA into tumor cells via nanoliposomes, thereby promoting SLC7A11 transcription. This results in a large amount of cysteine ​​being transported into the cell. Excessive cysteine ​​uptake leads to a significant depletion of the remaining intracellular NADPH, preventing the effective clearance of disulfide bonds and inducing disulfide death. Figure 4 ).

[0065] Example 5: NPs (ATF4) combined with BAY-876 effectively promote disulfide death in triple-negative breast cancer cells. To verify the effectiveness of NPs (ATF4) in combination with BAY-876, this invention first performed CCK8 assays, plate clone experiments, and immunofluorescence staining at the cellular level.

[0066] Experimental methods: Cell line: MDA-MB-231, source: human; cultured in DMEM complete medium.

[0067] Grouping: PBS+NPs(Ctrl): Treat cells using PBS and NPs(Ctrl); BAY-876+NPs(Ctrl): Cells were treated with BAY-876 25 nM and NPs(Ctrl); PBS+NPs(ATF4): Cells were treated with PBS and NPs(ATF4) at a concentration of 1 μg / mL. BAY-876+NPs(ATF4): Cells were treated with BAY-876 25 nM and NPs(ATF4) 1 μg / mL.

[0068] The dosage of NPs (ATF4) was 1 μg / mL, and the dosage of BAY-876 was 25 nM.

[0069] Cystine, glutamate, and NADPH assays: Cystine, glutamate, and NADPH were assayed using commercially available kits 24 hours after drug treatment.

[0070] CCK8 assay: 2000 cells per well in a 96-well plate, administered once daily for 4 consecutive days, with cell viability measured daily using CCK8.

[0071] Plate cloning assay: 6-well plates, cell seeding density of 60%, 7 days after drug administration, stained with crystal violet.

[0072] Compared with the single-drug group, the combination of NPs (ATF4) and BAY-876 significantly increased the cysteine ​​content and significantly decreased the glucose content of tumor cells. Figure 5 A, Figure 5 B). Compared with the single-drug group, the NADPH content in tumor cells of the BAY-876 combined with NPs (ATF4) group was significantly reduced ( Figure 5 C).

[0073] Meanwhile, rhodamine-phalloidin staining results showed that, compared with the single-drug group, the combination drug group exhibited disulfide death-like changes in fibromyalgin within tumor cells (with obvious contraction and aggregation-like morphological changes). Figure 5 F).

[0074] Compared with the single-drug group, the combination of NPs (ATF4) and BAY-876 significantly reduced the viability of tumor cells and had a more significant inhibitory effect on tumor cell growth. Figure 5 (D), and the plate colony formation assay results showed that, compared with the single-drug group, the colony-forming ability of tumor cells in the BAY-876 combined with NPs (ATF4) group was significantly reduced. Figure 5 E).

[0075] The above results suggest that the combination of NPs (ATF4) and BAY-876 can promote disulfide death in triple-negative breast cancer and can more effectively inhibit the malignant phenotype of tumor cells compared with monotherapy.

[0076] Example 6: NPs (ATF4) combined with BAY-876 effectively inhibited tumor growth in a triple-negative breast cancer model mouse. Furthermore, the effectiveness of this combination strategy was verified in animals.

[0077] Methods: 1×10⁻⁶ mg / L was injected in situ into the mammary fat pads of Balb / c Nude mice. 6 A triple-negative breast cancer mouse model was constructed using MDA-MB-231 cells.

[0078] Grouping: PBS+NPs(Ctrl): Processing with PBS and NPs(Ctrl); BAY-876+NPs(Ctrl): Treatment with BAY-876 3 mg / kg and NPs(Ctrl); PBS+NPs(ATF4): Treatment with PBS and NPs(ATF4) at 150ug / kg; BAY-876+NPs(ATF4): Treatment with BAY-876 3 mg / kg and NPs(ATF4) 150 ug / kg.

[0079] Administration methods: BAY-876 and PBS were administered via gavage; NPs (ATF4) and NPs (Ctrl) were injected intratumorally. Administration began on day 11 after tumor formation, administered every two days for a total of five doses. Tumor changes were monitored after administration.

[0080] Cystine, glucose, and NADPH assays: After reaching the ethical endpoint and receiving the samples, commercially available cystine, glutamate, and NADPH assay kits were used for testing.

[0081] Immunohistochemistry: Upon reaching the ethical endpoint, the samples were collected, embedded in paraffin, and prepared into paraffin sections, followed by immunohistochemical staining with Ki67.

[0082] Compared with the single-drug group, the tumor size in mice treated with NPs (ATF4) in combination with BAY-876 was significantly reduced. Figure 6 A) The growth rate of the tumor slowed down significantly. Figure 6 B), and the tumor weight was significantly reduced ( Figure 6 C).

[0083] Compared with the single-drug group, the glucose content in the tumor of mice in the combination of NPs (ATF4) and BAY-876 was significantly reduced. Figure 6 D) Cystine levels were significantly increased ( Figure 6 E), NADPH levels decreased significantly ( Figure 6 F).

[0084] Compared with the single-drug group, the NPs (ATF4) combined with BAY-876 group showed a significant decrease in the ki67 positivity rate in mouse tumors. Figure 6 G).

[0085] The results above suggest that, compared with BAY-876 monotherapy, the combination of NPs (ATF4) and BAY-876 can more effectively inhibit tumor growth in triple-negative breast cancer model mice, demonstrating the high feasibility of further clinical translation of this combination strategy.

[0086] Example 7: Mechanism analysis of dual inhibition of the tricarboxylic acid cycle in triple-negative breast cancer cells by NPs (ATF4) in combination with CB-839. The primary function of CB-839 is to inhibit the function of glutaminase GLS, preventing the metabolism of glutamine into glutamate within tumor cells. This reduced glutamate levels in tumor cells, which rely heavily on glutamate for the tricarboxylic acid (TCA) cycle, lead to a significant decrease in ATP production. However, tumor cells can compensate by generating glutamate through other pathways. As mentioned earlier, NPs (ATF4) deliver ATF4 mRNA into tumor cells via nanoliposomes, promoting SLC7A11 transcription and resulting in the uptake of large amounts of cysteine ​​by tumor cells. This process is accompanied by a significant efflux of glutamate. The combined use of these two drugs, while drastically reducing glutamate production, also leads to the excretion of large amounts of glutamate. This dual effect severely inhibits the energy supply of tumor cells, ultimately causing cell death. Figure 7 ).

[0087] Example 8: The combination of NPs (ATF4) and CB-839 can effectively enhance the inhibition of the tricarboxylic acid cycle in triple-negative breast cancer cells, leading to severe metabolic disorders. To verify the effectiveness of NPs (ATF4) in combination with CB-839, this embodiment also first conducted experiments at the cellular level.

[0088] Experimental methods: Cell line: MDA-MB-231, source: human; cultured in DMEM complete medium.

[0089] Grouping: PBS+NPs(Ctrl): Treat cells using PBS and NPs(Ctrl); CB-839+NPs(Ctrl): Cells were treated with CB-839 500 nM and NPs(Ctrl); PBS+NPs(ATF4): Cells were treated with PBS and NPs(ATF4) at 1 μg / mL; CB-839+NPs(ATF4): Cells were treated with CB-839 500 nM and NPs(ATF4) 1 μg / mL.

[0090] The dosage of NPs (ATF4) was 1 μg / mL, and the dosage of CB-839 was 500 nM.

[0091] Glutamate and ATP detection: 24 hours after drug administration, glutamate and ATP were detected using a commercially available detection kit.

[0092] CCK8: 2000 cells per well in a 96-well plate, administered once daily for 5 consecutive days, with cell viability assessed daily using CCK8.

[0093] Compared with the single-drug group, the NPs (ATF4) combined with CB-839 group showed a significant decrease in tumor cell viability. Figure 8 A). The glutamate content in tumor cells was significantly reduced in the combination drug group ( Figure 8 B), ATP content decreased significantly ( Figure 8 C).

[0094] The above results suggest that, compared with the single-drug group, the combination of NPs (ATF4) and CB-839 can further inhibit the tricarboxylic acid cycle in triple-negative breast cancer, resulting in a significant reduction in ATP production and causing tumor cells to exhibit more severe metabolic disorders.

[0095] Example 9: NPs (ATF4) combined with CB-839 can more effectively inhibit tumor growth in a triple-negative breast cancer model mouse. Furthermore, this embodiment validated the effectiveness of the combined strategy in animals.

[0096] Methods: Animal model: A triple-negative breast cancer mouse model was constructed by in situ injection of 1*10^6 MDA-MB-231 cells into the mammary fat pads of Balb / c Nude mice.

[0097] Grouping: PBS+NPs(Ctrl): This means processing with PBS and NPs(Ctrl); CB-839+NPs(Ctrl): This refers to treatment with CB-839 100 mg / kg and NPs(Ctrl). PBS+NPs(ATF4): This means treating the patient with PBS and NPs(ATF4) at a concentration of 1 μg / mL. CB-839+NPs(ATF4): This refers to treatment with CB-839 100 mg / kg and NPs(ATF4) 1 ug / mL.

[0098] Administration methods: CB-839 and PBS were administered by gavage; NPs (ATF4) and NPs (Ctrl) were injected intratumorally. Administration began on day 9 after tumor formation, once daily for a total of 5 doses.

[0099] Glutamate and ATP detection: After meeting the ethical endpoint, commercially available glutamate and ATP detection kits were used for detection.

[0100] Compared with the single-drug group, the tumor size in mice treated with NPs (ATF4) in combination with CB-839 was significantly reduced. Figure 9 A) The growth rate of the tumor was significantly reduced. Figure 9 B), and the tumor weight was significantly reduced ( Figure 9 C). Compared with the single-drug group, there was no significant difference in the body weight of mice ( Figure 9 D). Compared with the single-drug group, the NPs (ATF4) combined with CB-839 group showed a significant decrease in tumor glutamate content in mice. Figure 9 E), ATP content decreased significantly ( Figure 9 F).

[0101] The above results suggest that, compared with the single-drug group, the combination of NPs (ATF4) and CB-839 has a more significant inhibitory effect on tumors, proving that this combination strategy has the feasibility for further clinical translation.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An mRNA lipid nanoparticle, characterized in that, The mRNA lipid nanoparticles include lipid nanoparticles LNP and ATF4 mRNA; the lipid nanoparticles LNP encapsulate ATF4 mRNA; the nucleotide sequence of the ATF4 mRNA is shown in SEQ ID NO.

1.

2. The mRNA lipid nanoparticles as described in claim 1, characterized in that, The lipid nanoparticles (LNPs) include at least one of ionizable lipids, cofactor lipids, cholesterol, neutral cofactor phospholipids, and PEGylated lipids.

3. The mRNA lipid nanoparticles as described in claim 1, characterized in that, The average particle size of the mRNA lipid nanoparticles is 106-142 nm.

4. A method for preparing mRNA lipid nanoparticles as described in any one of claims 1-3, characterized in that, The preparation method includes the step of encapsulating ATF4 mRNA with lipid nanoparticles (LNP).

5. The use of the mRNA lipid nanoparticles as described in any one of claims 1-3 in combination with GLUT1 inhibitors and / or GLS inhibitors in the preparation of a medicament or formulation for the treatment of triple-negative breast cancer.

6. The application as described in claim 5, characterized in that, The GLUT1 inhibitor is BAY-876; the GLS inhibitor is CB-839.

7. The use of the mRNA lipid nanoparticles according to any one of claims 1-3 in the preparation of formulations that enhance the sensitivity of triple-negative breast cancer patients to GLUT1 inhibitors and / or GLS inhibitors.

8. The application as described in claim 7, characterized in that, The mRNA lipid nanoparticles enhance the sensitivity of tumor cells to GLUT1 inhibitors and / or GLS inhibitors by increasing the expression level of SLC7A11.

9. The application as described in claim 7, characterized in that, The GLUT1 inhibitor is BAY-876; the GLS inhibitor is CB-839.

10. A pharmaceutical composition for treating triple-negative breast cancer, characterized in that, It includes the following components: mRNA lipid nanoparticles as described in any one of claims 1-3, BAY-876, and CB-839.

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