Nanometer delivery carrier of IL-22BPmRNA and application thereof

Abstract

The invention belongs to the field of medicine preparation, and particularly relates to a nano delivery carrier of IL-22BPmRNA (Interleukin-22BPmRNA). The invention aims to overcome the defect of less types of ionizable phospholipids for preparing LNP delivery mRNA. The technical scheme for solving the technical problem is to provide a nano delivery carrier of IL-22BPmRNA, which is prepared on the basis of a novel ionizable phospholipid molecule capable of being used for preparing a nucleic acid delivery carrier. The nano delivery carrier of the IL-22BPmRNA, disclosed by the invention, is good in stability. Experiments show that therapeutic RNA can be efficiently delivered to various human and mouse tumor cells, high-level protein expression is realized, and the polypeptide has unique advantages and good application prospects in the field.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation, specifically relating to a nanodelivery carrier for IL-22BPmRNA and its application. Background Technology

[0002] Malignant tumors refer to new growths formed when cells, under the influence of tumorigenic factors, undergo gene alterations, lose normal regulation of their growth, and experience clonal abnormal proliferation. These new growths possess invasive and metastatic capabilities, causing adverse outcomes such as multiple organ failure, obstruction, hemorrhage, and cachexia. Current treatments for tumors mainly include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. To further regulate the genes of tumor cells at their root, gene therapy is considered a promising innovative treatment strategy. Based on gene therapy, effective tumor suppression can be achieved through strategies such as enhancing immunity, inducing cell death, and regulating the microenvironment. Among these, IL-22 binding protein (IL-22BP) is a natural antagonist of the cytokine IL-22. IL-22 is mainly produced by immune cells. After binding to the IL-22 receptor on tumor cells, it strongly activates the downstream STAT3 signaling pathway, initiating a series of gene expressions that promote cell cycle progression, proliferation, and anti-apoptosis. IL-22BP effectively inhibits the abnormal activation of STAT3 by blocking IL-22, thereby suppressing tumor cell growth and inducing apoptosis. Furthermore, IL-22 signaling recruits and activates myeloid-derived suppressor cells and regulatory T cells to strongly suppress the function of cytotoxic T cells. IL-22BP reduces the number and function of these suppressor cells by inhibiting IL-22. Therefore, increasing the level of IL-22BP in tumor sites can exert a powerful anti-tumor effect. Gene therapy based on IL-22BP mRNA can utilize human cells as factories for protein production, efficiently and continuously expressing large amounts of IL-22BP while avoiding the risk of insertional mutations. However, the mRNA molecular backbone carries a strong negative charge. The cell membrane is also composed of a negatively charged phospholipid bilayer, making it impossible for mRNA to approach and cross the cell membrane on its own. The human body's internal environment is filled with numerous powerful and ubiquitous ribonucleases that can rapidly cleave naked mRNA into fragments, rendering it completely ineffective within minutes of injection. Therefore, mRNA requires a delivery vector to function effectively.

[0003] Currently, the main mRNA vector systems are divided into two categories: viral vectors and non-viral vectors. Although viral vectors have high delivery efficiency, they have limited loading capacity, certain immunogenicity and insertion mutation risks, and complex production processes. In contrast, non-viral vectors have advantages such as simple production processes, short cycles, and ease of large-scale production, but their delivery efficiency is often lower, vector particles are easily cleared during in vivo circulation, and they are easily trapped in lysosomes within cells, leading to degradation.

[0004] In recent years, lipid nanoparticles (LNPs), as representatives of non-viral vectors, have received widespread attention due to their successful application in mRNA COVID-19 vaccines. Pfizer's BioNTech and Moderna's Spikevax are star products of LNP technology, demonstrating the feasibility and clinical value of the RNA-LNP technology platform.

[0005] The core components of LNPs include ionizable lipids, helper phospholipids, cholesterol, and PEG-lipids. Helper phospholipids help form the LNP's bilayer liposome structure, increasing its stability and integrity. Cholesterol improves the stability and fluidity of LNPs, allowing for better fusion with cell membranes. PEG-lipids form a hydrophilic protective layer on the LNP surface, preventing particles from adhering and agglomerating during preparation and storage, and also preventing them from evading rapid recognition and clearance by the immune system after entering the body. Ionizable lipids are crucial for encapsulating and delivering RNA. In an acidic environment, they are positively charged and can bind tightly to negatively charged RNA through electrostatic interactions, encapsulating it to form a stable complex. Ionizable lipids directly determine the efficiency, safety, and stability of RNA delivery by LNPs. However, patents for high-performance ionizable lipids (such as SM102 and ALC-0315) are monopolized by foreign companies such as Moderna and BioNTech. Therefore, developing new, highly efficient ionizable phospholipids to prepare mRNA nanocarriers is an important need in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the limitation of the limited types of ionizable phospholipids available for preparing LNP-delivered mRNA, and to provide a nanocarrier for IL-22BP mRNA prepared based on a new type of ionizable phospholipid molecule that can be used to prepare nucleic acid delivery carriers, thus providing a new and effective option for tumor treatment.

[0007] This invention provides a nanodelivery carrier for IL-22BP mRNA. The nanodelivery carrier is prepared by encapsulating messenger RNA encoding IL-22 binding protein (IL-22BP mRNA) in a lipid nanomedicine carrier; the lipid nanomedicine carrier comprises ionizable phospholipids, and further contains neutral phospholipids, cholesterol, and PEG-modified lipids; the ionizable phospholipids include compounds with the chemical formula Decanoic acid, 2-hexyl-,1,1'-[[[3-(1-pyrrolidinyl)propyl]imino]di-6,1-hexanediyll ester, or pharmaceutically acceptable salts, stereoisomers, deuterated derivatives, or prodrugs thereof.

[0008] The structure of the ionizable phospholipid in the aforementioned nano-delivery carrier is shown in the following formula:

[0009] .

[0010] The molar ratio of the components in the aforementioned nanodelivery carrier is: ionizable phospholipid 45-55 : cholesterol 35-40 : neutral phospholipid 8-10 : PEG-modified lipid 1-2. The neutral phospholipid is DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine). The PEG-modified lipid is DMG-PEG2000.

[0011] The molar ratio of each component in the above-mentioned nano-delivery carrier is as follows: ionizable phospholipid 50 : cholesterol 38.5 : DSPC 10 : DMG-PEG2000 1.5.

[0012] The IL-22BP mRNA in the aforementioned nanodelivery carrier is a nucleotide sequence encoding the IL-22BP protein described in SEQ ID No. 1 or SEQ ID No. 2.

[0013] The nucleotide sequence of the IL-22BP mRNA in the above-mentioned nanodelivery carrier is at least one of SEQ ID No. 3 or SEQ ID No. 4.

[0014] In the above-mentioned nanodelivery carrier, the mass ratio of ionizable phospholipids to IL-22BP mRNA is 20-25:1.

[0015] Preferably, the mass ratio of the ionizable phospholipid to IL-22BP mRNA in the above-mentioned nanodelivery carrier is 20-23:1. More specifically, the mass ratio of the ionizable phospholipid to IL-22BP mRNA is 21.32:1.

[0016] The aforementioned nanodelivery carrier is prepared by the following method:

[0017] a) Dissolve the ionizable phospholipids, cholesterol, DSPC, and DMG-PEG2000 in an organic solvent to form a lipid phase;

[0018] b) Dissolve the mRNA to be encapsulated in an acidic buffer solution to form an aqueous phase;

[0019] c) Mix the lipid phase with the aqueous phase;

[0020] d) Dialyze the mixed solution to remove the organic solvent and form the LNP formulation.

[0021] The aforementioned nanodelivery carrier meets at least one of the following criteria:

[0022] 1) The organic solvent mentioned in step a) is anhydrous ethanol;

[0023] 2) The acidic buffer solution mentioned in step b) is a sodium citrate buffer solution with a pH of 5.5;

[0024] 3) Step c) The mixing of the lipid phase and the aqueous phase of the mRNA is performed using a microfluidic device;

[0025] 4) Step d) Dialyze the mixed solution to remove the organic solvent and replace it with a neutral buffer solution (such as PBS, pH=7.4) to obtain the LNP formulation.

[0026] Furthermore, the procedure and method for microfluidic device processing in step c) above are as follows:

[0027] Pump A: Inner diameter 4.7 mm, specification 1 mL;

[0028] Pump B: Inner diameter 12.4 mm, specification 5 mL;

[0029] The liquid delivery flow rate ratio (A:B) is 1:3;

[0030] Total liquid delivery rate (V) A +V B = 8 mL / min.

[0031] This invention also provides the use of the above-described nanodelivery carrier in the preparation of a drug. Further, the drug is a drug for treating tumors. Even further, the tumor is a head and neck tumor.

[0032] Furthermore, this invention also provides a medicament for treating tumors, which is prepared by adding pharmaceutically acceptable auxiliary components to the aforementioned nanodelivery carrier. Further, the tumor is a head and neck tumor. Even further, the head and neck tumor is an oral cavity tumor. The oral cavity tumor is oral squamous cell carcinoma.

[0033] The beneficial effects of this invention are as follows:

[0034] The applicant has invented a novel nanodelivery carrier (lipid nanoparticle, LNP) for protecting and delivering IL-22BP mRNA. The ionizable phospholipid used in this nanodelivery carrier has a unique chemical structure, consisting of a hydrophilic nitrogen-containing head group and a hydrophobic fatty acid chain. Its head group has a protonable tertiary amine structure, exhibiting typical characteristics of ionizable phospholipids. Its 2-hexyldecanoic acid tail chain is hydrophobic and degradable. Its specific linker chain ensures molecular stability and biocompatibility. The nanodelivery carrier provided by this invention exhibits good stability, making it suitable for storage and administration. In vitro and in vivo experimental results show that the LNP of this invention can efficiently deliver mRNA to various human and mouse tumor cells, achieving high levels of protein expression, demonstrating broad-spectrum efficacy and unique advantages. Simultaneously, the IL-22BP mRNA nanodelivery carrier also significantly inhibited tumor growth in tumor-bearing mouse models by delivering nucleic acid drugs, and did not exhibit significant systemic toxicity at effective therapeutic doses, demonstrating high biosafety and promising application prospects in this field. Attached Figure Description

[0035] Figure 1 A schematic diagram of the chemical structure of the ionizable phospholipid 001 of this invention.

[0036] Figure 2 The nuclear magnetic resonance spectrum of the ionizable phospholipid 001 synthesized according to the present invention

[0037] Figure 3 Mass spectrum of the synthesized ionizable phospholipid 001 of this invention.

[0038] Figure 4 The transfection efficiency of human cell lines was evaluated by gradient testing of 001 LNP / EGFP mRNA prepared with ionizable phospholipid 001 molar ratios of 20%, 50%, 100%, and 200% (n=3, P*<0.05).

[0039] Figure 5The transfection efficiency of LNP / EGFP mRNA prepared from the ionizable phospholipid 001 of this invention on human cell lines was evaluated using the formulation from Moderna (n=3, P*<0.05, P**<0.01, P***<0.001, P****<0.0001), with 002LNP and 004LNP as controls.

[0040] Figure 6 The transfection efficiency of LNP / EGFP mRNA prepared from the ionizable phospholipid 001 of this invention on human cell lines was evaluated using the formula of Pfizer (n=3, P*<0.05, P**<0.01, P***<0.001, P****<0.0001), with 002LNP and 004LNP as controls.

[0041] Figure 7 The transfection efficiency of LNP / EGFP mRNA prepared from the ionizable phospholipid 001 of this invention on mouse cell lines was evaluated using the formulation from Moderna (n=3, P*<0.05, P**<0.01, P***<0.001, P****<0.0001), with 002LNP and 004LNP as controls.

[0042] Figure 8 The transfection efficiency of LNP / EGFP mRNA prepared from the ionizable phospholipids of this invention on mouse cell lines was evaluated using Pfizer's formulation (n=3, P*<0.05, P**<0.01, P***<0.001, P****<0.0001), with 002LNP and 004LNP as controls.

[0043] Figure 9 Using SM102 LNP as a control, the transfection efficiency of LNP / EGFP mRNA prepared by the ionizable phospholipids of this invention on human cell lines was evaluated (n=3, P*<0.05, P**<0.01, P***<0.001).

[0044] Figure 10 Using ALC-0315 LNP as a control, the transfection efficiency of LNP / EGFP mRNA prepared by the ionizable phospholipids of this invention on human cell lines was evaluated (n=3, P*<0.05, P**<0.01, P***<0.001).

[0045] Figure 11Using SM102 LNP as a control, the transfection efficiency of LNP / EGFP mRNA prepared by the ionizable phospholipids of this invention on mouse cell lines was evaluated (n=3, P***<0.001, P****<0.0001).

[0046] Figure 12 Using ALC-0315 LNP as a control, the transfection efficiency of LNP / EGFP mRNA prepared by the ionizable phospholipids of this invention on mouse cell lines was evaluated (n=3, P****<0.0001).

[0047] Figure 13 Example 5: In vivo antitumor activity of LNP / mIL-22BP in MOC1 tumor-bearing mouse model. a. In vitro images of tumors in each group of mice after treatment; b. Tumor volume / time change curve of mice (P*<0.05); c. Bar chart of tumor weight in each group of mice after treatment (P**<0.01). Detailed Implementation

[0048] To develop a novel nanoparticle delivery carrier (lipid nanoparticle, LNP) for the protection and delivery of IL-22BP mRNA, the preliminary work of this invention involved using a structure-feature-driven AI computational model to identify a group of ionizable phospholipids and other compounds as candidate compounds for preparing mRNA drug delivery carriers, and further experimental verification was conducted. Among the ionizable phospholipid compounds 001, 002, and 004 selected in the preliminary experiments, compound 001 was further optimized to exhibit better properties and is more suitable for preparing mRNA drug delivery carriers.

[0049] The chemical formula of ionizable phospholipid compound No. 001 is Decanoic acid, 2-hexyl-,1,1'-[[[3-(1-pyrrolidinyl)propyl]imino]di-6,1-hexanediyll ester. Its structure is shown below:

[0050] .

[0051] Based on this, this invention explores the preparation of nanodelivery carriers (lipid nanoparticles, LNPs) to protect and deliver IL-22BP mRNA, using compound 001 as a basis.

[0052] Specifically, the nanodelivery carrier of the present invention is prepared by encapsulating messenger RNA encoding IL-22 binding protein (IL-22BP mRNA) in a lipid nanomedicine carrier. The lipid nanomedicine carrier includes ionizable phospholipids, and further comprises neutral phospholipids, cholesterol, and PEG-modified lipids; the ionizable phospholipids include the above-mentioned compounds, or pharmaceutically acceptable salts, stereoisomers, deuterated derivatives, or prodrugs of the above-mentioned compounds.

[0053] The molar ratio of the components in the above-mentioned lipid nanoparticle drug carrier is: ionizable phospholipid 45-55 : cholesterol 35-40 : neutral phospholipid 8-10 : PEG-modified lipid 1-2. The neutral phospholipid can be a commonly used neutral phospholipid molecule in the art for preparing mRNA nanolipid carriers. Preferably, it is DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine). The PEG-modified lipid is preferably DMG-PEG2000.

[0054] In one embodiment of the present invention, a preferred molar ratio of the components was used: ionizable phospholipid 50 : cholesterol 38.5 : DSPC 10 : DMG-PEG2000 1.5.

[0055] In the LNP formulations mentioned above, the mass ratio of ionizable phospholipids to RNA is 20–25:1.

[0056] Furthermore, in the above-mentioned LNP preparation, the mass ratio of ionizable phospholipids to RNA is 22-23:1.

[0057] Preferably, the mass ratio of ionizable phospholipids to RNA in the above-mentioned LNP formulation is 22.31:1.

[0058] The IL-22BP mRNA is a nucleotide sequence encoding the IL-22BP protein with the amino acid sequence described in SEQ ID No. 1 or SEQ ID No. 2.

[0059] The amino acid sequence encoded by mouse IL-22BP mRNA is as follows:

[0060] MMPKHCLLGLLIILLSSATEIQPARVSLTPQKVRFQSRNFHNILHWQAGSSLPSNNSIYFVQYKMYGQSQWEDKVDCWGTTALFCDLTNETLDPYELYYGRVMTACAGRHSAWTR TPRFTPWWETKLDPPVVTITRVNASLRVLLRPPELPNRNQSGKNASMETYYGLVYRVFTINNSLEKEQKAYEGTQRAVEIEGLIPHSSYCVVAEMYQPMFDRRSPRSKERCVHIP .

[0061] The amino acid sequence encoded by human IL-22BP mRNA is as follows:

[0062] MMPKHCFLGFLISFFLTGVAGTQSTHESLKPQRVQFQSRNFHNILQWQPGRALTGNSSVYFVQYKIMFSCSMKSSHQKPSGCWQHISCNFPGCRTLAKYGQRQWKNKEDCWGTQELSCDLTSETSDIQEPYY GRVRAASAGSYSEWSMTPRFTPWWETKIDPPVMNITQVNGSLLVILHAPNLPYRYQKEKNVSIEDYYELLYRVFIINNSLEKEQKVYEGAHRAVEIEALTPHSSYCVVAEIYQPMLDRRSQRSEERCVEIP.

[0063] The nucleotide sequence of the IL-22BP mRNA in the above-mentioned nanodelivery carrier is at least one of SEQ ID No. 3 or SEQ ID No. 4.

[0064] Mouse-derived IL-22BP mRNA (SEQ ID No. 3):

[0065] ATGATGCCTAAGCATTGCCTTCTAGGTCTCCTCATCATACTCTTGAGCAGTGCAACAGAAATACAACCAGCTCGTGTATCTCTGACGCCCCAGAAGGTCCGATTTCAGTCCAGAAATTTCCACAATATTTTGCACTGGCAAGCAGGGAGCTCTCTCCCCAGCAACAACAGCATCTACTTTGTGCAGTACAAGATGTATGGACAGAGCCAATGGGAAGATAAAGTTGACTGCTGGGGGACCACGGCGCTCTTCTGTGACCTGACCAATGAAACCTTAGACCCATACGAGCTGTATTACGGGAGGGTGATGACGGCCTGTGCTGGACGCCACTCTGCCTGGACCAGGACACCCCGCTTCACTCCATGGTGGGAAACAAAACTAGATCCTCCGGTCGTGACTATAACCCGAGTTAACGCATCTTTGCGGGTGCTTCTCCGTCCTCCAGAGTTGCCAAATAGAAACCAAAGTGGAAAAAATGCATCCATGGAAACTTACTACGGCTTAGTATACAGAGTTTTCACAATCAACAATTCACTAGAGAAGGAGCAAAAAGCCTATGAAGGAACTCAGAGAGCTGTTGAAATTGAAGGTCTGATACCTCATTCCAGCTACTGCGTAGTGGCTGAAATGTACCAGCCCATGTTTGACAGAAGAAGCCCAAGAAGCAAGGAGAGATGTGTGCACATTCCATGA 。

[0066] Human IL-22BP mRNA (SEQ ID No.4):

[0067] ATGATGCCTAAACATTGCTTTCTAGGCTTCCTCATCAGTTTCTTCCTTACTGGTGTAGCAGGAACTCAGTCAACGCATGAGTCTCTGAAGCCTCAGAGGGTACAATTTCAGTCCCGAAATTTTCACAACATTTTGCAATGGCAGCCTGGGAGGGCACTTACTGGCAACAGCAGTGTCTATTTTGTGCAGTACAAAATC ATGTTCTCATGCAGCATGAAAAGCTCTCACCAGAAGCCAAGTGGATGCTGGCAGCACATTTCTTGTAACTTCCCAGGCTGCAGAACATTGGCTAAATATGGACAGAGACAATGGAAAAATAAAGAAGACTGTTGGGGTACTCAAGAACTCTCTTGTGACCTTACCAGTGAAACCTCAGACATACAGGAACCTTATTAC GGGAGGGTGAGGGCGGCCTCGGCTGGGAGCTACTCAGAATGGAGCATGACGCCGCGGTTCACTCCCTGGTGGGAAACAAAAATAGATCCTCCAGTCATGAATATAACCCAAGTCAATGGCTCTTTGTTGGTAATTCTCCATGCTCCAAATTTACCATATAGATACCAAAAGGAAAAAAATGTATCTATAGAAGATTAC TATGAACTACTATACCGAGTTTTTATAATTAACAATTCACTAGAAAAGGAGCAAAAGGTTTATGAAGGGGCTCACAGAGCGGTTGAAATTGAAGCTCTAACACCACACTCCAGCTACTGTGTAGTGGCTGAAATATATCAGCCCATGTTAGACAGAAGAAGTCAGAGAAGTGAAGAGAGATGTGTGGAAATTCCATGA .

[0068] In one embodiment of the present invention, the LNP formulation of the present invention can effectively inhibit the growth of typical head and neck tumors after delivering IL-22BP mRNA, and has good safety.

[0069] The LNP formulations described above can be prepared using commonly used LNP preparation methods in the art. Generally, existing microfluidic devices can be used for LNP preparation.

[0070] The commonly used methods mainly include the following steps:

[0071] a) Dissolve the ionizable phospholipids, cholesterol, DSPC, and DMG-PEG2000 in an organic solvent to form a lipid phase; wherein, the organic solvent may be anhydrous ethanol;

[0072] b) Dissolve the mRNA to be encapsulated in an acidic buffer to form an aqueous phase; the acidic buffer can be sodium citrate buffer with pH=5.5;

[0073] c) Mix the lipid phase with the aqueous phase; mixing may be performed using a microfluidic device;

[0074] d) Dialyze the mixed solution to remove the organic solvent and form the LNP formulation.

[0075] Generally, after removing the organic solvent, it is necessary to replace it with a neutral buffer solution (such as PBS, pH=7.4) to form the final LNP formulation.

[0076] In summary, this invention provides a novel lipid nanoparticle (LNP) for protecting and delivering IL-22BP mRNA. The core components of this LNP include ionizable phospholipids, helper phospholipids, cholesterol, and PEG-lipids. The helper phospholipids help form the LNP's bilayer liposome structure, increasing its stability and integrity. Cholesterol improves the stability and fluidity of the LNP, enabling better fusion with cell membranes. PEG-lipids form a hydrophilic protective layer on the LNP surface, preventing particles from adhering and agglomerating during preparation and storage, and also preventing them from evading rapid recognition and clearance by the immune system after entering the body. Ionizable phospholipid 001 is key to encapsulating and delivering mRNA. In an acidic environment, it carries a positive charge and can tightly bind to negatively charged mRNA through electrostatic interactions, encapsulating it tightly to form a stable complex. Lipid nanoparticles (LNPs) can effectively promote the further translational application of immunogene therapy targeting IL-22BP in the treatment of malignant tumors.

[0077] The specific implementation of the present invention will be further explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0078] Cholesterol, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0079] DSPC (1,2-distearate-sn-glycerol-3-phosphocholine) was purchased from CordenPharma, Switzerland.

[0080] DMG-PEG2000 (1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol-2000) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0081] ALC-0159 (methoxy polyethylene glycol bis(tetradecyl)acetamide) was purchased from MedChemExpress LLC, USA.

[0082] SM-102 (heptan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate)) was purchased from Cayman Chemical, USA.

[0083] ALC-0315 ([(4-hydroxybutyl)azadialkyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), purchased from MedChemExpress LLC.

[0084] Example 1: Screening and characterization of ionizable phospholipid 001

[0085] This embodiment provides an ionizable phospholipid 001 and its method of obtaining it. The ionizable phospholipid 001 is 2-hexyldecanoic acid, 1,1'-[[[3-(1-pyrrolidinyl)propyl]imino]di-6,1-hexanediyl ester, with the following chemical structural formula: Figure 1 As shown, its molecular structure consists of a hydrophilic nitrogen-containing head group and a hydrophobic fatty acid chain. Its head group possesses a protonable tertiary amine structure, exhibiting typical characteristics of ionizable phospholipids. The structure of ionizable phospholipid 001 is derived from an AI-assisted screening and construction strategy. This invention employs a structure-feature-driven AI computational model to identify ionizable phospholipid 001 and other compounds as candidate compounds, and further conducts synthesis and formulation validation.

[0086] The compound containing ionizable phospholipid 001 was prepared by a chemical synthesis company (Xi'an Ruixi Biotechnology Co., Ltd.), and its spectral data are as follows. Figure 2 and Figure 3 As shown, the pyrrolidine head structure of ionizable phospholipid 001 has an optimized pKa value; its 2-hexyldecanoic acid tail chain is hydrophobic and degradable; and its specific linking chain ensures molecular stability and biocompatibility.

[0087] Example 2: Determination and preparation of LNP component ratios

[0088] This embodiment explores whether ionizable phospholipid 001 can be used to prepare LNP, and the optimal ratio of ionizable phospholipid 001 in LNP. Ionizable phospholipid 001, cholesterol, DSPC, and DMG-PEG2000 provided by this invention were screened at different molar ratios, with the molar ratios of ionizable phospholipid 001, cholesterol, DSPC, and DMG-PEG2000 kept constant at (ionizable phospholipid 001: 10, 25, 50, 100): 38.5: 10: 1.5 for gradient testing.

[0089] The specific method is as follows: Ionizable phospholipid 001, cholesterol, DSPC, and DMG-PEG2000 in the above proportions were weighed and dissolved in ethanol as the lipid phase. Simultaneously, EGFP mRNA was dissolved in a 50 mM sodium citrate buffer (pH 5.5, aqueous phase), with a mass ratio of ionizable phospholipid to RNA of 21.32:1. Lipid nanoparticles were prepared by mixing using a microfluidic mixing device. The microfluidic device processing procedure and method are as follows:

[0090] Pump A: Inner diameter 4.7 mm, specification 1 mL;

[0091] Pump B: Inner diameter 12.4 mm, specification 5 mL;

[0092] The liquid delivery flow rate ratio (A:B) is 1:3;

[0093] Total liquid delivery rate (V) A +V B = 8 mL / min.

[0094] The lipid phase and aqueous phase were mixed at a flow rate ratio of 1:3 (lipid phase A: aqueous phase B) as described above, with the total flow rate controlled at 8 mL / min, to form coarsely dispersed lipid nanoparticles. The resulting mixture was then collected and placed in a dialysis bag with a molecular weight cutoff of 2–3 kDa. The mixture was dialyzed overnight at 4 °C in PBS buffer (pH 7.4), or dialyzed at room temperature for approximately 4 hours to remove ethanol and complete buffer system replacement, ultimately obtaining LNP / EGFP mRNA formulations with different molar ratios.

[0095] Cell transfection efficiency was evaluated for LNP formulations with different phospholipid ratios. Based on comprehensive analysis, the optimal formulation ratio for 001 LNP was determined to be a constant molar ratio of ionizable phospholipid 001, cholesterol, DSPC, and DMG-PEG2000 of 50:38.5:10:1.5 (see [reference]). Figure 4 ).

[0096] Example 3: In vitro cell transfection experiment with two candidate ionizable lipid molecules

[0097] To evaluate the mRNA delivery capability of LNPs prepared from ionizable phospholipid 001 in different cell lines, human and mouse cell lines were selected for in vitro transfection experiments. LNPs loaded with EGFP mRNA obtained in Example 2 were added to the cell culture system at a final concentration of 1 μg mRNA / well, with LNPs containing different candidate ionizable lipids (002, 004) used as controls.

[0098] The following two commercially available LNP formulations are used:

[0099] 1. Referencing Moderna's LNP formulation:

[0100] The molar ratios of ionizable phospholipids (001, 002, 004), cholesterol, DSPC, and DMG-PEG2000 were kept constant at 50:38.5:10:1.5. The mass ratio of ionizable phospholipids to RNA was 21.32:1.

[0101] 2. Referencing Pfizer's LNP formulation as follows:

[0102] The molar ratios of ionizable phospholipids (001, 002, 004), cholesterol, DSPC, and ALC-0159 were kept constant at 46.3:42.7:9.4:1.6. The mass ratio of ionizable phospholipids to RNA was 20.87 / 1.

[0103] The structural formula of 002 is:

[0104] .

[0105] The structural formula for 004 is:

[0106] .

[0107] The specific method is as follows: Human cell lines such as HCT-116 and AGS, as well as mouse cell lines such as MOC1 and SCC-7, were seeded into 24-well plates under suitable culture conditions. When the cell density reached approximately 70%–80% confluence, the corresponding LNP / EGFP mRNA formulation was added to each well, and the plates were incubated at 37 ℃ in a 5% CO2 incubator for 24 hours. After incubation, EGFP expression was observed using a fluorescence microscope, and the percentage of EGFP-positive cells was detected by flow cytometry to assess transfection efficiency.

[0108] Experimental results showed that, compared with LNP delivery systems prepared from ionizable phospholipids of different structures (002, 004), 001 LNP achieved more stable levels of EGFP expression in human cell lines (HeLa, HCT-116, HepG2, RKO, AGS, PSN-1, SK-OV-3, TPC-1, etc.). Figure 5 and Figure 6 (As shown). Simultaneously, relatively stable levels of EGFP expression were obtained in mouse cell lines (LL2, MOC1, SCC-7, primary mouse BMDC, etc.). Figure 7 and Figure 8 (As shown). The results show that the 001 LNP formulation provided by this invention for two different marketed LNP drugs exhibits relatively universal transfectivity and versatility in cell lines from different sources, and has good mRNA delivery performance.

[0109] Example 4: In vitro cell transfection experiment of the LNP of the present invention with positive controls SM-102 LNP and ALC-0315 LNP.

[0110] To evaluate the efficacy of the LNP formulation prepared by the ionizable phospholipid 001 of this invention and existing LNP formulations, this example uses various human and mouse cell lines for in vitro transfection experiments. The LNP loaded with EGFP mRNA obtained in Example 2 was added to the cell culture system to a final concentration of 1 μg mRNA / well. The control groups were Moderna's publicly disclosed lipid LNP formulation SM-102 LNP and Pfizer's publicly disclosed lipid LNP formulation ALC-0315 LNP.

[0111] The specific method is as follows: Human cell lines such as HCT-116 and AGS, as well as mouse cell lines such as MOC1 and SCC-7, were seeded into 24-well plates under suitable culture conditions. When the cell density reached approximately 70%–80% confluence, the corresponding LNP / EGFP mRNA formulation was added to each well, and the plates were incubated at 37 ℃ in a 5% CO2 incubator for 24 hours. After incubation, EGFP expression was observed using a fluorescence microscope, and the percentage of EGFP-positive cells was detected by flow cytometry to assess transfection efficiency.

[0112] Experimental results show that 001 LNP in human ( Figure 9 and Figure 10 (as shown) or mouse-derived cells ( Figure 11 and Figure 12 All three cell lines (as shown) exhibited relatively stable mRNA transfection capabilities. In human cell lines, the transfection efficiencies of 001 LNP and SM-102 LNP varied; 001 LNP showed a superior transfection advantage compared to ALC-0315 LNP. However, in the five mouse cell lines used in the experiment, the transfection efficiency of 001 LNP was significantly higher than that of SM-102 LNP, and the transfection efficiency of 001 LNP was also significantly higher than that of ALC-0315 LNP. These results indicate that the 001 LNP provided by this invention generally exhibits good mRNA delivery performance in cell lines from different sources.

[0113] Example 5: Preparation of IL-22BP mRNA nanoparticles and their therapeutic application in a mouse model of tumor treatment.

[0114] The mRNA encoding IL-22BP was obtained using the T7 polymerase method. First, the IL-22BP plasmid ORF fragment was transcribed using a T7 transcription kit and used as a PCR template for transcription. Then, the IL-22BP mRNA was purified using a purification kit.

[0115] The 001 LNP formulation encoding IL-22BP mRNA was prepared according to the formulations and methods described in Examples 2 and 3, and consisted of nanoparticles with a particle size of approximately 100-200 nm. The molar ratio of ionizable phospholipid 001, cholesterol, DSPC, and DMG-PEG2000 was 50:38.5:10:1.5. The mass ratio of ionizable phospholipid to IL-22BP mRNA was 21.32:1. To evaluate the efficacy of the IL-22BP mRNA nanoformulation in delivering mRNA in vivo, a mouse head and neck cancer subcutaneous xenograft model was used to verify the therapeutic effect. MOC1 cells were subcutaneously inoculated into female C57BL / 6 mice to establish a tumor model. After the tumor volume grew to approximately 100 mm³, the mice were randomly divided into two groups (n=4), and the specific grouping and administration regimens are as follows:

[0116] (1) Experimental group: 001 LNP preparation loaded with mouse IL-22BP mRNA was administered via subcutaneous injection around the tumor. The dose was 10 μg mRNA / mouse, once a day, for a total of 7 consecutive times.

[0117] (2) Control group: The same volume of PBS buffer as the experimental group was administered via subcutaneous injection.

[0118] During treatment, tumor volume changes were measured and recorded daily, while the mice's weight and general condition, such as mental status, were monitored to assess treatment efficacy and safety. After the treatment cycle ended, tumor volume changes in each group were analyzed.

[0119] Experimental results showed that the tumor growth rate in mice treated with 001 LNP was significantly slower than that in the control group (e.g., Figure 13 As shown in the figure, this indicates that the LNP formulation of the present invention has anti-tumor activity in vivo. During treatment, no significant adverse reactions were observed in the weight and condition of the mice, suggesting that the formulation has good in vivo safety.

[0120] The above experimental results show that the nucleic acid carrier and LNP formulation prepared from the ionizable phospholipids used in this invention have uniform particle size and a good low polydispersity index, exhibiting good stability. In vitro and in vivo experiments demonstrate that the LNP of this invention can efficiently deliver therapeutic RNA (such as IL-22BP mRNA) to various human and mouse tumor cells, achieving high levels of protein expression. It demonstrates superior performance compared to existing commercially available products in various cell lines and significantly inhibits tumor growth in tumor-bearing mouse models. Furthermore, the LNP formulation did not exhibit significant systemic toxicity at effective therapeutic doses, demonstrating high biosafety and promising application prospects in this field.

Claims

1. A nanodelivery carrier for IL-22BP mRNA, characterized in that: The drug is prepared by encapsulating messenger RNA encoding IL-22 binding protein (IL-22BP mRNA) in a lipid nanomedicine carrier; the lipid nanomedicine carrier includes ionizable phospholipids, and also contains neutral phospholipids, cholesterol, and PEG-modified lipids; the ionizable phospholipids include compounds with the chemical formula Decanoic acid, 2-hexyl-,1,1'-[[[3-(1-pyrrolidinyl)propyl]imino]di-6,1-hexanediyll ester, or pharmaceutically acceptable salts, stereoisomers, deuterated derivatives, or prodrugs thereof.

2. The nanodelivery carrier for IL-22BP mRNA according to claim 1, characterized in that: The structure of the ionizable phospholipid is shown in the following formula: 。 3. The nanodelivery carrier for IL-22BP mRNA according to claim 2, characterized in that: The molar ratio of each component is: ionizable phospholipid 45-55: cholesterol 35-40: neutral phospholipid 8-10: PEG-modified lipid 1-2; further, the neutral phospholipid is DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine); further, the PEG-modified lipid is DMG-PEG2000.

4. The nanodelivery carrier for IL-22BP mRNA according to claim 3, characterized in that: The molar ratio of each component is: ionizable phospholipid 48-52 : cholesterol 36-40 : neutral phospholipid 9-11 : PEG-modified lipid 1.3-1.7; further, the ionizable phospholipid is 50 : cholesterol 38.5 : DSPC 10 : DMG-PEG2000 1.

5.

5. The nanodelivery carrier for IL-22BP mRNA according to claim 1, characterized in that: The IL-22BP mRNA is a nucleotide sequence encoding the protein described in SEQ ID No. 1 or SEQ ID No.

2.

6. The nanodelivery carrier for IL-22BP mRNA according to claim 5, characterized in that: The nucleotide sequence of the IL-22BP mRNA is at least one of SEQ ID No. 3 or SEQ ID No.

4.

7. The nanodelivery carrier for IL-22BP mRNA according to any one of claims 1 to 6, characterized in that: The mass ratio of the ionizable phospholipid to IL-22BP mRNA is 20–25:1; further, the mass ratio of the ionizable phospholipid to IL-22BP mRNA is 20–23:

1.

8. The nanodelivery carrier for IL-22BP mRNA according to claim 7, characterized in that... Prepared by the following method: a) Dissolve the ionizable phospholipids, cholesterol, DSPC, and DMG-PEG2000 in an organic solvent to form a lipid phase; b) Dissolve the mRNA to be encapsulated in an acidic buffer solution to form an aqueous phase; c) Mix the lipid phase with the aqueous phase; d) Dialyze the mixed solution to remove the organic solvent and form the LNP formulation.

9. The LNP formulation according to claim 8, characterized in that... Meets at least one of the following criteria: 1) The organic solvent mentioned in step a) is anhydrous ethanol; 2) The acidic buffer solution mentioned in step b) is a sodium citrate buffer solution with a pH of 5.5; 3) Step c) The mixing of the lipid phase and the aqueous phase of the mRNA is performed using a microfluidic device; 4) Step d) Dialyze the mixed solution to remove the organic solvent and replace it with a neutral buffer solution (such as PBS, pH=7.4) to obtain the LNP formulation.

10. The use of the nanocarrier of IL-22BPmRNA according to any one of claims 1 to 9 in the preparation of a drug; further, the drug is a drug for treating tumors; even further, the tumor is a head and neck tumor.

11. A drug for treating tumors, characterized in that: It is prepared by adding pharmaceutically acceptable auxiliary components to the nanodelivery carrier of IL-22BPmRNA according to any one of claims 1 to 9; further, the tumor is a head and neck tumor.

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