Application of lipid nanoparticles in tumor treatment

Abstract

The present disclosure relates to the use of lipid nanoparticles in the treatment of tumors. In particular, the present disclosure provides a nano delivery system comprising: a first mRNA encoding an IFN-[beta]; a second mRNA encoding for IFN-[gamma]; a third mRNA encoding for TGF-[beta]; and a delivery vehicle molecule. The nano delivery system disclosed by the invention effectively delivers IFN-beta, IFN-gamma and TGF-beta by using LNP, not only retains low toxicity, but also can perform targeted therapy on pancreatic cancer.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to the role of lipid nanoparticles encapsulating mRNA encoding TGF-β, IFN-β, and IFN-γ (triplet lipid nanoparticles) in tumor treatment. Background Art

[0002] The current global high incidence of cancer is an important public health issue. Data show that the global cancer incidence and mortality are rising rapidly. Cancer brings huge medical expenses to patients, great pressure to the medical and health system, and a heavy burden to the national finances, making cancer one of the obstacles to the development of the world economy. For this reason, researchers from all countries are working hard to carry out scientific research and strive to overcome the cancer problem as soon as possible.

[0003] Pancreatic cancer is a malignant tumor with relatively high morbidity and mortality rates worldwide. Its incidence rate shows a continuous upward trend every year and has now risen from the 7th to the 3rd place among the causes of human cancer-related deaths.

[0004] Pancreatic ducialadenocarcinoma (PDAC) is the most common pathological type of pancreatic cancer, accounting for more than 90% of all histological types. Currently, the most effective treatment for PDAC is radical surgical resection, but more than 80% of patients have already experienced local progression or distant metastasis at the time of diagnosis and have lost the opportunity for surgery. Due to the highly invasive nature of the tumor, patients who are eligible for surgical resection are still prone to local recurrence and distant metastasis even after successful surgical resection. Adjuvant treatment for patients who have lost the opportunity for surgery and those who have completed surgical resection mainly relies on chemotherapy, in addition to radiotherapy, targeted therapy, and immunotherapy.

[0005] Cytokines represented by IL-2 have brought revolutionary breakthroughs in cancer treatment by enhancing the anti-tumor activity of the immune system and improving the tumor microenvironment. Traditional radiotherapy and chemotherapy are extremely harmful to the human body, and it is crucial to solve the problem of low-toxicity targeted treatment of pancreatic cancer. Summary of the invention

[0006] In view of the above-mentioned needs in the art, there is still a need to provide an improved treatment method.

[0007] The present disclosure has found that IFN-γ and IFN-β can induce tumor cell dormancy, and TGF-β plays an important role in the tumor microenvironment. Unexpectedly, it has been found that the simultaneous use of three cytokines, IFN-γ, IFN-β, and TGF-β (hereinafter referred to as IIT), can effectively treat the occurrence and development of pancreatic cancer. Lipid nanoparticles (LNP) are a delivery vehicle that can protect and deliver nucleic acid drugs. Using LNP to effectively deliver IIT not only retains the low toxicity of IIT but also enables targeted treatment of pancreatic cancer.

[0008] In the field of cancer treatment, a nano-delivery system refers to a technical means that uses nanotechnology to encapsulate or connect therapeutic drugs, genes, and other active substances to nanoscale carriers, and precisely delivers these active substances to tumor cells or tissues through various means to achieve efficient and low-toxic cancer treatment.

[0009] The nano-delivery system includes: a nano-carrier and a therapeutic payload carried by the nano-carrier, and also includes a targeting ligand. Nano-carriers are usually made of lipids, polymers, inorganic materials, etc., and have a nanoscale size (generally between 1 and 1000 nanometers). Such as liposomes, polymer nanoparticles, nano-micelles, etc., which can protect the loaded drugs or bioactive substances from the influence of the in vivo environment and improve the stability and bioavailability of the drugs. The therapeutic payload can be a chemotherapeutic drug, a targeted drug, an immunotherapeutic drug, a gene therapeutic drug, etc. The nano-delivery system can encapsulate these therapeutic payloads inside or connect them to the surface to achieve precise targeting of tumor cells. In order to achieve specific recognition and binding to tumor cells, the surface of the nano-delivery system is often modified with some targeting ligands, such as antibodies, polypeptides, nucleic acid aptamers, folic acid, etc. These targeting ligands can specifically bind to specific receptors or antigens overexpressed on the surface of tumor cells, guiding the nano-delivery system to preferentially accumulate in tumor tissues and achieve active targeted delivery.

[0010] For the present disclosure, there is provided a nano-delivery system, the nano-delivery system comprising:

[0011] (i) a first mRNA encoding IFN-β;

[0012] (ii) a second mRNA encoding IFN-γ;

[0013] (iii) a third mRNA encoding TGF-β; and

[0014] (iv) a delivery mediator molecule.

[0015] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are encapsulated in the delivery mediator molecule (encapsulated in a nano-carrier formed by the delivery mediator molecule as an example).

[0016] In some embodiments, the first mRNA, the second mRNA, and the third mRNA are on different strands.

[0017] In other embodiments, the first mRNA, the second mRNA, and the third mRNA are on the same strand.

[0018] In some embodiments, the first mRNA accounts for 5 to 15 molar fractions; the second mRNA accounts for 3 to 9 molar fractions; and the third mRNA accounts for 1 to 3 molar fractions. Molar fractions refer to the relative proportions of each component in terms of moles. For example, the first mRNA accounts for 10 molar fractions; the second mRNA accounts for 6 molar fractions; and the third mRNA accounts for 2 molar fractions, then the molar ratio of the first mRNA, the second mRNA, and the third mRNA is 5:3:1, and so on.

[0019] In an exemplary technical solution, the mRNA delivered by the nanodelivery system of the present disclosure is linear.

[0020] The mRNA delivered by the nano delivery system of the present disclosure is a therapeutic mRNA. A typical therapeutic mRNA structure generally includes the following parts:

[0021] 1) 5' cap structure: located at the 5' end of the mRNA, usually a modified guanosine nucleotide, connected to the first nucleotide of the mRNA by a 5'-5' triphosphate bond.

[0022] 2) 5' untranslated region (5'UTR): It is a sequence that does not encode protein and is located between the 5' cap structure and the coding region in the mRNA molecule. It generally contains multiple cis-acting elements, such as ribosome binding sites, internal ribosome entry sites (IRES), etc.

[0023] 3) Coding region: The genetic information required to encode a specific protein (such as the cytokines IFN-β, IFN-γ, and TGF-β in the present application), which consists of a series of consecutive codons.

[0024] 3) Origin of replication (ori): It is the smallest cis-acting region that supports autonomous replication of plasmids. It is rich in AT bases and is easy to unwind to initiate plasmid replication. The origin of replication regulates the copy number of the plasmid. Common origins include but are not limited to ColE1, pMB1, pSC101, R6K, 15A, etc.

[0025] 4) Promoter: It can be recognized by RNA polymerase and transcribe DNA into RNA. Commonly used RNA polymerases include T7, T3, and SP6, corresponding to T7 promoter, T3 promoter, SP6 promoter, etc.

[0026] 5) Restriction site: A linear restriction site is usually designed downstream of the mRNA coding sequence of the plasmid template. Commonly used IIS type restriction endonucleases such as BspQI, BsaI, etc. can cut downstream of the target gene to produce RNA transcripts of a specific length without leaving any excess nucleotides to ensure the integrity of the polyA tail.

[0027] 6) Kozak consensus sequence: usually located in the 5'UTR near the coding region, usually near the start codon. Its function is to help the ribosome accurately recognize the start codon, improve the accuracy and efficiency of translation initiation, and ensure the correct initiation of protein translation.

[0028] 7) Coding region: Immediately following the Kozak sequence is the region that encodes the protein.

[0029] 8) 3' untranslated region (3'UTR): located between the coding region and the polyadenylation tail, it does not encode proteins but contains a variety of regulatory elements, such as AU-rich elements (AREs), microRNA (miRNA) binding sites, etc.

[0030] 9) Polyadenylic acid tail (polyA tail): a sequence consisting of multiple adenylic acid residues located at the 3' end of mRNA, usually between tens and hundreds of nucleotides in length. In a specific example, the polyA tail in the mRNA used in the present disclosure consists of 101 adenine nucleotides.

[0031] In some specific embodiments, the mRNA encoding IFN-β, IFN-γ, and TGF-β of the present disclosure comprises: T7 promoter, 5'UTR, Kozak consensus sequence, coding region (encoding IFN-β, IFN-γ, or TGF-β), 3'UTR, and polyA tail. The mRNA may be in the form of a plasmid.

[0032] The nanocarrier can be in the form of LNP. As an example, LNP is composed of ionizable lipids, auxiliary lipids, cholesterol or its derivatives and pegylated lipids. Ionizable lipids are neutral at physiological pH and positively charged in the acidic endosomal environment, which helps mRNA escape from the endosomal into the cytoplasm. Auxiliary lipids and cholesterol help maintain the stability and membrane fluidity of LNP, and pegylated lipids can prolong the circulation time of LNP in the body.

[0033] Any suitable delivery vehicle molecule may be used. For example, the delivery vehicle molecule may be a lipid-based vehicle (such as a lipid nanoparticle) or a polymer-based nanoparticle.

[0034] In some embodiments, the delivery vehicle molecule is selected from the group consisting of: ionizable lipids, helper lipids, cholesterol or its derivatives, pegylated lipids, and combinations thereof.

[0035] In some embodiments, the delivery vehicle molecule comprises an ionizable lipid, a helper lipid, cholesterol or a derivative thereof, a pegylated lipid, and combinations thereof.

[0036] Ionizable lipids are a class of lipid molecules that can change their charge state in different pH environments. At physiological pH (about 7.4), they are usually neutral, while in acidic environments (such as endosomes, pH about 5 - 6), they can be protonated and carry a positive charge. When preparing LNPs, ionizable lipids form complexes with negatively charged nucleic acids (such as mRNA, siRNA, etc.) through electrostatic interactions under neutral or alkaline conditions, thus efficiently encapsulating the nucleic acids inside the LNPs. After entering the cell, the LNPs are endocytosed into endosomes. The acidic environment of the endosomes protonates the ionizable lipids, causing them to carry a positive charge and interact with the negative charges on the endosomal membrane, leading to the instability and rupture of the endosomal membrane, promoting the release of the nucleic acids from the endosomes into the cytoplasm, avoiding degradation by lysosomes, and achieving effective gene delivery. Exemplary ionizable lipids include, but are not limited to: N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); 3-(N-(N′,N′-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol); dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyl-oxy-3-dimethylammonium propane; 1,2-dialkoxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC); 1,2-distearoyl-oxy-N,N-dimethyl-3-aminopropane (DSDMA); 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylammonium (DMRIE); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP); 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 2,3-dioleyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA); 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA); 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA); dioctadecylamidoglycyl spermine (DOGS); 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane (CLinDMA); 2-[5′-(cholest-5-en-3-β-oxy)-3′-oxapentyloxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienyloxy)propane (CpLinDMA); N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA);1,2-N,N′-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP); 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP); 1,2-N,N′-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP); 1,2-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLinCDAP); 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA); 2,2-dilinoleyl Oleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); triheptadyl-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA); N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylamine bromide (DMRIE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propylamine bromide (GAP-DMORIE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy) )-1-propylamine bromide (GAP-DLRIE); (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylamine bromide (GAP-DMRIE); N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylamine bromide (βAE-DMRIE); N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleyloxy)propane-1-amine (DOBAQ); 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene] -1-yloxy]propane-1-amine (Octyl-CLinDMA); 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP); 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP); N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE);N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amine bromide (DMORIE); di((Z)-non-2-en-1-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl) dioctanoate (ATX); N,N-dimethyl-2,3-bis(dodecyloxy)propane-1-amine (DLDMA); N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amine amine (DMDMA); di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319); N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propanamide (lipidoid 98N12-5); 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200). In a specific embodiment, the ionizable lipid is AA3-Dlin.;

[0037] Auxiliary lipids are a class of lipid components that together with ionizable lipids constitute LNPs. They usually have a stable chemical structure and specific physical properties, and work synergistically with other lipid components to optimize the performance of LNPs. Auxiliary lipids can regulate the fluidity and stability of lipid membranes, so that LNPs form uniform and stable nanoparticle structures, preventing particle aggregation and fusion. Certain auxiliary lipids (such as DOPE) have a special molecular structure, which can undergo phase transitions in the acidic environment of endosomal membranes to form non-double-layer structures, which help LNPs to fuse with endosomal membranes and promote the release of nucleic acids. Auxiliary lipids can also reduce the toxicity of ionizable lipids, improve the biocompatibility of LNPs, and reduce adverse reactions to the body. Exemplary auxiliary lipids are, for example, but not limited to, dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylcholine (DSPC). Exemplary helper lipids include, but are not limited to, hydrogenated soybean phosphatidylcholine (HSPC); 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC); 1,2-dioleoyl-sn-glycero-3-phosphate (DEPA); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DEPC); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DEPE); 1,2-dioleoyl-phosphatidylglycerol (DEPG); 1,2-dilinoleoyl 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLOPC); 1,2-Dilauroyl-sn-glycero-3-phosphate (DLPA); 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE); 1,2-Dilauroyl-sn-glycero-3-phosphatidylglycerol (DLPG); 1,2-Dilauroyl-sn-glycero-3-phosphoserine (DLPS); 1,2- Dimyristoyl-sn-glycero-3-phosphate (DMPA); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG); 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS); 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA ); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG); 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS); 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA); 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE); 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG); 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DPPS); 1,2-Distearoyl-sn-glycero-3-phosphate (DSPA); 1,2-Distearoyl-sn-glycero-3-phosphocholine ( DSPC); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG); 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS); 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC); 1-myristoyl-2-stearoyl -sn-glycero-3-phosphocholine (MSPC); 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE); 1-stearoyl-2-oleoyl-sn-glyceryl-3-phosphocholine (SOPC); 1-stearoyl-2-palmitoyl-sn-glyceryl-3-phosphocholine (SPPC). In a specific embodiment, the auxiliary lipid is DOPA. ;

[0038] Cholesterol is a natural lipid molecule with a rigid sterol ring structure and is one of the important components of biological membranes. In LNP, cholesterol, as an important lipid component, has an important impact on the performance of LNP. Cholesterol can be inserted into the lipid bilayer to regulate the fluidity and rigidity of the lipid membrane. At physiological temperature, cholesterol can reduce the fluidity of the lipid membrane, making LNP more stable; and in the acidic environment of the endosomal body, it can maintain a certain fluidity, which is conducive to endosomal escape. Cholesterol can interact with cholesterol receptors on the cell membrane, promote the binding and uptake of LNP and cells, and improve the delivery efficiency of nucleic acid drugs. Exemplary cholesterol or its derivatives include, but are not limited to: cholesterol, sitosterol, coprostanol, saposterol, rapeseed sterol, ergosterol, tomatine, ursolic acid, α-tocopherol, stigmasterol, avenasterol, ergocalciferol, campesterol. In a specific embodiment, cholesterol is used.

[0039] PEGylated lipids are a class of amphiphilic molecules formed by chemically linking polyethylene glycol (PEG) to lipid molecules. PEG is a hydrophilic polymer with good biocompatibility and water solubility. PEGylated lipids can form a hydrophilic polymer shell on the surface of LNP, reduce the nonspecific binding of LNP to plasma proteins, avoid being recognized and taken up by the reticuloendothelial system (RES), thereby extending the half-life of LNP in the blood circulation and improving its stability and targeted delivery ability in the body. During the preparation of LNP, PEGylated lipids can adjust the surface properties and charge distribution of the particles, control the particle size and dispersibility of LNP, and form uniform and stable nanoparticles of LNP. Exemplary PEGylated lipids include, but are not limited to, DMPE-PEG1000, DPPE-PEG1000, DSPE-PEG1000, DOPE-PEG1000, Ceramide-PEG2000, DPPE-PEG2000, Azido-PEG2000, DSPE-PEG2000-Mannose, Ceramide-PEG5000, DSPE-PEG5000, DSPE-PEG2000amine, ALC-0159, dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG In some embodiments, the PEGylated lipid is DSPE-PEG2000, 1,2-dioleoyl-rac-glycerol (DOG-PEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-aminopolyethylene glycol 2000 (DOPE-PEG2000). In a specific embodiment, the PEGylated lipid is DSPE-PEG2000.

[0040] In LNPs for nucleic acid drugs, the ratio range of ionizable lipids, auxiliary lipids, cholesterol or its derivatives, and pegylated lipids varies depending on factors such as the route of administration, the site of administration, and the type of nucleic acid encapsulated. The commonly used ratio ranges in this field are:

[0041] The molar ratio of ionizable lipid to total lipid (i.e., the nanocarrier) is 30% to 55% (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55% or any range therebetween);

[0042] The molar ratio of the helper lipid to the total lipid is 10% to 40% (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40% or any range therebetween);

[0043] The molar ratio of cholesterol or its derivatives to the total lipids is 20% to 50% (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or any range therebetween);

[0044] The molar ratio of PEGylated lipids to total lipids is 0.4% to 5% (e.g., 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 or any range therebetween).

[0045] It should be noted that the above ratio range is only for general reference, and the technician can determine it according to the needs and in combination with common knowledge. In a specific embodiment, the molar ratio of the exemplary ionizable lipid, auxiliary lipid, cholesterol, and pegylated lipid is 40:40:25:0.5.

[0046] In some embodiments, the mass ratio of nanocarrier to mRNA (the sum of the first, second and third) is 10:1 to 30:1 (e.g., 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1), more preferably 20:1.

[0047] The present disclosure also provides a pharmaceutical composition, which comprises the nano-delivery system of the present disclosure and a pharmaceutically acceptable carrier.

[0048] A pharmaceutically acceptable carrier refers to a substance or material that can effectively deliver drugs to the site of action, has good biocompatibility and stability under physiological conditions, has no obvious toxicity and adverse reactions to the body, and can improve the physicochemical properties, pharmacokinetic and pharmacodynamic properties of the drug to a certain extent.

[0049] Nano delivery system or pharmaceutical composition of the present disclosure can be conveniently presented in unit dosage form. Nano delivery system or pharmaceutical composition of the present disclosure can be formulated into any suitable dosage form, such as but not limited to, injection, tablet, capsule, gel, etc. Nano delivery system or pharmaceutical composition of the present disclosure can also be formulated into a suspension in an aqueous, non-aqueous or mixed medium. Nano delivery system or pharmaceutical composition of the present disclosure includes but is not limited to solutions, emulsions, foams and liposome-containing preparations. Pharmaceutical composition of the present disclosure may include one or more penetration enhancers, diluents, excipients, etc.

[0050] In some embodiments, the nanodelivery system of the present disclosure or a pharmaceutical composition containing the same is administered to a subject in a therapeutically effective amount.

[0051] The term "therapeutically effective amount" may refer to the amount of an active ingredient or pharmaceutical composition that induces an animal or human to exhibit a biological or medical response considered by a researcher, veterinarian, doctor or other clinician, and such an amount may include the amount of an active ingredient or pharmaceutical composition that is used to induce relief of a disease or condition to be treated. The therapeutically effective dose and the number of administrations of the active ingredient disclosed herein may vary depending on the desired effect. The term "subject" refers to an animal, preferably a mammal. According to a specific embodiment, the subject is a mammal, including, for example, camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, mice, primates (e.g., humans). In a specific embodiment, the subject is a human. In a specific embodiment, the subject is susceptible to, suspected of having, or has had a malignant tumor.

[0052] The term "administering" may refer to providing a predetermined substance to a subject by any appropriate method.

[0053] The administration amount can be administered in various administration doses and methods by dispensing the composition according to the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and disease severity, for example once a day or multiple times a day.

[0054] The administration route of the pharmaceutical composition of the present disclosure can be administered by any general route, as long as it can reach the target tissue. It can be administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, endothelially, intranasally, intrapulmonaryly, rectal, intracavitary, intraperitoneally and intrathecally, but is not limited thereto. In a preferred embodiment, the drug is administered parenterally, preferably by intraperitoneal injection.

[0055] The present disclosure also provides use of the nano-delivery system of the present disclosure in preparing a drug for treating or preventing pancreatic cancer.

[0056] The present disclosure also provides use of a combination of IFN-β, IFN-γ, and TGF-β in preparing a medicament for treating or preventing pancreatic cancer.

[0057] In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0058] The term "treat" refers to eliminating the disease, arresting the progression of the disease, slowing the progression of the disease, reducing the duration of one or more symptoms associated with the disease, improving or reversing at least one measurable parameter associated with the disease, or increasing the survival of subjects suffering from the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1A to Figure 1B IIT-LNP was shown to inhibit tumor growth.

[0060] FIG. 2A to FIG. 2B IIT-LNP was shown to prolong the survival of tumor-inoculated mice.

[0061] FIG. 3A to FIG. 3B IIT-LNP was shown to reduce the cell viability of tumor cells.

[0062] Figure 4 It was shown that IIT-LNP induced the cleavage of GSDME and promoted the death of tumor cells.

[0063] FIG. 5A to FIG. 5B IIT-LNPs were shown to inhibit the growth of tumor cells in PDX-pancreatic cancer and could even induce the regression of tumor cells. DETAILED DESCRIPTION

[0064] Preparation Example 1. Preparation of encoding plasmid

[0065] The sequences of IFN-γ, IFN-β and TGF-β were respectively based on the NCBI reference sequences NM_000619.3, NM_002176.4 and NM_000660.7, which were codon optimized as needed. The coding sequence of each gene was cloned into an in vitro transcribed mRNA (IVT-mRNA) production template plasmid carrying a T7 promoter, 5' and 3' untranslated region (UTR) elements, a Kozak consensus sequence and a 101 polyadenylic acid tail (polyA).

[0066] IVT-mRNA was produced using the linearized IVT template plasmid and the MEGAScript T7 kit and formulated with nucleoside-modified m1Ψ-5′-triphosphate (TriLink, N-1081, San Diego, CA) instead of UTP.

[0067] The three mRNAs of IFN-γ, IFN-β and TGF-β were mixed at a molar ratio of 5:3:1. The mass ratio of lipid:mRNA was 20:1.

[0068] Preparation Example 2. Preparation of Nanodelivery System

[0069] 1. Preparation of LNP (lipid nanoparticles): Dissolve in ethanol at a molar ratio of 40:40:25:0.5 (AA3-DLin:DOPE:cholesterol:DSPE-PEG2000).

[0070] 2. In 25 mM sodium acetate buffer solution, the lipid mixture was mixed with the mRNA obtained in Preparation Example 1 at a ratio of 20:1 (AA3-DLin:mRNA, weight ratio). The preparation was placed in an ultrafiltration tube (Millipore, UFC8100), centrifuged at 1000 g, and collected to obtain a nano delivery system (hereinafter referred to as IIT-LNP).

[0071] Example 1. IIT-LNP inhibits tumor growth

[0072] BXPC-3 pancreatic cancer cells were purchased from the Cell Resource Center of Peking Union Medical College. All human pancreatic cancer tissue samples were provided by the Cancer Hospital of the Chinese Academy of Medical Sciences and the Cancer Hospital of Peking University Health Science Center. Ethical approval was granted by the Medical Ethics Committee of Peking Union Medical College (ZS2023038). All protocols adhered to the Declaration of Helsinki and the Declaration of the World Medical Association, and informed consent forms were signed by all individuals or their family members.

[0073] Experimental Materials Male wild-type NOD-SCID mice were purchased from the Medical Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China), and the animals were maintained under sterile conditions in the animal facility of the Chinese Academy of Medical Sciences. All studies involving mice were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences (ACUC-A02-2024-026).

[0074] 1. Model establishment

[0075] In this disclosure, two pancreatic ductal adenocarcinoma (PDAC) cell lines, BXPC-3 and ASPC-1, were selected because they represent different molecular characteristics and biological behaviors in PDAC. BXPC-3 has high invasiveness, while ASPC-1 exhibits unique characteristics in metabolic reprogramming, which is suitable for studying the metabolic adaptability of tumors. These two cell lines are common cell lines in PDAC research and provide reliable models for studying treatment mechanisms and treatment effects.

[0076] 2. Experimental Procedure

[0077] NOD-SCID mice were inoculated subcutaneously on the ventral side with 1 × 10 6 Eight days after tumor inoculation, mice were randomly divided into two groups (n=6 per group) based on tumor size and body weight. One group was intraperitoneally injected with empty lipid nanoparticles (Blank control); the other group was intraperitoneally injected with IIT-LNP targeting pancreatic cancer cells. The injection was once every two days. Starting from the eighth day after tumor inoculation, the tumor size was measured every two days, and the tumor growth curve was recorded.

[0078] 3. Experimental results

[0079] The tumor size of the BXPC-3 pancreatic tumor in mice treated with IIT-LNP was statistically significantly smaller than that in the Blank group ( Figure 1A The tumor size of the mouse ASPC-1 pancreatic tumor in the IIT-LNP treatment group was statistically significantly smaller than that in the Blank group ( Figure 1B )

[0080] Example 2. IIT-LNP prolongs the survival of tumor-bearing mice

[0081] 1. Experimental Procedure

[0082] NOD-SCID mice were inoculated subcutaneously on the ventral side with 1 × 10 6 Eight days after tumor inoculation, mice were randomly divided into two groups (n=6 in each group) based on tumor size and body weight. One group was intraperitoneally injected with empty lipid nanoparticles (Blank); the other group was intraperitoneally injected with IIT-LNP targeting pancreatic cancer cells. The injections were performed once every two days. The long-term survival curve of mice was recorded.

[0083] 2. Experimental results

[0084] In BXPC-3 pancreatic tumor mice, the survival time of mice in the IIT-LNP treatment group was statistically significantly prolonged compared with the Blank group ( Figure 2A In mouse ASPC-1 pancreatic tumors, the survival of mice in the IIT-LNP treatment group was statistically significantly prolonged compared with the Blank group ( Figure 2B ).

[0085] Example 3. IIT-LNP reduces cell viability of tumor cells

[0086] 1. Experimental Procedure

[0087] NOD-SCID mice were inoculated subcutaneously on the ventral side with 1 × 10 6 BXPC3 pancreatic cancer cells or ASPC-1 pancreatic cancer cells were inoculated with the tumor for 8 days. The mice were randomly divided into 2 groups (n=6 in each group) based on tumor size and body weight. One group was intraperitoneally injected with empty lipid nanoparticles (Blank); the other group was intraperitoneally injected with IIT-LNP targeting pancreatic cancer cells; the injections were given once every two days. The mice were killed 30 days after tumor inoculation, and the subcutaneous tumor tissue was peeled off and cut into pieces, transferred to a 50ml centrifuge tube, and 20ml of culture medium and 2mg / ml type IV collagenase were added. Digestion was performed at 37°C and 120rpm for 2-4h. After digestion, the tumor cell suspension was obtained by filtration. Tumor cells were separated by Percoll, and the cell viability of tumor cells was detected using a cell viability detection kit.

[0088] 2. Experimental results

[0089] In mouse BXPC-3 pancreatic tumors, the cell viability of tumor cells in the IIT-LNP treatment group was statistically significantly decreased compared with the Blank group ( Figure 3A In mouse ASPC-1 pancreatic tumors, the cell viability of tumor cells in the IIT-LNP treatment group was statistically significantly decreased compared with the Blank group ( Figure 3B ).

[0090] Example 4. IIT-LNP induces tumor cell death

[0091] 1. Experimental Procedure

[0092] NOD-SCID mice were inoculated subcutaneously on the ventral side with 1 × 10 6 BXPC3 pancreatic cancer cells or ASPC-1 pancreatic cancer cells were inoculated with the tumor for 8 days. The mice were randomly divided into 2 groups (n=6 in each group) based on tumor size and body weight. One group was intraperitoneally injected with empty lipid nanoparticles (Blank); the other group was intraperitoneally injected with IIT-LNP targeting pancreatic cancer cells; the injections were given once every two days. The mice were killed 30 days after tumor inoculation, and the subcutaneous tumor tissue was peeled off and cut into pieces, transferred to a 50ml centrifuge tube, and 20ml of culture medium and 2mg / ml type IV collagenase were added. Digestion was performed at 37°C and 120rpm for 2-4h. After digestion, the tumor cell suspension was obtained by filtration. Tumor cells were separated by Percoll, and the growth of the tumor was observed using cell death and viability dyes.

[0093] 2. Experimental results

[0094] Compared with the Blank group, the tumor cells in the IIT-LNP treatment group showed significant death ( Figure 4 ).

[0095] Example 5. IIT-LNP inhibits the growth of tumor cells in PDX-pancreatic cancer and can even induce tumor cell regression

[0096] 1. Experimental Procedure

[0097] Two human pancreatic cancer tumor tissues (F0 generation) were washed three times in PBS containing penicillin / streptomycin, and the entire specimen was cut into tissues of approximately 5×5 mm in size. A single tumor tissue was orthotopically implanted into pancreatic cancer of anesthetized NSG mice to generate the F1 generation. After implantation of the F1 generation tumor, the tumor grew to 1 cm 3 When the tumor was 5×5 mm, it was removed and cut into multiple tissues (3×3 mm) and transplanted into new NOD-SCID mice to produce the next generation. After 3 generations, the mice were randomly divided into different groups. After the F4 generation tumor grew to 5×5 mm, the mice were randomly divided into 2 groups (n=6 in each group). One group was intraperitoneally injected with empty lipid nanoparticles (Blank); the other group was intraperitoneally injected with IIT-LNP (50 μg / kg) targeting pancreatic cancer cells; the injection was once every two days, and the tumor growth curve was recorded.

[0098] 2. Experimental results

[0099] Compared with the Blank group, IIT-LNP inhibited the proliferation of PDX-pancreatic cancer ( Figure 5A :#1 Patient, Figure 5B : Patient #2) and can even induce tumor cell regression ( FIG. 5A to FIG. 5B ).

[0100] The results of the above examples show that IIT-LNP has a killing effect on pancreatic cancer tumor cells and is a new strategy for tumor immunotherapy. In vivo studies have found that compared with the control group, IIT-LNP can inhibit tumor growth and improve survival rate; compared with the control group, IIT-LNP can reduce the cell viability of tumor cells; IIT-LNP can induce the shearing of GSDME and promote the death of tumor cells.

[0101] Compared with other drugs in clinical trials, IIT-LNP is essentially a cytokine and is patient-friendly. IIT-LNP can promote GSDME shearing, thereby inhibiting the growth of tumor cells, inducing tumor cell death, and reducing the survival rate of tumor cells.

Claims

1. A nano delivery system, comprising: (i) a first mRNA encoding IFN-β; (ii) a second mRNA encoding IFN-γ; (iii) a third mRNA encoding TGF-β; and (iv) a delivery vehicle molecule, in: The first mRNA, the second mRNA, and the third mRNA are encapsulated in a nanocarrier formed by the delivery vehicle molecule; The first mRNA, the second mRNA and the third mRNA are on the same strand or on different strands; The first mRNA accounts for 5 to 15 molar fractions; The second mRNA accounts for 3 to 9 molar fractions; The third mRNA accounts for 1 to 3 molar fractions; Preferably, the molar ratio of the first mRNA, the second mRNA and the third mRNA is 5:3:

1.

2. The nano-delivery system according to claim 1, wherein the delivery vehicle molecule is selected from any one or a combination of the following: ionizable lipids, helper lipids, cholesterol or cholesterol derivatives, pegylated lipids; Preferably, the molar ratio of the ionizable lipid is 30% to 55% based on the total lipids; Preferably, the molar ratio of the helper lipid is 10% to 40% based on the total lipids; Preferably, the molar ratio of cholesterol or its derivatives is 20% to 50% based on total lipids; Preferably, the molar ratio of the PEGylated lipid is 0.4% to 5% based on the total lipids.

3. The nano-delivery system according to claim 1, wherein: The mRNA is linear or circular; preferably, the mRNA is linear; The mRNA further comprises any one element or a combination thereof selected from the group consisting of: a promoter, a 5'UTR, a 3'UTR, a Kozak consensus sequence, and a poly(A) tail. The nano-delivery system according to claim 1 , which is in the form of an injection. 5 . The nano-delivery system according to claim 1 , wherein the mass ratio of the nano-carrier to the mRNA is 10:1 to 30:1, preferably 20:

1.

6. A pharmaceutical composition comprising: The nano delivery system according to any one of claims 1 to 5; A pharmaceutically acceptable carrier.

7. Use of the nano delivery system according to any one of claims 1 to 5 in the preparation of a medicament for treating or preventing pancreatic cancer.

8. Use of a combination of IFN-β, IFN-γ and TGF-β in the preparation of a medicament for treating or preventing pancreatic cancer.

9. The use according to claim 7 or 8, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

10. A method for inhibiting the growth of cancer cells, inducing the death of cancer cells, or reducing the survival rate of cancer cells in vitro, comprising the steps of: Contacting the cancer cell with an effective amount of the nanodelivery system of any one of claims 1 to 5; Preferably, the cancer cells are pancreatic cancer cells; More preferably, the cancer cells are pancreatic ductal adenocarcinoma cells.

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