Multivalent ionizable lipid-polypeptide and preparation method and application thereof

Abstract

The application discloses a kind of multivalent state ionizable lipid-polypeptide and its preparation method and application, with poly (beta-benzyl-L-aspartic acid), alkylamine compound, amine compound containing tertiary amine group as raw material, the above-mentioned multivalent state ionizable lipid-polypeptide is prepared by reaction, realize mRNA stable loading and tumor-specific transfection.MILP is effectively encapsulated into nanocomposite (MILP@mRNA & MILP@pDNA) by the synergistic effect of electrostatic, hydrophobic and hydrogen bond interaction, these complexes exhibit stability to lyophilization and long-term storage.Compared with SM-102 lipid-based lipid nanoformulation, the MILP@mRNA complex of the application realizes efficient cell uptake, significant endosome escape ability, and efficient transfection in various cell types.The application provides an innovative tumor-specific transfection strategy, which is expected to greatly expand the application range of mRNA therapy and provide an application direction for precision medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a nucleic acid delivery carrier, in particular to a multi-valence ionizable lipid-polypeptide and a preparation method and application thereof, which can be used for tumor-targeted delivery of mRNA. BACKGROUND

[0002] Messenger RNA (mRNA) technology has shown great potential in preventing and treating various diseases due to its ability to rapidly synthesize a variety of proteins, including cytokines, antibodies, antigens, and gene editing proteins [Hou, X.; Zaks, T.; Langer, R.; Dong, Y., Lipid nanoparticles for mRNA delivery, Nat. Rev. Mater. 6 (2021) 1078-1094. Barbier, A. J.; Jiang, A. Y.; Zhang, P.; Wooster, R.; Anderson, D. G., The clinical progress of mRNA vaccines and immunotherapies, Nat. Biotechnol. 40 (2022) 840-854. Huang, X.; Kong, N.; Zhang, X.; Cao, Y.; Langer, R.; Tao, W., The landscape of mRNA nanomedicine, Nat. Med. 28 (2022) 2273-2287. Rohner, E.; Yang, R.; Foo, K. S.; Goedel, A.; Chien, K. R., Unlocking the promise of mRNA therapeutics, Nat. Biotechnol. 40 (2022) 1586-1600], and the successful implementation of mRNA therapy largely depends on efficient targeted delivery systems [Zhang, Y.; Hu, Y.; Tian, H.; Chen, X., Opportunities and Challenges for mRNA Delivery Nanoplatforms, J. Phys. Chem. Lett. 13 (2022) 1314-1322. Huang, P.; Deng, H.; Wang, C.; Zhou, Y.; Chen, X., Cellular Trafficking of Nanotechnology-Mediated mRNA Delivery, Adv. Mater.36 (2024) e2307822], this system must be able to protect mRNA from degradation and overcome physiological barriers to ensure efficient protein expression in target cells. Although lipid nanoparticle (LNP) technology has played a key role in the rapid development of mRNA drugs, its liver enrichment and non-specific delivery characteristics limit therapeutic efficacy and can induce non-target organ toxicity such as hepatitis and liver injury [Loughrey, D.; Dahlman, J. E., Non-liver mRNA Delivery, Acc. Chem. Res. 55 (2022) 13-23]. To achieve non-liver delivery of mRNA drugs, researchers have adopted various strategies, including optimizing the structure of ionizable lipids, introducing selective organ targeting molecules (SORT) as the fifth component of LNP, and modifying targeting groups on the surface of LNP. In addition to LNP, cationic polymers such as poly(beta-amino ester) [PBAE], charge-alterable release transporters (CART), polyaspartamide, and poly(TPAE-co-butyryl chloride) have also been developed for the extraliver delivery of mRNA, achieving selective protein expression in the lung and spleen by adjusting the charge ratio of PEG-lipid, mRNA, and polymer, as well as the length and molar ratio of alkyl chains [Chen, J.; Ye, Z.; Huang, C.; Qiu, M.; Song, D.; Li, Y.; Xu, Q., Lipid nanoparticle-mediated lymph node-targeting delivery of mRNA cancer vaccine elicits robust CD8(+) T cell response, Proc. Natl. Acad. Sci. USA 119 (2022) e2207841119. Qiu, M.; Tang, Y.; Chen, J.; Muriph, R.; Ye, Z.; Huang, C.; Evans, J.; Henske, E. P.; Xu, Q., Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis, Proc. Natl. Acad. Sci. USA 119 (2022) e2116271119. He, Z.; Le, Z.; Shi, Y.; Liu, L.; Liu, Z.; Chen, Y., A Multidimensional Approach to Modulating Ionizable Lipids for High-Performing and Organ-Selective mRNA Delivery, Angew. Chem. Int. Ed. 62 (2023) e202310401. Zhang, X.; Su, K.; Wu, S.; Lin, L.; He, S.; Yan, X.; Shi, L.; Liu, S., One-Component Cationic Lipids for Systemic mRNA Delivery to Splenic T Cells, Angew. Chem. Int. Ed. 63 (2024) e202405444. Cheng, Q.; Wei, T.; Farbiak, L.; Johnson, L. T.; Dilliard, S. A.; Siegwart, D. J., Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing, Nat. Nanotechnol. 15 (2020) 313-320. Dilliard, S. A.; Cheng, Q.; Siegwart, D. J., On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles, Proc. Natl. Acad. Sci. USA 118 (2021) e2109256118] Localized drug delivery, especially intratumoral administration, has become an important strategy for tumor treatment, as it can bypass physiological barriers, improve efficacy, and reduce toxicity in non-target organs. Studies have shown that LNP and PBAE-based nanocarriers can effectively deliver mRNA encoding IL-12, IL-15 and 4-1BBL after intratumoral administration, resulting in high levels of protein expression, immune cell recruitment and anti-tumor immunity at the tumor site. Although these local delivery systems significantly increase local mRNA concentration and improve efficacy, the side effects of nanoparticle distribution in other organs and mRNA expression further limit their clinical application [Bitounis, D.Jacquinet, E.; Rogers, M. A.; Amiji, M. M., Strategies to reduce the risks of mRNA drug and vaccine toxicity, Nat. Rev. Drug Discovery 23 (2024) 281-300]. Therefore, it is particularly urgent to develop a delivery system that can achieve tumor-specific distribution and selective expression of mRNA drugs. SUMMARY

[0003] The present application develops a multi-valence ionizable lipid-polypeptide (MILP) to achieve stable encapsulation of mRNA and tumor-specific transfection. MILP can be synthesized by one-step aminolysis of poly(β-benzyl L-aspartic acid) with small molecules with alkane (Cx) and tertiary amine (Ny), and its hydrophobicity, pKa and function can be adjusted by changing the structure, length and ratio of selected alkane and tertiary amine. MILP has excellent biocompatibility, multifunctional structure and function, and can be widely used in the fields of drug delivery and regenerative medicine. With the help of multi-valence electrostatic, hydrophobic and hydrogen bond interactions, MILP alone can provide strong affinity for mRNA stable complexation, cell uptake, endosome disruption, tumor residence and distribution. In mice carrying subcutaneous tumor xenografts, MILP@mIL-12 induced mRNA-specific distribution and expression at the tumor site, reshaped the tumor immune environment and produced a significant anti-tumor response.

[0004] The present application adopts the following technical solutions.

[0005] A multi-valence ionizable lipid-polypeptide, the chemical structural formula of which is as follows:

[0006]

[0007] wherein m is 0-10, n is 5-100, and z is 0-1 and does not include 0; preferably, m is 1-8, n is 10-80, and z is 0.1-0.8; further preferably, m is 2-7, n is 20-50, and z is 0.3-0.7; the oblique line represents random copolymerization, which is a conventional representation method;

[0008] wherein Ny is a tertiary amine-containing group, Cx is an alkyl group, and z represents the molar fraction of the alkyl group.

[0009] Preferably, in the tertiary amine-containing group, the tertiary amine is a linear or cyclic tertiary amine; and in the alkyl group, the alkyl group is a linear or cyclic alkyl group.

[0010] Further preferably, in the tertiary amine-containing group, the number of carbon atoms is 1-10; and in the alkyl group, the number of carbon atoms is 1-30.

[0011] More preferably, the tertiary amine-containing group has 2-8 carbon atoms; and the alkyl group has 3-20 carbon atoms.

[0012] The application discloses a preparation method of the multivalent ionizable lipid-polypeptide, and comprises the following steps: taking poly(beta-benzyl-L-aspartic acid), an alkyl amine compound and an amine compound containing a tertiary amine group as raw materials, and preparing the multivalent ionizable lipid-polypeptide through reaction.

[0013] In the application, the alkyl group in the alkyl amine compound is linear or cyclic, preferably, the alkyl amine compound has 1-30 carbon atoms, and more preferably, the alkyl amine compound has 3-20 carbon atoms.

[0014] In the application, the tertiary amine in the amine compound containing a tertiary amine group is linear or cyclic; preferably, the amine compound containing a tertiary amine group has 1-10 carbon atoms; and more preferably, the amine compound containing a tertiary amine group has 2-8 carbon atoms.

[0015] In the application, the reaction time is 5-50 hours, preferably 10-40 hours, and more preferably 15-30 hours; and the reaction temperature is 0 DEG C- room temperature.

[0016] In the application, the poly(beta-benzyl-L-aspartic acid) is prepared from an amine compound and beta-benzyl-L-aspartic acid; and the chemical structural formula of the poly(beta-benzyl-L-aspartic acid) is as follows:

[0017]

[0018] In the application, m and n are selected according to the multivalent ionizable lipid-polypeptide.

[0019] The application discloses a multivalent ionizable lipid-polypeptide nucleic acid complex, which comprises the multivalent ionizable lipid-polypeptide and nucleic acid.

[0020] In the application, the nucleic acid comprises RNA and DNA; and the RNA comprises mRNA as an example.

[0021] The application discloses a multivalent ionizable lipid-polypeptide, a preparation method and application thereof, and relates to the field of medicine. The application discloses a novel multivalent ionizable lipid-polypeptide (MILP) which realizes robust formation of mRNA complexes and tumor-specific transfection. MILP effectively encapsulates mRNA and plasmid DNA into 80-nanometer nanoparticles through the synergistic effect of electrostatic, hydrophobic and hydrogen bonding interactions. These particles exhibit stability to lyophilization and long-term storage. Compared with a SM-102 lipid-based lipid nanoparticle formulation (LNP@mRNA), the MILP@mRNA complex realizes more efficient cell uptake, significant endosome escape ability and high-efficiency transfection in various cell types. In particular, MILP@mLuc exhibits unique tumor residence and distribution characteristics due to its multivalent orientation of strong affinity and transcellular transport capacity. After intratumoral injection, MILP@mLuc realizes specific protein expression in tumor cells and macrophages, which is in sharp contrast to the non-specific distribution and expression of LNP@mLuc in major organs such as tumors and livers. In addition, MILP@mIL-12 induces significant tumor microenvironment remodeling and anti-tumor immune response in subcutaneous Lewis lung cancer and 4T1 tumor xenograft models through specific and efficient cytokine expression. Therefore, MILP provides an innovative tumor-specific transfection strategy, which is expected to greatly expand the application range of mRNA therapy and provide new possibilities for future precision medicine.

[0022] The application discloses a preparation method of the multivalent ionizable lipid-polypeptide nucleic acid complex, which comprises the following steps: mixing the multivalent ionizable lipid-polypeptide and nucleic acid in a solvent to obtain the multivalent ionizable lipid-polypeptide nucleic acid complex.

[0023] In the application, the solvent comprises a buffer solution.

[0024] In the application, the mass ratio of the multivalent ionizable lipid-polypeptide and nucleic acid is (1-50):1; preferably, the mass ratio of the multivalent ionizable lipid-polypeptide and nucleic acid is (5-20):1.

[0025] Preferably, the preparation method of the multivalent ionizable lipid-polypeptide nucleic acid complex comprises the following steps: vortexing the multivalent ionizable lipid-polypeptide and nucleic acid in a solvent, and then incubating and standing to obtain the multivalent ionizable lipid-polypeptide nucleic acid complex.

[0026] The application discloses a freeze-dried powder, which is obtained by freeze-drying the above-mentioned multi-valence ionizable lipid-polypeptide nucleic acid complex.

[0027] The application discloses a medicine, and an active ingredient of the medicine comprises the above-mentioned multi-valence ionizable lipid-polypeptide nucleic acid complex.

[0028] As common sense, the medicine can further comprise a pharmaceutically acceptable carrier in addition to the active ingredient, and the carrier is a conventional technology.

[0029] The application discloses an application of the above-mentioned multi-valence ionizable lipid-polypeptide nucleic acid complex in the preparation of a medicine.

[0030] The application discloses an application of the above-mentioned multi-valence ionizable lipid-polypeptide nucleic acid complex in the preparation of an anti-tumor medicine.

[0031] The application discloses an application of the above-mentioned multi-valence ionizable lipid-polypeptide in the preparation of a medicine or a nucleic acid carrier.

[0032] In the application, the tumor comprises a solid tumor, such as lung cancer, breast cancer, melanoma and the like.

[0033] In the application, the multi-valence ionizable lipid-polypeptide (MILP) is simple to synthesize and is used for robust mRNA complexation and tumor-selective transfection. By simulating the multi-valence electrostatic, hydrophobic and hydrogen bond interactions in numerous biochemical processes, the MILP provides strong affinity for nucleic acid (mRNA, DNA) complexation, cell binding and endosome disruption. The complex formed with mRNA (MILP@mRNA) shows high stability for long-term storage and freeze-drying, 8 times higher cell uptake efficiency than the existing SM-102 ionizable lipid-based LNP, high-efficiency transfection in different cells, and tumor-limited distribution and specific protein expression after intratumoral injection. In the subcutaneous LLC and 4T1 tumor xenograft models, MILP@mIL-12 specifically induced the expression of IL-12 at the tumor site, significantly reshaped the tumor immune environment, and significantly enhanced the anti-tumor activity, while significantly prolonging the survival time. Therefore, the MILP is expected to broaden the application range of nucleic acid drugs by providing simulation of biological multi-valence interactions, robust mRNA complexation, site-specific delivery and high-efficiency transfection. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1Characterization of poly(β-benzyl-L-aspartate) (PBLA), where (A) is the proton nuclear magnetic resonance spectrum (400 MHz, DMSO-d6), (B) is the matrix-assisted laser desorption / ionization time-of-flight mass spectrum, and (C) is the gel permeation chromatogram.

[0035] Figure 2 Preparation and characterization of multivalent ionizable lipid-polypeptide (MILP) and mRNA complex (MILP@mRNA). (A) Synthetic route of lipid-polypeptide and small molecule structure for the synthesis of P(zCxNy), where “z” (0.4, 0.6) represents the molar fraction of alkane (Cx). (B) Complex formation of MILP and mRNA (MILP@mRNA). (C) Particle size and zeta potential of MILP@mGFP determined by dynamic light scattering (n = 3). (D) Representative particle size distribution and (E) transmission electron microscopy (TEM) image of MILP11@mGFP formed by P(0.6C8cN6M). Scale bar, 100 nm. (F) Size change of MILP11@mRNA in different solutions, control (sodium acetate buffer, 5 mM), PBS + NaCl (200 mM), urea (200 mM), Tween 20 (5 mM), and mixed solution (PBS + NaCl, urea, and Tween 20) treated at 37°C (n = 3). (G) Percentage of GFP-positive cells after 293T cells were treated with MILP@mGFP for 24 hours (mGFP: 1.0 μg / mL, n = 3). (H) Transfection efficiency of MILP11@mGFP and Lipo2000@mGFP after incubation with different cells for 24 hours (mGFP: 1.0 μg / mL, n = 3). (I-J) Stability of MILP11@mGFP stored at 4°C (I) and freeze-dried using sucrose (Sur, 3-12%, w / v) as a protective agent (J), with EGFP expression in 293T cells as an indicator (mGFP: 1.0 μg / mL, n = 3).

[0036] Figure 3 Proton nuclear magnetic resonance spectrum (400 MHz, CDC13 / CD30D) of lipid-polypeptides P(0.4CxN6) and P(0.6CxN6).

[0037] Figure 4NMR spectra of lipids-polypeptide (MILP) (A) P(0.6C8cN1), (B) P(0.6C8cN5), (C) P(0.6C8cN6), (D) P(0.6C8cN6M) (E) P(0.6C8cN6P), and (F) P(0.6C8cN7) (400 MHz, CDC13 / CD30D).

[0038] Figure 5 Efficiency of mRNA encapsulation for different complexes (MILP@mGFP) (n = 3).

[0039] Figure 6 Results of different cells transfected with MILP11@mGFP for 24 hours (mGFP: 1.0 pg / mL, n = 3).

[0040] Figure 7 Results of 293T cells transfected with different complexes (MILP@pGFP) for 72 hours (n = 3).

[0041] Figure 8 Cell viability of different cells treated with MILP@mGFP for 24 hours, mGFP: 1.0 pg / mL (n = 4).

[0042] Figure 9 Correlation of transfection efficiency with cellular uptake and endosome escape ability of complex MILP@pGFP in vitro (A) Cellular uptake and endosome escape characterization (n = 3). Cellular uptake of 293T cells to different complexes was characterized by flow cytometry. Endosome escape ability of polymers was evaluated by hemolysis analysis of MILP at pH 5.5. Correlation analysis of transfection efficiency of MILP@mGFP with cellular uptake efficiency (B) and endosome escape ability (C), correlation significance was evaluated by Spearman method, and statistically significant correlation data sets were represented by fitting lines. (n = 5).

[0043] Figure 10 Hemolysis of ionizable lipid-polypeptide (MILP) under different pH conditions (n = 3).

[0044] Figure 11Biological distribution and expression of mRNA after intratumoral injection in subcutaneous LLC model. (A) Bioluminescence imaging of mice at different time points using IVIS imaging system (n = 3). (B) Ratio of fluorescence intensity of tumor to abdomen. LNP@mLuc derived from SM-102 was used as control. (C) In vitro imaging of biodistribution (Cy5) and luciferase expression (Luc) of MILP11@mLuc and LNP@mLuc in tumor and major organs at 6 hours post-injection (mLuc-Cy5: 0.5 mg / kg). (D-E) Cellular uptake (D) and mGFP expression (E) of MILP11@mGFP by different cells (LLC cells, CD45 - CD44 + ; macrophages (Mφ), CD45 + F4 / 80 + ) (n = 3). Tumor was dissected and flow cytometry analysis was performed 12 hours after intratumoral injection of MILP11@mGFP (mGFP: 0.5 mg / kg). (F-G) Cellular uptake of MILP11@mLuc and LNP@mLuc by LLC cells at different time points. (mLuc-Cy5: 1.0 μg / mL, n = 3). Percentage of Cy5 + LLC cells (F) and mean fluorescence intensity (MFI) of Cy5 + LLC cells (G) were measured by flow cytometry. (H-I) Cell-to-cell transport of MILP11@mLuc-Cy5 and LNP@mLuc-Cy5 in LLC cells (n = 3). Percentage of Cy5 + LLC cells (H) and MFI of Cy5 + cells (I) were analyzed by flow cytometry in different batches.

[0045] Figure 12 MILP11@mIL-12 produced strong anti-tumor effect in subcutaneous LLC tumor xenograft model. (A) IL-12 secretion by LLC cells 24 hours after transfection with MILP11@mIL-12 and Lipo2000@mIL-12 (n = 3). (B) Ratio of IL-12 in tumor to liver and (C) IL-12 expression in peripheral blood after intratumoral injection of MILP11@mIL-12 and LNP@mIL-12 in LLC tumor-bearing mice. Data are presented as mean ± standard deviation, n = 3. (D) Schematic diagram of MILP11@mIL-12 and aPD-1 dosing regimen. When tumor volume reached approximately 100 mm 3At the time of administration, MILP11@mIL-12 and MILP11@mLuc (10 μg per mouse per dose) were injected intratumorally, followed by αPD-1 (20 μg per mouse per dose) administered via tail vein the next day. (EF) Individual and mean tumor volume changes over time (n = 6). (G) Kaplan-Meier survival curves of mice carrying LLC tumors (n = 6).

[0046] Figure 13 This study demonstrates the strong antitumor effect of MILP11@mIL-12 in a subcutaneous 4T1 tumor xenograft model (n = 6). (A) Schematic diagram of the MILP11@mIL-12 and αPD-1 administration regimen. When the tumor volume reached approximately 100 mm³, MILP11@mIL-12 was administered intratumorally (10 μg per mouse per dose), and αPD-1 (20 μg per mouse per dose) was administered via tail vein the following day. (B) Curve showing the change in mean 4T1 tumor volume over time. (C) Kaplan-Meier survival curves of mice carrying 4T1 tumors. (D) Weight change in mice carrying 4T1 tumors after treatment, n = 6. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Survival rates were analyzed using Kaplan-Meier techniques combined with the Mantel-Cox test. (ns, no significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0047] Figure 14 To regulate the tumor microenvironment (TME) in an LLC xenograft model using MILP11@mIL-12. Figure 4 The treatment regimen shown in D was followed by tumor dissection two days after the last treatment. The tumor tissue was digested to isolate all cells for flow cytometry analysis. (A) Schematic diagram of the mechanism by which MILP11@mIL-12 regulates TME (n = 5). (B) CD45 in live tumor cells. + Percentage of cells. (C) CD8 in tumor sections. + Immunostaining of T cells (red). Scale bar: 50 μm. (D)CD45 + CD8 in cells + Percentage of T cells. (E)CD8 + Activated T cells (CD25) in T cells + (F)CD8 percentage. + IFN-γ in T cells + Percentage of cells. (G)CD4 +Percentage of regulatory T cells (Tregs, FOXP3 + ) in T cells. (H) Percentage of MDSCs (CD11b + Gr-1 + ) in live cells. (I) Ratio of M2 / M1 macrophages. (J) Percentage of CD80 + cells of DCs in TDLNs. (K) Percentage of MHCII+ cells of DCs in TDLNs. Statistical analysis was performed by one-way ANOVA and Tukey's multiple comparison test.

[0048] Figure 15 Schematic of multivalent ionizable lipid-polypeptide (MILP) for robust mRNA complex formation and tumor-restricted transfection. MILP can robustly encapsulate mRNA into sub-100 nm MILP@mRNA nanoparticles by utilizing multivalent electrostatic, hydrophobic, and hydrogen bonding interactions. In the mildly acidic tumor microenvironment (TME), MILP@mRNA is partially protonated and exhibits unique tumor-residence and distribution properties by multivalent-directed strong affinity and transcellular transport. Subsequently, MILP@mRNA achieves efficient endocytosis and endosome escape by multivalent perturbation of cell and endosome membranes, resulting in a strong mRNA transfection effect. DETAILED DESCRIPTION

[0049] mRNA-based therapies are emerging to revolutionize the treatment of various diseases including cancer, considering that mRNA can be translated into functional proteins that either directly inhibit tumor progression or trigger and potentiate immune responses, however, LNP systems still face several challenges such as non-selective delivery, potential adverse reactions caused by PEG and cationic lipids, complex composition based on four lipids, and unsatisfactory stability, which requires ultralow-temperature transportation and storage. The present invention discloses a multivalent ionizable lipid-polypeptide (MILP) based on polyaspartamide derivatives to achieve robust complexation and tumor-selective transfection of mRNA. The present invention employs different ratios of different tertiary amines and alkanes to form nucleic acid complexes through electrostatic and hydrophobic interactions to achieve the hydrophobicity, pKa, and functionality of the lipid-polypeptide. After screening, the best-performing lipid-polypeptide P(0.6C8cN6M) is named MILP11, which effectively encapsulates mRNA or DNA and provides in vitro transfection efficiency comparable to commercial Lipo2000, indicating that MILP can effectively deliver nucleic acids to the cytoplasm and nucleus. In particular, compared with LNP prepared with four lipids, MILP@mRNA / MILP@pDNA is constructed with one component by simple vortex, which facilitates the preparation and scale-up of nucleic acid drugs. By mimicking the multivalent electrostatic, hydrophobic, and hydrogen bonding interactions in many biochemical processes, MILP@mRNA shows high encapsulation efficiency (>90%), monodisperse small size, and significant stability to lyophilization and long-term storage (about 5 weeks). In contrast, mRNA-1273 and BNT162b2 need to be stored at -15 to -20°C and -60 to -80°C, respectively, and then thawed for administration.

[0050] The technical progress of the present application is illustrated by the following specific experiments, the reagents used are existing products, the specific preparation operation and performance test are conventional technology; unless otherwise specified, the present application is carried out in a conventional environment; the modeling and data statistical analysis method of the present application is the existing technology, the data is expressed by mean ± SD, and the analysis is carried out by Prism 8.1. Unless otherwise specified, comparisons between multiple groups use one-way ANOVA and Tukey post-test. Significant differences between groups were determined by one-way ANOVA (Tukey's multiple comparison test). Kaplan-Meier survival curves were analyzed by log-rank test. NS, no significant meaning; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Cells and animals: LLC cells and 4T1 cells were purchased from the China Academy of Type Culture Collection (Shanghai, China), 6-week-old female C57BL / 6 mice (Beijing Charles River Experimental Animal Technology Co., Ltd.) and 6-week-old female Balb / c mice (Shanghai Jihui Experimental Animal Care Co., Ltd.) were bred under specific pathogen-free conditions at Soochow University; all animal experiments were approved by the Animal Care and Use Committee of Soochow University, and all animal experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals.

[0051] 1-Octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate (SM-102), 1,2-dioctadecanoyl-sn-glycero-3-phophocholine (DSPC), Cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol)-2000 (DMG-PEG) were from AVT (Shanghai) Pharmaceutical Technology Co., Ltd. 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Bioteke) D-Fluorescein potassium salt (Meiren Bio) and Lysozyme Tracker Green DND-26 (Yixing Biotech) were used as received. EGFP mRNA was purchased from Cynbio (used for in vitro cell experiments unless otherwise specified). Luc mRNA and IL-12 mRNA were from Bodmed (Suzhou). Label IT nucleic acid labeling kit, Cy5 (Mirus Bio Corporation, Madison, WI, USA). Live / Dead Cell Viability Kit (Thermo Fisher Scientific) and Interleukin 12 (IL-12) enzyme-linked immunosorbent assay kit (ELISA kit, Invitrogen) were used according to the manufacturer's instructions.

[0052] To the ethanol solution of MILP (1.5 mg / mL, 2.0 mL), hydrochloric acid solution (0.1 M) was added in increments of 2.0-3.0 µL. The pH value was measured using a pH probe, and the pKa was determined using the half-equivalent point titration method.

[0053] The CD spectra of MILP were measured using a J-815 CD spectrometer (JASCO, Easton, MD, USA). P(0.6C8cN6M) was dissolved in NMP (100 mg / mL) and diluted to 0.1 mg / mL with sodium acetate buffer (20 mM, pH 4.0) and dialyzed against water overnight in 1.0 kDa MWCO cassettes. Then, the sample solution was placed in a quartz sample cell with a light path of 1 millimeter. According to the measured apparent ellipticity, the average residue molar ellipticity was calculated according to the following formula: Ellipticity ([θ], unit deg cm 2 dmol -1 ) = (millidegrees x average residue weight) / (path length, unit millimeters x polypeptide concentration, unit mg mL -1). CD spectra were measured at room temperature.

[0054] Nano ITC was used to measure the interaction between MILP11 and mRNA. Briefly, MILP11 solution in a syringe was injected into the sample cell containing mRNA solution (1.0 μL for the first injection, and then 24 injections of 2.0 μL each). The interval time for each injection was 120 seconds, the system temperature was set at 25 °C, and the solution was stirred at 300 rpm to ensure complete mixing. To further understand the interaction between MILP11 and negative cell membranes, ITC was determined using cell membrane mimetic phospholipid liposomes as a model. The process of titrating MILP11 into liposomes was similar to that of titrating polypeptides into mRNA.

[0055] LLC cells were stained with LysoTracker Green DND-26 and DAPI to detect the subcellular localization and intracellular transport of MILP11@mRNA. Cells (1 × 10 5 cells / well) were seeded in 35 mm glass culture dishes overnight, and then incubated with MILP11@mLuc-Cy5 (mLuc-Cy5, 2.0 μg / mL) for different time points. After light washing with PBS for three times, the endosomal / lysosomal organelles and nuclei of LLC cells were stained with LysoTracker Green DND-26 (diluted 20000 times with PBS) and DAPI (5.0 μg / mL), respectively. The treated cells were imaged with CLSM, and the co-localization of mLuc-Cy5 with endosomal / lysosomal organelles was analyzed with ImageJ. The Pearson correlation coefficient was used to measure the co-localization between the red (mLuc-Cy5) channel and the yellow (endosome marker) channel (n = 5).

[0056] All animal experiments were performed according to the approved protocols by the Experimental Animal Center and the Animal Care and Use Committee of Soochow University. To study mRNA expression, MILP11 (MILP11@mLuc) and LNP (LNP@mLuc) loaded mRNA encoding firefly luciferase were injected intratumorally at a dose of 10 μg mRNA / mouse. Bioluminescence imaging of injected mice was performed at different time points using the IVIS imaging system after intraperitoneal injection of D-luciferin for 10 minutes. Then, the region of interest (ROI) was selected and the total flux was quantified using Living Image 4.3 software (PerkinElmer).

[0057] To investigate biodistribution, MILP11 (MILP11@mLuc-Cy5) and LNP@mLuc-Cy5 were injected intratumorally into mice at a dose of 10 μg mRNA per mouse. Twelve hours post-injection, major organs and tumors were harvested, and biodistribution analysis was performed using an IVIS imaging system.

[0058] To carry LLC tumor (approximately 100 mm) 3 Mice were intratumorally injected with either MILP11@mIL-12 or LNP@mIL-12 at a dose of 10 μg mRNA per mouse. The mice were then divided into two groups (n = 3). One group was used for blood analysis; 100 μL of blood was collected from the orbital cavity at specified time points, centrifuged at 1000 g for 5 minutes, and plasma samples were collected. The other group of mice was used for tissue analysis; mice were sacrificed at specified time points, and organs and tumors were collected. Tissue samples were ground in tissue protein extraction reagent (RIPA lysis buffer containing 1% PMSF), centrifuged at 10000 g for 10 minutes, and the supernatant was collected. The concentration of IL-12 in plasma and tissue samples was determined using an ELISA kit.

[0059] The in vivo antitumor effects of MILP11@mIL-12 were evaluated in LLC and 4T1 breast cancer models. The LLC model was established as described above, and the LLC subcutaneous tumor model was established by using 1×10 6 5 × 10 LLC cells (PBS plus 30% Methylmatrigel) were subcutaneously injected into the right abdomen of female C57 mice (6 weeks old). The 4T1 breast cancer model was established by injecting 5 × 10 5 Four T1 cells (PBS plus 30% tritrixate) were subcutaneously injected into the right abdomen of female Balb / c mice (6 weeks old). Tumor volume reached 75–100 mm. 3 On day 0, mice were randomly assigned to groups (n = 6) and treated with PBS, mIL-12, αPD-1, MILP11@mIL-12, or MILP11@mIL-12+αPD-1, respectively. Mouse weight and tumor volume were measured every 2 or 3 days. Tumor volume was calculated using the formula: Tumor volume = 1 / 2 × longest diameter × (shortest diameter)². A mouse was considered dead when its tumor volume increased to 1500 mm³ or when it died during treatment.

[0060] Mice (n = 5) bearing LLC tumors (-100 mm 3 Three days after the last treatment, tumor tissues and TDLNs were harvested, ground, centrifuged to obtain single-cell suspensions, and red blood cells were lysed with red blood cell lysis solution (ACK). Before surface staining, samples were incubated with a live / dead kit for 10 minutes and then blocked with Fc Block for 20 minutes; then stained with corresponding antibodies for 30 minutes. All samples were analyzed by flow cytometry (BD FACSVerse), and data were processed by FlowJo 10 version. Example One MILP Preparation

[0061] Multivalent ionizable lipid-polypeptide (MILP) was synthesized by one-step aminolysis reaction of poly( -benzyl-L-aspartic acid) (PBLA) with small molecules containing linear or cyclic tertiary amines (Ny) and alkanes (Cx), and the product was named P(zCxNy), where z represents the molar fraction of alkanes.

[0062] PBLA was synthesized by ring-opening polymerization of BLA-NCA with n-butylamine as an initiator. Briefly, n-butylamine (16 μL, 134.0 μmol) in DCM was added to BLA-NCA (1.0 g, 4.0 mmol) dissolved in DCM / DMF (9:1 v / v), and then polymerization was carried out at 37 °C under nitrogen for 72 hours. The resulting polymer was precipitated in hexane / ethyl acetate (6:4, v / v) and dried under reduced pressure overnight; yield: 85.0%. According to the integral area ratio of the proton signals of the phenyl group (-COOCH2C6H5, δ = 7.2-7.3) to the methyl group (CH3(CH2)3NH, δ = 0.8) in the H NMR spectrum, the degree of polymerization of PBLA was calculated to be 31 with a narrow molecular weight distribution (Mw / Mn = 1.11), and the molecular weight was determined by GPC to be 3.1 kDa. 1 H NMR spectra of the phenyl group (-COOCH2C6H5, δ = 7.2-7.3) to the methyl group (CH3(CH2)3NH, δ = 0.8) in the H NMR spectrum, the degree of polymerization of PBLA was calculated to be 31 with a narrow molecular weight distribution (Mw / Mn = 1.11), and the molecular weight was determined by GPC to be 3.1 kDa. 1 H NMR (Figure 1A), MALDI-TOF (Figure IB), and GPC (Figure 1C) were performed.

[0063] MILPs were synthesized by aminolysis of PBLA. As an example, P(zCscN6M) was synthesized by dissolving 50 mg of PBLA in NMP and cooling to 4 °C. Then, the PBLA solution was added dropwise to a mixture of 2-cyclohexylethylamine (Csc) (267.0 mg, 2.1 mmol) and 3-(2-methyl-1-piperidinyl)propylamine (N6-1) (226.0 mg, 1.4 mmol) under nitrogen at 4 °C. The mixture was stirred at 4 °C for 24 h, precipitated in diethyl ether, and dried under vacuum overnight.

[0064] Other MILPs and P(N6M) polymers were synthesized using the same synthetic protocol.

[0065] P(N6-1) was synthesized according to the procedure for P(zCxNy) and MILP)@mRNA was synthesized according to the procedure for MILP)@mRNA.

[0066] Figure 2 A shows the structures of Nyand Cx. Using these structures, 13 MILP compounds were synthesized with z values of 0.4 and 0.6, respectively (Table 1). 1 The proton signal at 7.3 ppm, which is attributed to the benzyl group, completely disappeared in the H NMR spectrum, while the characteristic signals at 0.9-1.7 ppm were clearly visible, confirming the successful construction of P(zCxNy) polypeptides. Figure 3 and Figure 4 ).

[0067] Table 1 Characterization of MILP polymers

[0068]

[0069] Example 2 mRNA complex formation

[0070] MILP or P(N6M) (100 mg / mL) dissolved in N-methyl-2-pyrrolidone (NMP) was diluted to 10 mg / mL in sodium acetate buffer (20 mM, pH 4.0). Then, the resulting MILP solution was mixed with an equal volume of mRNA solution at different MILP / mRNA mass ratios (5, 10, 15, and 20), vortexed for 15 s, and then incubated at room temperature for 20 min to obtain MILP@mRNA or P(N6M)@mRNA.

[0071] According to the above method, mRNA was replaced by pDNA to obtain MILP@pDNA.

[0072] The mRNA used for the following tests was mGFP and the DNA was pGFP, with a mass ratio of 20 / 1, unless otherwise specified.

[0073] In in vivo studies, MILP@mRNA was dialyzed in a 1000 kDa MWCO dialysis bag with cold PBS at 4°C for 120 minutes, and the mRNA encapsulation efficiency was quantified using the Quant-iT RiboGreen RNA assay kit (n = 3).

[0074] MILP@mRNA was mixed with sucrose (3-12% (w / v) final concentration) as a cryoprotective agent, snap-frozen in liquid nitrogen, and then processed in a freeze-drying system (Christ ALPHA 1-2 LD plus). The freeze-dried MILP@mRNA was stored at -80°C until use. Upon application, the freeze-dried NPs were resuspended in sterile water to restore their original concentration.

[0075] Complexation of MILP with mRNA was achieved by simple vortexing in sodium acetate buffer (AcNa, pH 4.0) Figure 2 B). The MILP@mRNA complexes formed had an average diameter of less than 80 nm and a positive zeta potential Figure 2 C). Specifically, the complex formed by P(0.6C8cN6M) (denoted as MILP11@mRNA) exhibited a small size of about 60 nm, monodispersity, and a spherical morphology, which were evaluated by conventional DLS and TEM measurements Figure 2 D, Figure 2 E). The encapsulation efficiency of mRNA was about 90% Figure 5 . As shown in Figure 2 F, the addition of Tween 20, sodium chloride, or urea to the complex caused the MILP@mRNA particle size to gradually increase, while the combined use of these agents produced a particle size change of more than ten times Figure 1 F), which supported the occurrence of hydrophobic (Tween 20), ionic (sodium chloride), and hydrogen bonding (urea) interactions during the complexation process. In contrast, the complex formed by P(N6-1) with mRNA (P(N6-1)@mGFP) showed a larger average diameter of 276 nm due to the lack of lipids. In addition, isothermal titration calorimetry (ITC) showed that the binding constant (Ka) of P(0.6C8cN6M) was much higher than that of P(N6M) (Ka1: 4.2 x 10 8 vs 1.0 x 10 8 ; Ka2: 2.9 x 10 6 vs 2.4 x 10 5) (Table 2), indicating multiple strong interactions between P(0.6C8cN6M) and mRNA. Circular dichroism (CD) spectra showed that P(0.6C8cN6M) was an a-helix structure. MILP@mRNA showed little size change in PBS within 12 h, in contrast, the complexes treated with proteinase K (12.0 units / mL) showed obvious swelling within 12 h, with an average size of about 600 nm, indicating that they were enzymatically degradable.

[0076] Table 2 Binding constants of P(0.6C8cN6M) or P(N6M) to mRNA

[0077]

[0078] Example Three In vitro performance of MILP

[0079] The transfection efficiency of MILP@mRNA was evaluated in 293T, LLC, U87-MG and SKOV3 cells using mRNA and pDNA encoding GFP (mGFP and pGFP) as reporter genes by fluorescence microscopy and flow cytometry. Cells were cultured in DMEM medium containing 10% FBS, and the culture density in 48-well plates was 50000-70000 cells / well. After 12 h, MILP@mGFP or MILP@pGFP (1.0 μg / mL, n = 3) was added to each well. After 6 h of culture, the medium was replaced with fresh medium, and the cells were cultured in the fresh medium and observed by fluorescence microscopy at different time points (24, 48, 72 h). After the sequential addition of 0.25% trypsin-EDTA and 0.5 mL PBS, the cells were collected by centrifugation and then resuspended in 0.3 mL PBS for flow cytometry analysis.

[0080] In 293T cells, the transfection efficiency of MILP with mRNA complex encoding green fluorescent protein (GFP) (MILP@mGFP) was evaluated, see Fig. 6A and Fig. 6B, by confocal microscopy imaging and flow cytometry (FACS) analysis, respectively. The results showed that the transfection efficiency of MILP@mGFP was higher than that of Lipofectamine 2000. Figure 2G), most of P(0.6CxNy) had a molar fraction of alkane 0.6, showing more than 60% GFP positive cells, in sharp contrast to the low transfection efficiency (<30%) of P(0.4CxNy) group. In addition, higher GFP expression could be obtained in P(0.4CxNy) by increasing the alkane chain length from C8 to C12, but P(0.6CxNy) formed by C12 alkane showed poor solubility, which was not suitable for complexing with nucleic acid. Notably, P(0.6C8cN6) showed superior protein expression in P(0.6CxN6) group, indicating that appropriate hydrophobicity and structure played an important role in mRNA transfection. Further, P(0.6C8cN6M) as alkane with mRNA complex presented the best transfection efficiency, about 85% GFP positive cells, close to Lipo2000, much higher than branched PEI 25k (<10%). Therefore, MILP11@mRNA formed by P(0.6C8cN6M) was selected for further in vitro and in vivo studies.

[0081] The transfection ability of MILP11@mGFP in cancer cells was evaluated. Notably, about 80% transfection efficiency was achieved in different cancer cells (LLC, U87, SKOV3), among which human U87 and SKOV3 represented about 95% GFP positive cells (MFI ~ 1000) at MILP11 / mRNA ratio of 20 / 1 (N / P ~ 20 / 1) (Fig. 6A). Figure 2 H). Even at low MILP11 / mRNA mass ratio of 5 / 1 (N / P ~ 2.5 / 1), MILP11@mGFP provided a decent transfection efficiency of 80% and 60% in U87 and SKOV-3 cells, respectively (Fig. 6B). Figure 6 ). Meanwhile, MILP11@mGFP still induced 70% GFP positive cells (MFI ~ 1000) in 293T cells after long-term storage (5 weeks) at 4°C (I) and 75% MFI, in sharp contrast to the little transfection of MC3-LNP@mGFP after storage for one week under the same condition. Figure 2

[0082] In addition, the stability of MILP11@mGFP after lyophilization was evaluated using sucrose as a protective agent. When the sucrose concentration was greater than 3% (m / v), the transfection efficiency of lyophilized MILP11@mGFP was comparable to that of fresh samples (J). In addition, the size and zeta potential of lyophilized MILP11@mGFP showed minor changes at low sucrose concentration of 3-6%, while obvious nanoparticle aggregation was observed when the sucrose concentration increased to 12%. Figure 2 J). In addition, the size and zeta potential of lyophilized MILP11@mGFP showed minor changes at low sucrose concentration of 3-6%, while obvious nanoparticle aggregation was observed when the sucrose concentration increased to 12%.

[0083] ​The MILP11@mRNA in the application will greatly promote the transportation and clinical application of mRNA drugs in the preservation of mRNA activity after long-term storage and freeze-drying.

[0084] In addition, MILP can effectively compress pDNA, and the MILP@pGFP nanoparticles formed show a transfection efficiency comparable to that of Lipo2000 complex in 293T cells Figure 7 ), indicating that MILP has completed effective intranuclear delivery of pDNA.

[0085] The CCK8 detection method was used to evaluate the viability of different cells treated with MILP@mRNA. LLC, U87-MG or SKOV3 cells were seeded into 96-well plates at a density of 15000-20000 cells / well, placed in DMEM medium containing 10% FBS, and incubated with MILP@mGFP (1.0 μg / mL, n = 4). After 6 hours of culture, the culture medium was removed, and the cells were incubated in fresh culture medium for another 18 hours, then 10 μL of CCK8 solution was added, and the cells were incubated for another 3 hours.

[0086] Therefore, MILP becomes a multifunctional platform for the effective delivery of different nucleic acids including mRNA and DNA in cells. Importantly, different cells (293T, LLC, U87, SKOV) co-incubated with MILP@mGFP for 24 hours all showed more than 80% cell viability Figure 8 ), indicating good cell compatibility.

[0087] Example Four MILP-mediated cellular uptake and endosome escape

[0088] The cellular uptake efficiency of MILP@mRNA was evaluated with 293T cells and LLC cells. The cells were seeded into 24-well plates at a density of 100000 cells / well for 12 hours, then incubated with MILP@mLuc-Cy5. At intervals, the cells were washed twice with PBS, digested with trypsin, and then centrifuged at 1,000 rpm for 3 minutes. The collected cells were resuspended in 0.3 mL of PBS and analyzed by flow cytometry, n = 3. See Figure 9Compared to MILP@mRNA formed from P(0.4CxN6), nanoparticles based on P(0.6CxN6) showed significantly higher cellular uptake. The C8 alkane group showed greater uptake in 293T cells, with cyclic C8c being superior to linear C8 alkanes. Lipid-peptides with 6- and 7-membered cyclic tertiary amines (N6, N6M, N6P, and N7) achieved better cellular uptake. Interestingly, correlation analysis between transfection efficiency and cellular uptake efficiency showed a significant positive correlation between mRNA transfection and uptake efficiency (r = 0.74 and P = 0.0002). Figure 9 B).

[0089] Red blood cells (RBCs) were isolated from freshly collected whole blood from mice, centrifuged at 3000 rpm for 5 minutes, and then washed five times with PBS buffer. The RBCs were then resuspended in 20 mM HEPES (pH 7.4) or 20 mM MES (pH 5.5) containing 150 mM NaCl. MILP solution (5.0 μg / mL) was added to the RBC suspension in 96-well plates. After incubation at 37 °C for 1 hour, the plates were centrifuged at 3000 rpm for 5 minutes. The amount of hemoglobin released was measured at 540 nm using a microplate reader (n = 3). RBC suspensions incubated in PBS and Triton X-100 solution (1.0 wt.%) served as negative and positive controls, respectively. Hemolysis rate = 100% × (OD - OD) PBS ) / (OD Triton - OD PBS ), where OD, OD Triton and OD PBS The absorbance values ​​are those of RBCs after treatment with MILP, Triton, and PBS, respectively. The hemolysis assay was used to evaluate the membrane-damaging ability of the carriers, further reflecting the efficiency of endosome escape from different carriers. Under acidic conditions (pH 5.5), P(0.6CxN6), with more hydrophobic segments, exhibited significantly greater hemolytic activity than P(0.4CxN6). The hemolysis rate of P(0.6CxN6) with C6, C8, and C8c alkanes was 11-17%. Figure 9 A) ensuring their active escape from endosomes. Meanwhile, MILPs with different tertiary amines all exhibited significant hemolytic activity, with MILPs from N6, N6M, and N7 showing a hemolysis rate of approximately 15%. Notably, endosome escape (reflected in the hemolysis rate) is generally positively correlated with mRNA transfection, such as... Figure 9 As shown in C. Importantly, MILP typically shows a hemolysis rate of less than 5% at pH 7.4 ( Figure 10), which confirmed their hemocompatibility under physiological conditions. The pH-dependent hemolysis switch mainly comes from the protonation of the tertiary amine in the lipid-polypeptide, with a pKa value of 6.3 ~ 7.0, which helps to build an efficient and safe mRNA delivery system.

[0090] Example Five MILP@mRNA Enables Tumor-specific biodistribution and expression

[0091] In a mouse model carrying LLC tumor, the in vivo biodistribution and protein expression of MILP11@mLuc were studied. As a control group, LNP@mLuc composed of ionizable SM-102 lipid was used, with an average particle size of about 100 nm and a neutral surface charge. Through in vivo bioluminescence imaging technology, it was found that MILP11@mLuc specifically promoted the expression of luciferase in LLC ( Figure 11 A, Figure 11 B)and 4T1 tumors after intratumoral injection, in sharp contrast to the dispersed distribution of luciferase fluorescence in tumors and abdomen in the LNP@mLuc group. Through in vitro fluorescence imaging technology, tumor-specific protein expression of MILP11@mLuc was also clearly observed ( Figure 11 C). At the same time, Cy5-labeled mLuc (mLuc-Cy5) was used to monitor biodistribution. Consistent with the protein expression results, MILP11@mLuc was only distributed in the tumor after 6 hours of intratumoral administration, while LNP@mLuc detected strong Cy5 fluorescence in the tumor and major organs (liver, lung, kidney and spleen) ( Figure 11 C). Among the existing clinically applied delivery carriers, lipid nanoparticles based on SM-102 or ALC0315 can detect mRNA non-specific distribution and expression in normal organs, especially the liver, after local or systemic injection. Notably, MILP11@mLuc showed little protein expression after intramuscular and subcutaneous injection, almost undetectable, confirming the low transfection efficiency of MILP11@mRNA in skin and muscle tissue. In addition, histological analysis showed that MILP11@mLuc did not induce apoptosis and necrosis of adjacent skin tissue. The biodistribution and transfection efficiency of MILP11@mRNA were further evaluated in LLC cells and tumor-associated macrophages (TAMs, Mφ), considering that they account for the majority of the tumor (about 90%). Notably, about 48.5% of LLC cells (CD45 - CD44 + ), 55.7% of TAMs (CD45 + F4 / 80 + ) and 37.0% of total cells isolated from the tumor were Cy5 positive ( Figure 11D), confirming the wide distribution of MILP11@mRNA in tumors and efficient cellular uptake. Accordingly, significant GFP expression was observed in LLC cells (25.7%) and TAMs (18.2%), while GFP expression in total cells was less (17.8%) due to the lower transfection efficiency of MILP11@mRNA in other cells ( + cells (17.8%) due to the lower transfection efficiency of MILP11@mRNA in other cells ( Figure 11 E).

[0092] Example Six MILP@mIL-12 for Anti-tumor Activity in vivo (4T1 and LLC tumor models)

[0093] Interleukin 12 (IL-12) is a potent pro-inflammatory cytokine that promotes T cell proliferation and activation, effectively stimulates the production of interferon-gamma (IFN-g), and is therefore considered a promising anti-cancer agent; unfortunately, the clinical application of IL-12 is limited by its short half-life, insufficient local concentration in tumors, and immune-related adverse events. The present application uses a complex encapsulating IL-12 mRNA (MILP11@mIL-12) to improve the immunosuppressive tumor environment. Notably, MILP11@mIL-12 significantly expressed IL-12 in LLC cells in vitro ( Figure 12 A), and IL-12 was enriched in tumors within 6-12 hours after administration, with protein expression in tumors more than 9 times higher than in the liver ( Figure 12 B). In contrast, LNP@mIL-12 produced similar IL-12 levels in tumors and livers under the same conditions ( Figure 12 B). Importantly, MILP11@mIL-12 produced much less IL-12 in blood than LNP@mIL-12 (250 vs 1500 pg / mL) ( Figure 12 C), indicating that MILP11@mIL-12 is less likely to cause systemic immunotoxicity. At the same time, mice receiving MILP11@mIL-12 treatment had little change in body weight, normal liver and kidney function and tissue structure (no difference between groups), and MILP11@mIL-12 showed excellent biosafety. In LLC tumor-bearing xenograft mice, MILP11@mIL-12 significantly inhibited tumor growth and greatly prolonged survival time, while free mIL-12 had little effect on tumor inhibition and mouse survival, indicating the key role of the MILP11 delivery system. In addition, non-therapeutic MILP11@mLuc had no obvious inhibitory effect on tumor growth ( Figure 12 E, Figure 12 F, Figure 12G). In combination with aPD-1, MILP11@mIL-12 + aPD-1 group showed better efficacy in LLC tumor inhibition and survival rate than MILP11@mIL-12 alone group, with 50% of mice survived during the experiment, while no mice survived in MILP11@mIL-12 alone group, with median survival time (MST) of 25 days (p < 0.0001) Figure 12 G). Similarly, MILP11@mIL-12 + aPD-1 group showed more significant tumor inhibition and prolonged survival in 4T1 tumor-bearing mice than MILP11@mIL-12 group (MST 33 vs 29) (p < 0.0001) Figure 13 ). Therefore, intratumoral delivery of MILP11@mRNA has superior tumor inhibition effect and good safety, and has significant potential in the treatment of various malignant tumors such as breast cancer, melanoma and lung cancer.

[0094] Example Seven MILP@mIL-12 Mediated Tumor Microenvironment Modulation

[0095] The product of the present application can also promote the production of IFN-γ, activate dendritic cells, and re-regulate the immunosuppressive TME (Tumor Microenvironment) Figure 14 A). After three doses of treatment, as shown in Figure 12 D, tumor and its adjacent tumor-draining lymph nodes (TDLNs) were collected on day 9 for flow cytometry immunological analysis. MILP11@mIL-12 and MILP11@mIL-12 + aPD-1 both significantly increased the infiltration of CD45 + cells ( Figure 14 B) and CD8 + T cells ( Figure 14 C, Figure 14 D), as well as the population of activated CD8 + T cells (CD8 + CD25 + T) ( Figure 14 E) and IFN-γ + CD8 + T cells ( Figure 14 F). At the same time, after treatment with MILP11@mIL-12 or MILP11@mIL-12 + aPD-1, the population of immunosuppressive cells in the tumor, including Tregs (CD4 + FOXP3 + ) and MDSCs (CD11b + Gr-1 + ) was significantly reduced ( Figure 14 G, Figure 14H). Compared with free mIL-12, MILP11@mIL-12 promoted the polarization of macrophages to M1 phenotype and showed a significantly lower M2 / M1 ratio. In TDLNs, both MILP11@mIL-12 and MILP11@mIL-12+PD-1 groups detected a significant increase in CD80 + DCs and a slight increase in MHCII + DCs and a slight increase in MHCII Figure 14 J, Figure 14 K). These results suggest that MILP11@mIL-12 combined with a-PD-1 can enhance the infiltration of anti-tumor immune cells and improve the immunosuppressive TME.

[0096] Specific and efficient delivery of mRNA to target lesions will greatly benefit the treatment of various diseases. Existing LNP@mRNA tends to accumulate in the liver after administration, which often limits the enrichment of the drug at the target site and causes liver damage; the present application solves this problem. As an example, see Figure 15 In mice carrying subcutaneous LLC tumors, MILP@mRNA achieved tumor tissue-selective retention and expression through strong affinity and transcellular transport of multivalent state, and provided specific protein expression in tumor cells and macrophages after intratumoral injection, in sharp contrast to the indiscriminate distribution and expression of LNP@mRNA in tumor tissues and normal organs. Tumor-limited distribution and protein expression not only maximize therapeutic effect, but also minimize adverse reactions to normal organs caused by mRNA and carriers.

[0097] Cytokines, as important mediators of cell communication, can provide direct anti-proliferative activity against tumor cells or indirect cytotoxic activity through stimulation of immune cells. Although two cytokines (interferon-a and IL-2) have been approved for the treatment of hairy cell leukemia, advanced renal cell carcinoma and metastatic melanoma, they usually show slight clinical benefits, mainly due to short half-life and severe systemic adverse toxicity. The present invention effectively solves this problem, MILP@mIL-12 has more than 9 times higher protein expression in tumor than in liver, which is in sharp contrast to the LNP@mIL-12 formulation under the same conditions, showing comparable IL-12 levels in tumor and liver. Importantly, MILP@mIL-12 produces much less IL-12 in blood than LNP@mIL-12 (250 vs 1500 pg / mL), and has little change in body weight, normal liver and kidney function and tissue structure, indicating that MILP@mIL-12 is less likely to cause systemic immunotoxicity. At the same time, MILP@mIL-12 significantly reshapes the tumor immune environment by inducing infiltration and activation of effector T cells and DCs, secretion of IFN-g and inhibition of the presence of immunosuppressive cells (Tregs, MDSCs), showing significant therapeutic potential for subcutaneous LLC and 4T1 tumor xenografts.

[0098] Further, in mice carrying subcutaneous tumor xenografts, MILP@mIL-12 combined with aPD-1 provides the greatest tumor growth inhibition and significantly prolonged survival time, the combination of cytokines and immune checkpoint therapy can produce a synergistic anti-cancer mechanism and maximize the therapeutic effect.

Claims

1. A multivalent ionizable lipid-peptide, the chemical structural formula of which is as follows: ； in, m is 0–10, n is 10–80, and z is 0.1–0.8; Ny is a group containing tertiary amines; the tertiary amine is a linear or cyclic tertiary amine, and the number of carbon atoms in the group containing tertiary amines is 5 to 10; Cx represents an alkyl group, which can be linear or cyclic; the number of carbon atoms in the alkyl group is 3 to 20. z represents the mole fraction of alkyl groups.

2. The multivalent ionizable lipid-peptide according to claim 1, characterized in that, m ranges from 1 to 8.

3. The method for preparing the multivalent ionizable lipid-peptide according to claim 1 includes the following steps: using poly(β-benzyl-L-aspartic acid), alkylamine compounds, and amine compounds containing tertiary amine groups as raw materials, the above-mentioned multivalent ionizable lipid-peptide is prepared by reaction.

4. The method for preparing multivalent ionizable lipid-peptide according to claim 3, characterized in that, The reaction time was 5–50 hours; the reaction temperature was 0℃–room temperature; poly(β-benzyl-L-aspartic acid) was prepared using amine compounds and β-benzyl-L-aspartic acid as raw materials; the chemical structural formula of the poly(β-benzyl-L-aspartic acid) is as follows: 。 5. A multivalent ionizable lipid-peptide-nucleic acid complex, comprising the multivalent ionizable lipid-peptide of claim 1 and nucleic acid.

6. A method for preparing the multivalent ionizable lipid-peptide-nucleic acid complex according to claim 5, comprising the following steps: mixing the above-mentioned multivalent ionizable lipid-peptide and nucleic acid in a solvent to obtain the above-mentioned multivalent ionizable lipid-peptide-nucleic acid complex.

7. The method for preparing the multivalent ionizable lipid-peptide-nucleic acid complex according to claim 6, characterized in that, The mass ratio of multivalent ionizable lipids, polypeptides, and nucleic acids is (1-50):

1.

8. A lyophilized powder obtained by lyophilizing the multivalent ionizable lipid-peptide nucleic acid complex of claim 5.

9. A drug whose active ingredient comprises the multivalent ionizable lipid-peptide-nucleic acid complex of claim 5.

10. The use of the multivalent ionizable lipid-peptide of claim 1 in the preparation of a drug or nucleic acid carrier, or in the preparation of a drug.

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