Multivalent ionizable lipid-polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a multivalent ionizable lipid-polypeptide as well as a preparation method and application thereof, poly (beta-benzyl-L-aspartic acid), an alkylamine compound and an amine compound containing a tertiary amine group are taken as raw materials, the multivalent ionizable lipid-polypeptide is prepared through reaction, and stable mRNA loading and tumor specific transfection are realized. The MILP effectively encapsulates mRNA and plasmid DNA into nano-composites (MILP (at) mRNAamp; MILP (at) pDNA) through the synergistic effect of electrostatic, hydrophobic and hydrogen bond interaction, and the composites show stability for freeze-drying and long-term storage. Compared with a lipid nano formula based on SM-102 lipid, the MILP (at) mRNA compound disclosed by the invention has the advantages that efficient cellular uptake, remarkable endosome escape ability and efficient transfection in various cell types are realized. The invention provides an innovative tumor specific transfection strategy, the application range of mRNA treatment is expected to be greatly expanded, and an application direction is provided for precise medical treatment.

Description

Technical Field

[0001] The present invention belongs to biomedical technology and relates to a nucleic acid delivery vector, specifically a multivalent ionizable lipid-polypeptide and a preparation method and application thereof, which can be used for tumor-targeted delivery of mRNA. Background Art

[0002] Messenger RNA (mRNA) technology has shown great potential in the prevention and treatment of 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, AJ; Jiang, AY; Zhang, P.; Wooster, R.; Anderson, DG, 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, KS; Goedel, A.; Chien, KR, Unlocking the promise of mRNA therapeutics, Nat. Biotechnol. 40 (2022) 1586-1600], the successful implementation of mRNA therapy depends largely 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 effective 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 nonspecific delivery characteristics limit the therapeutic effect and may cause non-target organ toxicity such as hepatitis and liver damage [Loughrey, D.; Dahlman, JE, Non-liver mRNA Delivery, Acc. Chem. Res. 55 (2022) 13-23]. In order to achieve non-liver delivery of mRNA drugs, researchers have adopted a variety of 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 LNPs, cationic polymers such as poly(β-amino ester) [PBAE], charge-variable release transporters (CARTs), polyasparagine, and poly(TPAE-co-butyryl chloride) have also been developed for extrahepatic delivery of mRNA, and selective protein expression in the lung and spleen has been achieved by adjusting the charge ratio of PEG-lipid, mRNA to polymer, and alkyl chain length and molar ratio [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, EP; 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., AMultidimensional 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, Delivery to SplenicT Cells, Angew. Chem. Int. Ed. 63 (2024) e202405444. Cheng, Q.; Wei, T.; Farbiak, L.; Johnson, LT; (2020) 313-320. Dilliard, SA; Cheng, Q.; Siegwart, DJ, On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles, Proc. Natl. Acad. Sci. USA 118 (2021) e2109256118]. Local regional drug delivery, especially intratumoral drug delivery, has become an important strategy for cancer treatment because it can bypass physiological barriers, improve efficacy and reduce non-target organ toxicity. 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 concentrations and improve efficacy, the side effects caused by the distribution of nanoparticles in other organs and mRNA expression further limit their clinical application [Bitounis, D.; Jacquinet, E.; Rogers, MA; Amiji, MM, 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 of the invention

[0003] The present invention develops a multivalent ionizable lipid-peptide (MILP) to achieve stable mRNA encapsulation and tumor-specific transfection. MILP can be synthesized by a one-step aminolysis reaction of poly(β-benzyl L-aspartic acid) with small molecules bearing alkanes (Cx) and tertiary amines (Ny), and its hydrophobicity, pKa and functionality can be adjusted by changing the structure, length and ratio of the selected alkanes and tertiary amines. MILP has excellent biocompatibility, multifunctional structure and function, and can be widely used in the fields of drug delivery and regenerative medicine. Utilizing multivalent electrostatic, hydrophobic and hydrogen bonding interactions, MILP alone can achieve stable mRNA complexation, cellular uptake, endosomal disruption, tumor retention and distribution with strong affinity. In mice bearing subcutaneous tumor xenografts, MILP@mIL-12 induced specific distribution and expression of mRNA at the tumor site, reshaped the tumor immune environment and produced significant anti-tumor responses.

[0004] The present invention adopts the following technical scheme.

[0005] A multivalent ionizable lipid-peptide, the chemical structure of which is as follows:

[0006] Wherein, m is 0 to 10, n is 5 to 100, and z is 0 to 1 excluding 0; preferably, m is 1 to 8, n is 10 to 80, and z is 0.1 to 0.8; more preferably, m is 2 to 7, n is 20 to 50, and z is 0.3 to 0.7; the slash represents random copolymerization, which is a conventional representation method; Wherein, Ny is a tertiary amine-containing group, Cx is an alkyl group, and z represents the molar fraction of the alkyl group.

[0007] Preferably, in the tertiary amine-containing group, the tertiary amine is a linear or cyclic tertiary amine; and the alkyl group is a linear or cyclic alkyl group.

[0008] More preferably, the tertiary amine-containing group has 1 to 10 carbon atoms, and the alkyl group has 1 to 30 carbon atoms.

[0009] More preferably, the tertiary amine-containing group has 2 to 8 carbon atoms, and the alkyl group has 3 to 20 carbon atoms.

[0010] The present invention discloses a method for preparing the above-mentioned multivalent ionizable lipid-polypeptide, comprising the following steps: using poly (β-benzyl-L-aspartic acid), an alkylamine compound, and an amine compound containing a tertiary amine group as raw materials, and reacting to prepare the above-mentioned multivalent ionizable lipid-polypeptide.

[0011] In the present invention, in the alkylamine compound, the alkyl group is a linear or cyclic alkyl group. Preferably, the alkylamine compound has 1 to 30 carbon atoms, and more preferably, has 3 to 20 carbon atoms.

[0012] In the present invention, in the amine compound containing a tertiary amine group, the tertiary amine is a linear or cyclic tertiary amine; preferably, the amine compound containing a tertiary amine group has 1 to 10 carbon atoms; more preferably, the tertiary amine group has 2 to 8 carbon atoms.

[0013] In the present invention, the reaction time is 5 to 50 hours, preferably 10 to 40 hours, and more preferably 15 to 30 hours; the reaction temperature is 0° C. to room temperature.

[0014] In the present invention, poly(β-benzyl-L-aspartic acid) is prepared using amino compounds and β-benzyl-L-aspartic acid as raw materials; the chemical structure of the poly(β-benzyl-L-aspartic acid) is as follows:

[0015] Wherein, the selection of m and n refers to the above-mentioned multivalent ionizable lipid-peptide.

[0016] The invention discloses a multivalent ionizable lipid-polypeptide nucleic acid complex, comprising the multivalent ionizable lipid-polypeptide and nucleic acid.

[0017] In the present invention, nucleic acid includes RNA and DNA; as an example, RNA includes mRNA.

[0018] The present invention discloses a multivalent ionizable lipid-peptide and a preparation method and application thereof. The multivalent ionizable lipid-peptide is prepared by reaction using poly(β-benzyl-L-aspartic acid), an alkylamine compound, and an amine compound containing a tertiary amine group as raw materials. mRNA drugs show great potential in the treatment of infectious diseases, oncology, and rare diseases. However, the extrahepatic delivery of mRNA is still a great challenge. The present invention discloses a novel multivalent ionizable lipid-peptide (MILP) to achieve robust formation of mRNA complexes and tumor-specific transfection. MILP effectively encapsulates mRNA and plasmid DNA into 80-nanometer nanoparticles through the synergistic effects of electrostatic, hydrophobic, and hydrogen-bond interactions, and these particles show stability to freeze-drying and long-term storage. Compared with the lipid nanoparticle formulation (LNP@mRNA) based on SM-102 lipids, the MILP@mRNA complex of the present invention achieves more efficient cellular uptake, significant endosomal escape ability, and efficient transfection in multiple cell types. Of particular note, MILP@mLuc exhibits unique tumor retention and distribution properties due to its multivalent-guided strong affinity and transcellular transport capabilities. After intratumoral injection, MILP@mLuc achieved specific protein expression in tumor cells and macrophages, in sharp contrast to the nonspecific distribution and expression of LNP@mLuc in major organs such as tumors and liver. In addition, MILP@mIL-12 induced significant tumor microenvironment remodeling and antitumor immune responses 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 that is expected to greatly expand the application range of mRNA therapy and provide new possibilities for future precision medicine.

[0019] The present invention discloses a method for preparing the above-mentioned multivalent ionizable lipid-polypeptide nucleic acid complex, comprising the following steps: mixing the above-mentioned multivalent ionizable lipid-polypeptide and nucleic acid in a solvent to obtain the above-mentioned multivalent ionizable lipid-polypeptide nucleic acid complex.

[0020] In the present invention, the solvent includes a buffer solution.

[0021] In the present invention, the mass ratio of multivalent ionizable lipid-polypeptide to nucleic acid is (1-50):1; preferably, the mass ratio of multivalent ionizable lipid-polypeptide to nucleic acid is (5-20):1.

[0022] Preferably, the method for preparing the above-mentioned multivalent ionizable lipid-polypeptide nucleic acid complex comprises the following steps: vortexing the above-mentioned multivalent ionizable lipid-polypeptide and nucleic acid in a solvent, and then incubating them statically to obtain the above-mentioned multivalent ionizable lipid-polypeptide nucleic acid complex.

[0023] The present invention discloses a lyophilized powder obtained by lyophilizing the above-mentioned multivalent ionizable lipid-polypeptide nucleic acid complex. The specific lyophilization method is a conventional technique and can be carried out in the presence of a lyophilization protective agent.

[0024] The present invention discloses a medicine, the active ingredient of which includes the above-mentioned multivalent ionizable lipid-polypeptide nucleic acid complex; further, the above-mentioned multivalent ionizable lipid-polypeptide nucleic acid complex of the present invention can be used alone as an active ingredient to prepare the medicine, and can also be used together with other agents with pharmaceutical activity as an active ingredient to prepare the medicine.

[0025] As is common sense, the above-mentioned drugs may include, in addition to active ingredients, pharmaceutically acceptable carriers, specifically according to conventional techniques.

[0026] The invention discloses the application of the multivalent ionizable lipid-polypeptide nucleic acid complex in the preparation of medicines.

[0027] The invention discloses the application of the multivalent ionizable lipid-polypeptide nucleic acid complex in the preparation of anti-tumor drugs.

[0028] The invention discloses the application of the multivalent ionizable lipid-polypeptide in the preparation of medicine or nucleic acid carrier.

[0029] In the present invention, tumors include solid tumors, such as lung cancer, breast cancer, melanoma, etc.

[0030] In the present invention, multivalent ionizable lipid-peptides (MILPs) are synthesized simply for robust mRNA complexation and tumor-selective transfection. By mimicking multivalent electrostatic, hydrophobic, and hydrogen bonding interactions in numerous biochemical processes, MILPs provide strong affinity for complexation, cell binding, and endosomal disruption of nucleic acids (mRNA, DNA). The complexes formed with mRNA (MILP@mRNA) showed high stability for long-term storage and lyophilization, 8-fold higher cellular uptake efficiency than existing SM-102 ionizable lipid-based LNPs, efficient transfection in different cells, and tumor-limited distribution and specific protein expression after intratumoral injection. In subcutaneous LLC and 4T1 tumor xenograft models, MILP@mIL-12 specifically induced IL-12 expression at the tumor site, significantly reshaped the tumor immune environment, and significantly enhanced antitumor activity, while significantly prolonging survival time. Therefore, MILPs are expected to broaden the application range of nucleic acid drugs by providing simulated biological multivalent interactions to achieve robust complexation, site-specific delivery, and efficient transfection of mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1Characterization of poly(β-benzyl-L-aspartic acid) (PBLA), including (A) H NMR spectrum (400 MHz, DMSO-d6), (B) matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, and (C) gel permeation chromatography.

[0032] Figure 2 Preparation and characterization of multivalent ionizable lipid-peptide (MILP)-mRNA complexes (MILP@mRNA). (A) Synthesis route of lipid-peptide and small molecule structure used to synthesize P(zCxNy), where “z” (0.4, 0.6) represents the mole fraction of alkane (Cx). (B) Complex formation of MILP with 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 from P(0.6C8cN6M). Scale bar, 100 nm. (F) Size changes 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 solutions (PBS + NaCl, urea and Tween 20) treated at 37°C (n = 3). (G) Percentage of GFP-positive cells in 293T cells after 24 h of MILP@mGFP treatment (mGFP: 1.0 μg / mL, n = 3). (H) Transfection efficiency of MILP11@mGFP and Lipo2000@mGFP after 24 h of incubation with different cells (mGFP: 1.0 μg / mL, n = 3). (IJ) Stability of MILP11@mGFP stored at 4°C (I) and lyophilized 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).

[0033] Figure 3 H NMR spectra (400 MHz, CDCl3 / CD3OD) of lipid-peptide P(0.4CxN6) and P(0.6CxN6).

[0034] Figure 4H NMR spectra (400 MHz, CDCl3 / CD3OD) of lipid-peptides (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).

[0035] Figure 5 is the mRNA encapsulation efficiency of different complexes (MILP@mGFP) (n = 3).

[0036] Figure 6 The results of different cells transfected with MILP11@mGFP for 24 hours (mGFP: 1.0 μg / mL, n = 3).

[0037] Figure 7 Results of 293T cells transfected with different complexes (MILP@pGFP) for 72 h (n = 3).

[0038] Figure 8 Cell viability of different cells after being treated with MILP@mGFP for 24 hours, mGFP: 1.0 μg / mL (n = 4).

[0039] Fig. 9 To comprehensively evaluate the correlation between the transfection efficiency of the complex MILP@pGFP and its cellular uptake and endosomal escape ability in vitro. (A) Characterization of cellular uptake and endosomal escape (n = 3). The uptake of different complexes by 293T cells was characterized by flow cytometry. The endosomal escape ability of the polymer was evaluated by hemolysis analysis of MILP at pH 5.5. Correlation analysis of the transfection efficiency of MILP@mGFP with cellular uptake efficiency (B) and endosomal escape ability (C). The Spearman method was used to evaluate the significance of the correlation. The statistically significant correlation data sets are represented by fitted lines. (n = 5).

[0040] Fig.10 Figure 2 shows the hemolysis of ionizable lipid-peptide (MILP) under different pH conditions (n ​​= 3).

[0041] Fig.11Figure 2. Biodistribution and expression of mRNA after intratumoral injection in a subcutaneous LLC model. (A) Bioluminescence imaging of mice at different time points using an IVIS imaging system (n = 3). (B) Ratio of fluorescence intensity between tumor and abdomen. SM-102-derived LNP@mLuc was used as a control. (C) In vitro imaging of the biodistribution (Cy5) and luciferase expression (Luc) of MILP11@mLuc and LNP@mLuc in tumors and major organs 6 hours after injection (mLuc-Cy5: 0.5 mg / kg). (D-E) Analysis of different cells (LLC cells, CD45 - CD44 + ; Macrophages (Mφ), CD45 + F4 / 80 + ) Cellular uptake of MILP11@mGFP (D) and mGFP expression (E) (n = 3). Tumors were dissected and analyzed by flow cytometry 12 h after intratumoral injection of MILP11@mGFP (mGFP: 0.5 mg / kg). (FG) Cellular uptake of MILP11@mLuc and LNP@mLuc by LLC cells at different time points. (mLuc-Cy5: 1.0 μg / mL, n = 3). Cy5 expression was measured by flow cytometry. + The percentage of LLC cells (F) and Cy5 + Mean fluorescence intensity (MFI) of LLC cells (G). (HI) Cellular transport of MILP11@mLuc-Cy5 and LNP@mLuc-Cy5 in LLC cells (n = 3). Flow cytometry analysis of Cy5 + Percentage of LLC cells (H) and Cy5 + MFI of cells (I).

[0042] Fig.12 MILP11@mIL-12 produces a strong antitumor effect in a subcutaneous LLC tumor xenograft model. (A) IL-12 secretion of LLC cells 24 hours after transfection with MILP11@mIL-12 and Lipo2000@mIL-12 (n = 3). (B) The ratio of IL-12 in tumor to liver after intratumoral injection of MILP11@mIL-12 and LNP@mIL-12 in LLC tumor-bearing mice, and (C) IL-12 expression in peripheral blood. Data are expressed as mean ± SD, n = 3. (D) Schematic diagram of the MILP11@mIL-12 and αPD-1 dosing regimen. When the tumor volume reached approximately 100 mm 3MILP11@mIL-12 and MILP11@mLuc were injected intratumorally (10 μg per mouse / per dose) at 4 pm, and αPD-1 (20 μg per mouse / per dose) was administered via the tail vein the next day. (EF) Curves of individual and average tumor volume changes over time (n = 6). (G) Kaplan-Meier survival curves of LLC tumor-bearing mice (n = 6).

[0043] Fig.13 MILP11@mIL-12 produced a strong antitumor effect in a subcutaneous 4T1 tumor xenograft model (n = 6). (A) Schematic diagram of the dosing regimen of MILP11@mIL-12 and αPD-1. When the tumor volume reached approximately 100 mm³, MILP11@mIL-12 was injected intratumorally (10 μg per mouse per dose) and αPD-1 (20 μg per mouse per dose) was administered via the tail vein the next day. (B) Curve of the mean volume of 4T1 tumors over time. (C) Kaplan-Meier survival curve of mice bearing 4T1 tumors. (D) Body weight changes of mice bearing 4T1 tumors after treatment, n = 6. Statistical analysis was performed using one-way ANOVA combined with Tukey's multiple comparison test. Survival was analyzed by the Kaplan-Meier technique combined with the log-rank (Mantel-Cox) test. (ns, not significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0044] Fig.14 MILP11@mIL-12 regulates the tumor microenvironment (TME) in LLC xenograft models. Figure 4 The regimen shown in D was used for administration. Two days after the last treatment, the tumors were dissected and the tumor tissues were digested to separate all cells for flow cytometry analysis. (A) Schematic diagram of the mechanism of MILP11@mIL-12 regulating TME (n = 5). (B) CD45 in live tumor cells + (C) The percentage of CD8 + Immunostaining of T cells (red). Scale bar: 50 μm. (D) CD45 + CD8 + The percentage of T cells. (E) CD8 + Activated T cells (CD25 + ) percentage. (F) CD8 + IFN-γ in T cells + The percentage of cells. (G) CD4 +Regulatory T cells (Tregs, FOXP3 + ) percentage. (H) MDSCs (CD11b + Gr-1 + ) percentage. (I) Ratio of M2 / M1 macrophages. (J) CD80 of DCs in TDLNs + (K) The percentage of MHCII+ cells in DCs in TDLNs. Statistical analysis was performed by one-way ANOVA and Tukey's multiple comparison test.

[0045] Fig.15 Schematic diagram of robust mRNA complex formation and tumor-limited transfection by multivalent ionizable lipid-peptides (MILPs). Utilizing multivalent electrostatic, hydrophobic, and hydrogen-bonding interactions, MILPs are able to robustly encapsulate mRNA into sub-100 nm MILP@mRNA nanoparticles. In the mildly acidic tumor microenvironment (TME), MILP@mRNA is partially protonated and exhibits unique tumor retention and distribution properties through multivalent-directed strong affinity and transcellular transport. Subsequently, efficient endocytosis and endosomal escape of MILP@mRNA are achieved through multivalent perturbations of cell and endosomal membranes, resulting in robust mRNA transfection. DETAILED DESCRIPTION

[0046] Advances in mRNA-based therapeutics 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 enhance immune responses. However, LNP systems still face several challenges, such as non-selective delivery, potential adverse reactions caused by PEG and cationic lipids, complex compositions based on four lipids, and unsatisfactory stability, which requires ultra-low temperature transportation and storage. The present invention discloses multivalent ionizable lipid-peptides (MILPs) based on polyasparagine derivatives to achieve robust complexation and tumor-selective transfection of mRNA. The present invention uses different tertiary amines and alkanes in different ratios to form nucleic acid complexes in combination with electrostatic and hydrophobic interactions to achieve the hydrophobicity, pKa and functionality of lipid-peptides. After screening, the best performing lipid-peptide P (0.6C8cN6M) is called MILP11, which effectively encapsulates mRNA or DNA and provides in vitro transfection efficiency comparable to commercial Lipo2000, indicating that MILPs can effectively deliver nucleic acids to the cytoplasm and nucleus. In particular, compared with LNPs prepared with four lipids, MILP@mRNA / MILP@pDNA is constructed with one component by simple vortexing, facilitating 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 remarkable 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.

[0047] The following is a detailed description of the technical progress of the present invention. The reagents used are existing products, and the specific preparation operations and performance tests are all conventional techniques. Unless otherwise specified, the present invention is carried out in a conventional environment. The modeling and statistical analysis methods of the present invention are existing techniques. The data are expressed as mean ± SD and analyzed using Prism 8.1. Unless otherwise specified, comparisons between multiple groups were performed using one-way analysis of variance and Tukey's post-test. The significant differences between the groups were determined by one-way analysis of variance (Tukey's multiple comparison test). The Kaplan-Meier survival curves were analyzed by the log-rank test. NS, not significant; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Cells and animals: LLC cells and 4T1 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Six-week-old female C57BL / 6 mice (Charles River Laboratory Animal Technology Co., Ltd., Beijing) and six-week-old female Balb / c mice (Jihui Laboratory Animal Care Co., Ltd., Shanghai) were housed at Soochow University under pathogen-free conditions. All animal experiments were approved by the Institutional 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.

[0048] 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, Bio-Tianmei Biotechnology Co., Ltd.), D-luciferin potassium salt (MeiLun Biotechnology Co., Ltd.), and lysozyme tracking green DND-26 (YiSheng Biotechnology Co., Ltd.) were used as received. EGFP mRNA was purchased from Cynbio (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 BioCorporation, 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 instructions.

[0049] To a solution of MILP in ethanol (1.5 mg / mL, 2.0 mL) was added hydrochloric acid solution (0.1 M) in increments of 2.0 to 3.0 µL. The resulting pH was measured using a pH probe, and the pKa was determined using the semiequivalent point titration method.

[0050] CD spectra of MILP were measured using a J-815 CD spectrometer (JACS, Easton, MD, USA). P(0.6C8cN6M) was dissolved in NMP (100 mg / mL), diluted to 0.1 mg / mL in sodium acetate buffer (20 mM, pH 4.0), and dialyzed against water overnight in a 1.0 kDa MWCO cartridge. Then, a quartz sample cell with a pathlength of 1 mm was used to place the sample solution. Based on the measured apparent ellipticity, the average residual molar ellipticity was calculated as follows: Ellipticity ([θ], unit: deg cm 2 dmol -1 ) = (millimeter × average residue weight) / (path length, mm × peptide concentration, mg mL -1). CD spectra were measured at room temperature.

[0051] Nano-ITC was used to measure the interaction between MILP11 and mRNA. Briefly, MILP11 solution was injected from a syringe into a sample cell containing mRNA solution (1 injection of 1.0 μL, followed by 24 injections of 2.0 μL each). The interval between each injection was 120 seconds, the system temperature was set to 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 measured using cell membrane-mimicking phospholipid liposomes as a model. The process of titrating MILP11 into liposomes is similar to the process of titrating peptides into mRNA.

[0052] LLC cells were stained with LysoTracker Green DND-26 and DAPI to detect the subcellular localization and intracellular trafficking of MILP11@mRNA. 5 Cells were seeded into 35 mm glass dishes overnight and then incubated with MILP11@mLuc-Cy5 (mLuc-Cy5, 2.0 μg / mL) for different time points. After three washes with PBS, the endosomal / lysosomal organelles and nuclei of LLC cells were stained with LysoTracker Green DND-26 (20,000-fold diluted with PBS) and DAPI (5.0 μg / mL), respectively. The treated cells were imaged by CLSM, and the colocalization of mLuc-Cy5 with endosomal / lysosomal organelles was analyzed by ImageJ. The colocalization between the red (mLuc-Cy5) channel and the yellow (endosome marker) channel was measured by Pearson's correlation coefficient (n = 5).

[0053] All animal experiments were performed in accordance with protocols approved by the Experimental Animal Center and the Institutional Animal Care and Use Committee of Soochow University. To study mRNA expression, MILP11 (MILP11@mLuc) and LNP (LNP@mLuc) loaded with 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 10 minutes after intraperitoneal injection of D-luciferin using the IVIS imaging system. Then, the region of interest (ROI) was selected and the total flux was quantified using Living Image 4.3 software (PerkinElmer).

[0054] To study biodistribution, MILP11 (MILP11@mLuc-Cy5) and LNP@mLuc-Cy5 were injected intratumorally at a dose of 10 μg mRNA per mouse. 12 hours after injection, major organs and tumors were harvested and biodistribution analysis was performed using an IVIS imaging system.

[0055] Towards LLC tumors (approximately 100 mm 3 Mice with 10 μg mRNA were injected intratumorally with MILP11@mIL-12 or LNP@mIL-12 at a dose of 10 μg mRNA per mouse. Then, the mice were divided into two groups (n = 3). One group was used for blood analysis, and 100 μL of blood was collected from the orbit at the specified time points, and plasma samples were collected after centrifugation at 1000 g for 5 minutes. The other group of mice was used for tissue analysis, and the mice were killed at the specified time points, and organs and tumors were collected. Tissue samples were ground in tissue protein extraction reagent (RIPA lysis buffer containing 1% PMSF), and the supernatant was collected after centrifugation at 10,000 g for 10 minutes. The concentration of IL-12 in plasma and tissue samples was determined using an ELISA kit.

[0056] The in vivo antitumor effect of MILP11@mIL-12 was evaluated in the LLC model and 4T1 breast cancer model. The LLC model was established as described above. The LLC subcutaneous tumor model was established by adding 1×10 6 LLC cells (PBS plus 30 volume fraction of matrigel) were subcutaneously injected into the right abdomen of C57 mice (female, 6 weeks old). The 4T1 breast cancer model was established by injecting 5×10 5 4T1 cells (PBS plus 30 volume fraction of Matrigel) were subcutaneously injected into the right abdomen of Balb / c mice (female, 6 weeks old). When the tumor volume reached 75∼100 mm 3 At day 0, mice were randomly divided into groups (n = 6) and treated with PBS, mIL-12, αPD-1, MILP11@mIL-12, or MILP11@mIL-12+αPD-1. The body weight and tumor volume of mice were measured every 2 or 3 days. The tumor volume was calculated as follows: tumor volume = 1 / 2 × longest diameter × (shortest diameter) 2. Mice were considered dead when the tumor volume increased to 1500 mm3 or when the mice died during treatment.

[0057] LLC tumor-bearing mice (approximately 100 mm) were treated with PBS, mIL-12, αPD-1, MILP11@mIL-12, and MILP11@mIL-12+αPD-1 (mIL-12: 10 μg per mouse, αPD-1: 20 μg per mouse). 3 ) mice (n = 5). Three days after the last treatment, tumor tissues and TDLNs were harvested, ground, centrifuged to obtain single-cell suspensions, and erythrocytes were lysed with erythrocyte lysis buffer (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 the corresponding antibodies for 30 minutes. All samples were analyzed by flow cytometry (BD FACSVerse), and the data were processed by FlowJo version 10. Example 1 MILP preparation

[0058] Multivalent ionizable lipid-peptides (MILPs) are synthesized by a one-step aminolysis reaction of poly(β-benzyl-L-aspartic acid) (PBLA) with small molecules containing linear or cyclic tertiary amines (Ny) and alkanes (Cx). The product is named P(zCxNy), where z represents the molar fraction of the alkane.

[0059] PBLA was synthesized by ring-opening polymerization of BLA-NCA using n-butylamine as 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 the polymerization reaction was carried out at 37°C in a nitrogen environment 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 1 The integrated area ratio of the proton signals of phenyl (-COOCH2C6H5, δ = 7.2-7.3) and methyl (CH3(CH2)3NH, δ = 0.8) in the H NMR spectrum was used to calculate the degree of polymerization of PBLA to be 31 with a narrow molecular weight distribution (Mw / Mn = 1.11). 1 The product was characterized by H NMR ( Figure 1A ), MALDI-TOF ( Figure 1B ), and GPC ( Figure 1C ).

[0060] MILP is synthesized by aminolysis of PBLA. Taking the synthesis of P(0.6C8cN6M) as an example, 50 mg of PBLA was dissolved in NMP and cooled to 4°C. Then, the PBLA solution was added dropwise to a mixture of 2-cyclohexylethylamine (C8c) (267.0 mg, 2.1 mmol) and 3-(2-methyl-1-piperidinyl)propylamine (N6-1) (226.0 mg, 1.4 mmol). The mixture was stirred at 4°C for 24 hours under nitrogen, precipitated in diethyl ether, and dried overnight under vacuum.

[0061] Other MILP and P(N6M) polymers were synthesized using the same synthetic scheme.

[0062] The synthesis method of P(N6-1) refers to that of P(zCxNy), and the preparation method of P(N6-1)@mRNA refers to that of MILP)@mRNA.

[0063] Figure 2 A shows the structures of Ny and 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 in the H NMR spectrum completely disappeared, which was attributed to the benzyl group. At the same time, the characteristic signals at 0.9-1.7 ppm were clearly visible. These results confirmed the successful construction of the P(zCxNy) peptide ( Figure 3 and Figure 4 ).

[0064] Table 1 Characterization of MILP polymers

[0065] Example 2: mRNA complex formation 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 obtained 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 seconds, and then incubated at room temperature for 20 minutes to obtain MILP@mRNA or P(N6M)@mRNA.

[0066] Referring to the above method, mRNA was replaced with pDNA to obtain MILP@pDNA.

[0067] Unless otherwise specified, the mRNA used in the following tests is mGFP and the DNA is pGFP, with a mass ratio of 20 / 1.

[0068] In the in vivo study, MILP@mRNA was dialyzed against cold PBS at 4°C for 120 min in a dialysis bag with 1000 kDa MWCO, and the mRNA encapsulation efficiency was quantified using the Quant-iT RiboGreen RNA Assay Kit ( n =3).

[0069] MILP@mRNA was mixed with sucrose as a cryoprotectant (final concentration of 3%-12% (w / v)), snap-frozen in liquid nitrogen, and then processed in a freeze-drying system (Christ ALPHA 1-2 LD plus). The lyophilized MILP@mRNA was stored at -80°C until use. When used, the lyophilized NPs were resuspended in sterile water to restore their original concentration.

[0070] Complexation of MILP with mRNA was achieved by simple vortexing in sodium acetate buffer (AcNa, pH 4.0) ( Figure 2 B). The average diameter of the formed MILP@mRNA complex is less than 80 nanometers and has a positive zeta potential ( Figure 2 C). Specifically, the complex formed by P(0.6C8cN6M) (denoted as MILP11@mRNA) exhibited a small size of approximately 60 nm, monodispersity, and spherical morphology, which were assessed by conventional DLS and TEM measurements ( Figure 2 D, Figure 2 E). The mRNA encapsulation efficiency is about 90% ( Figure 5 ).like Figure 2 As shown in F, the addition of Tween 20, NaCl, or urea to the complex resulted in a gradual increase in the particle size of MILP@mRNA, while the combined use of these reagents resulted in a more than tenfold change in particle size ( Figure 1 F), which supports the occurrence of hydrophobic (Tween 20), ionic (NaCl), and hydrogen bonding (urea) interactions during the complexation process. In contrast, the complex formed by P(N6-1) and 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: 4.2×10 8 vs 1.0×10 8 ; Ka2: 2.9×10 6 vs 2.4×10 5) (Table 2), indicating that multiple strong interactions occurred between P(0.6C8cN6M) and mRNA. Circular dichroism (CD) spectra showed that P(0.6C8cN6M) was an α-helical structure. In contrast, the MILP@mRNA complexes treated with proteinase K (12.0 units / mL) showed significant swelling within 12 hours, with an average size of approximately 600 nm, indicating that they were enzymatically degradable.

[0071] Table 2 Binding constants of P(0.6C8cN6M) or P(N6M) and mRNA

[0072] Example 3 In vitro performance of MILP The transfection effect of MILP@mRNA was evaluated by fluorescence microscopy and flow cytometry in 293T, LLC, U87-MG, and SKOV3 cells using mRNA and pDNA encoding GFP (mGFP and pGFP) as reporter genes. Cells were cultured in DMEM containing 10% FBS at a density of 50,000 to 70,000 cells / well in 48-well plates. 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, and cells were cultured in fresh medium and observed by fluorescence microscopy at different time points (24, 48, and 72 h). After adding 0.25% trypsin-EDTA and 0.5 mL PBS, the cells were collected by centrifugation and resuspended in 0.3 mL PBS for flow cytometric analysis.

[0073] The transfection efficiency of MILP complexed with mRNA encoding green fluorescent protein (GFP) (MILP@mGFP) was evaluated in 293T cells, as shown by confocal microscopy imaging and flow cytometry ( Figure 2G), most P(0.6CxNy)s had an alkane mole fraction of 0.6 and showed more than 60% GFP-positive cells, in sharp contrast to the low transfection efficiency (<30%) of the P(0.4CxNy) group. In addition, higher GFP expression could be obtained by increasing the alkane chain length from C8 to C12 in P(0.4CxNy), but P(0.6CxNy) formed with C12 alkanes showed poor solubility and was not suitable for complex formation with nucleic acids. Notably, P(0.6C8cN6) showed superior protein expression in the P(0.6CxN6) group, indicating that appropriate hydrophobicity and structure play an important role in mRNA transfection. Further, P(0.6C8cN6M) with C8c as the alkane presented the best transfection efficiency with mRNA complex, approximately 85% GFP-positive cells, close to Lipo2000 and 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.

[0074] The transfection capacity of MILP11@mGFP in cancer cells was evaluated. Notably, a transfection efficiency of about 80% was achieved in different cancer cells (LLC, U87, SKOV3), with human U87 and SKOV3 representing about 95% of GFP-positive cells at a MILP11 / mRNA ratio of 20 / 1, respectively ( Figure 2 H). Even at a low MILP11 / mRNA mass ratio of 5 / 1 (N / P ~ 2.5 / 1), MILP11@mGFP provided a good transfection efficiency of 80% and 60% in U87 and SKOV-3 cells, respectively ( Figure 6 At the same time, after long-term storage (5 weeks) at 4°C, MILP11@mGFP still induced 70% of GFP-positive cells in 293T cells ( Figure 2 I) and 75% MFI, in stark contrast to the fact that there was little transfection of MC3-LNP@mGFP after one week of storage under the same conditions.

[0075] In addition, the stability of MILP11@mGFP after freeze-drying was evaluated using sucrose as a protective agent. When the sucrose concentration was greater than 3% (m / v), the transfection efficiency of freeze-dried MILP11@mGFP was comparable to that of fresh samples ( Figure 2 J). In addition, the size and zeta potential of freeze-dried MILP11@mGFP showed little change at low sucrose concentrations of 3–6%, whereas obvious nanoparticle aggregation was observed when the sucrose concentration was increased to 12%.

[0076] The maintenance of mRNA activity after long-term storage and freeze-drying by MILP11@mRNA in the present invention will greatly promote the transportation and clinical application of mRNA drugs.

[0077] In addition, MILP was able to effectively compact pDNA, and the resulting MILP@pGFP nanoparticles showed comparable transfection efficiency to that of Lipo2000 complexes in 293T cells ( Figure 7 ), indicating that MILP achieved efficient intranuclear delivery of pDNA.

[0078] The viability of different cells treated with MILP@mRNA was evaluated by CCK8 assay. LLC, U87-MG or SKOV3 cells were seeded into 96-well plates at a density of 15,000 to 20,000 cells / well in DMEM medium containing 10% FBS and cultured with MILP@mGFP (1.0 μg / mL, n = 4). After 6 hours of culture, the medium was removed and the cells were cultured in fresh medium for another 18 hours, and then 10 μL of CCK8 solution was added and cultured for another 3 hours.

[0079] Therefore, MILP has become a versatile platform for the efficient delivery of different nucleic acids, including mRNA and DNA, in cells. Importantly, different cell types (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.

[0080] Example 4 MILP-mediated cellular uptake and endosomal escape The efficiency of cellular uptake of MILP@mRNA was evaluated using 293 T cells and LLC cells. Cells were seeded at a density of 100,000 cells / well in 24-well plates for 12 hours and then incubated with MILP@mLuc-Cy5. At intervals, cells were rinsed twice with PBS, trypsinized, and centrifuged at 1,000 rpm for 3 minutes. The collected cells were resuspended in 0.3 mL PBS and analyzed by flow cytometry, n = 3. See Fig. 9A, Compared with MILP@mRNA formed by P(0.4CxN6), nanoparticles based on P(0.6CxN6) showed significantly higher cellular uptake, the C8 alkane group showed more uptake in 293T cells, while cyclic C8c was superior to linear C8 alkane, and lipid-peptides with 6- and 7-membered tertiary amines (N6, N6M, N6P, and N7) achieved better cellular uptake. Interestingly, the correlation analysis between transfection efficiency and cellular uptake efficiency showed that mRNA transfection was significantly positively correlated with uptake efficiency (r = 0.74 and P = 0.0002) ( Fig. 9 B).

[0081] Red blood cells (RBCs) were isolated from whole blood freshly collected from mice, centrifuged at 3000 rpm for 5 min, and then washed five times with PBS buffer. Then, RBCs were suspended 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 a 96-well plate. After incubation at 37 °C for 1 hour, centrifugation was performed at 3000 rpm for 5 min. The released hemoglobin content was measured at a wavelength of 540 nm using a microplate reader (n = 3). RBC suspensions incubated in PBS and Triton X-100 solution (1.0 wt.%) were used 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 ​​of RBC after MILP, Triton and PBS treatment are shown respectively. The ability of the carrier to damage the membrane was evaluated by hemolysis test, which further reflects the efficiency of endosomal escape of different carriers. Under acidic conditions (pH 5.5), P(0.6CxN6) with more hydrophobic segments showed significantly greater hemolysis than P(0.4CxN6). The hemolysis ratio of P(0.6CxN6) with C6, C8 and C8c alkanes was 11-17% ( Fig. 9 A), ensuring their active escape from endosomes. At the same time, MILPs with different tertiary amines all showed obvious hemolysis, among which MILPs from N6, N6M and N7 showed a hemolysis ratio of about 15%. It is worth noting that endosomal escape (reflected in the hemolysis ratio) is usually positively correlated with mRNA transfection, such as Fig. 9 C. Importantly, MILP generally showed less than 5% hemolysis at pH 7.4 ( Fig.10), confirming their blood compatibility under physiological conditions. The pH-dependent hemolytic switch mainly comes from the protonation of tertiary amines in lipid-peptides, with a pKa value of 6.3 to 7.0, which helps to build an efficient and safe mRNA delivery system.

[0082] Example 5 MILP@mRNA achieves tumor-specific biodistribution and expression The in vivo biodistribution and protein expression of MILP11@mLuc were investigated in a mouse model bearing LLC tumors. As a control group, LNP@mLuc composed of ionizable SM-102 lipids with an average particle size of approximately 100 nanometers and a neutral surface charge was used. In vivo bioluminescence imaging revealed that after intratumoral injection, MILP11@mLuc was expressed in LLC ( Fig.11 A, Fig.11 The expression of luciferase was specifically promoted in 4T1 tumors and 4T1 tumors, which was in sharp contrast to the dispersed distribution of luciferase fluorescence in the tumor and abdomen of the LNP@mLuc group. The tumor-specific protein expression of MILP11@mLuc was also clearly observed by in vitro fluorescence imaging technology ( Fig.11 C). Meanwhile, 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) ( Fig.11 C). Among the delivery vehicles currently in clinical use, lipid nanoparticles based on SM-102 or ALC0315 can detect non-specific distribution and expression of mRNA in normal organs (especially the liver) after local or systemic injection. Notably, after intramuscular and subcutaneous injection, MILP11@mLuc showed little protein expression, which was almost undetectable, confirming the low transfection efficiency of MILP11@mRNA in skin and muscle tissues. In addition, histological analysis showed that MILP11@mLuc did not induce cell apoptosis and necrosis in adjacent skin tissues. 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 tumors (approximately 90%). It is worth noting that approximately 48.5% of LLC cells (CD45 - CD44 + ), 55.7% of TAMs (CD45 + F4 / 80 + ) and 37.0% of total cells isolated from tumors were Cy5 positive ( Fig.11D), confirming the widespread distribution and efficient cellular uptake of MILP11@mRNA in tumors. Correspondingly, significant GFP expression was observed in LLC cells (25.7%) and TAMs (18.2%), while GFP expression in total cells was 7.8% and 1.2% respectively. + Fewer cells (17.8%), due to the low transfection efficiency of MILP11@mRNA in other cells ( Fig.11 E).

[0083] Example 6 In vivo anti-tumor activity of MILP@mIL-12 (4T1 and LLC tumor models) Interleukin 12 (IL-12) is a powerful proinflammatory cytokine that promotes T-cell proliferation and activation and effectively stimulates interferon-γ (IFN-γ) production, and is therefore considered a promising anticancer agent; unfortunately, the clinical application of IL-12 is limited by its short half-life, insufficient local concentration within tumors, and immune-related adverse events. The present invention uses a complex (MILP11@mIL-12) that encapsulates IL-12 mRNA to improve the immunosuppressive tumor environment. Notably, MILP11@mIL-12 significantly expressed IL-12 in LLC cells in vitro ( Fig.12 A), and IL-12 was enriched in the tumor within 6-12 hours after administration, and the protein expression in the tumor was more than 9 times higher than that in the liver ( Fig.12 B). In contrast, LNP@mIL-12 produced similar IL-12 levels in tumors and liver under the same conditions ( Fig.12 B). Importantly, MILP11@mIL-12 produced much less IL-12 in the blood than LNP@mIL-12 (250 vs 1500 pg / mL) ( Fig.12 C), indicating that MILP11@mIL-12 is unlikely to cause systemic immunotoxicity. At the same time, mice treated with MILP11@mIL-12 showed 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 mice bearing LLC tumor xenografts, MILP11@mIL-12 significantly inhibited tumor growth and greatly prolonged survival, while free mIL-12 had little effect on tumor suppression and mouse survival, indicating the key role of the MILP11 delivery system. In addition, non-therapeutic MILP11@mLuc had no significant inhibitory effect on tumor growth ( Fig.12 E. Fig.12 F. Fig.12G). Combined with αPD-1, the MILP11@mIL-12+αPD-1 group showed better effects in LLC tumor inhibition and survival rate than the MILP11@mIL-12 group alone, with 50% of mice surviving during the experiment, while no mice survived in the MILP11@mIL-12 group alone, with a median survival time (MST) of 25 days ( Fig.12 G). Similarly, the MILP11@mIL-12+αPD-1 group showed more significant tumor suppression and prolonged survival (MST 33 vs 29) in 4T1 tumor-bearing mice than the MILP11@mIL-12 group ( Fig.13 ). Therefore, intratumoral delivery of MILP11@mRNA has superior tumor suppression effect and good safety, and has significant potential in the treatment of various malignant tumors such as breast cancer, melanoma, and lung cancer.

[0084] Example 7 MILP@mIL-12-mediated Tumor Microenvironment Regulation The product of the present invention can also promote the production of IFN-γ, activate dendritic cells, and re-regulate the immunosuppressive TME ( Fig.14 A). After three doses of treatment, if Fig.12 As shown in D, tumors and adjacent tumor draining lymph nodes (TDLNs) were collected on day 9 for flow cytometry immune analysis. Both MILP11@mIL-12 and MILP11@mIL-12+αPD-1 significantly increased CD45 + cell( Fig.14 B) and CD8 + T cells ( Fig.14 C, Fig.14 D) Infiltration and activation of CD8 + T cells (CD8 + CD25 + T)( Fig.14 E) and IFN-γ + CD8 + T cells ( Fig.14 At the same time, after MILP11@mIL-12 or MILP11@mIL-12+αPD-1 treatment, the immunosuppressive cell populations in the tumor, including Tregs (CD4 + FOXP3 + ) and MDSCs (CD11b + Gr-1 + ) decreased significantly ( Fig.14 G, Fig.14H). Compared with free mIL-12, MILP11@mIL-12 promoted the polarization of macrophages toward the M1 phenotype and showed a significantly lower M2 / M1 ratio. In TDLNs, CD80 was detected in both MILP11@mIL-12 and MILP11@mIL-12+PD-1 groups. + Significant increase in DC and MHCII + A slight increase in DC ( Fig.14 J, Fig.14 K). These results suggest that MILP11@mIL-12 combined with α-PD-1 can enhance the infiltration of anti-tumor immune cells and improve the immunosuppressive TME.

[0085] 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 invention solves this problem. As an example, see Fig.15 In mice bearing subcutaneous LLC tumors, MILP@mRNA achieved tumor tissue selective retention and expression through multivalent-directed strong affinity and transcellular transport, and provided specific protein expression in tumor cells and macrophages after intratumoral injection, which was in sharp contrast to the nonspecific distribution and expression of LNP@mRNA in tumor tissues and normal organs. Tumor-limited distribution and protein expression can not only maximize the therapeutic effect, but also minimize the adverse reactions to normal organs caused by mRNA and carriers.

[0086] Cytokines, as important mediators of cell communication, can provide antiproliferative activity directly against tumor cells or indirect cytotoxic activity by stimulating immune cells. Although two cytokines (interferon-α and IL-2) have been approved for the treatment of hairy cell leukemia, advanced renal cell carcinoma, and metastatic melanoma, they generally show mild clinical benefits, mainly due to short half-lives and severe systemic adverse toxicity. The present invention effectively solves this problem, and the protein expression of MILP@mIL-12 in tumors is more than 9 times higher than that in the liver, which is in sharp contrast to the LNP@mIL-12 formulation that shows comparable IL-12 levels in tumors and liver under the same conditions. Importantly, MILP@mIL-12 produces much less IL-12 in the blood than LNP@mIL-12 (250 vs 1500 pg / mL), and there is little change in body weight, and liver and kidney function and tissue structure are normal, indicating that MILP@mIL-12 is unlikely to cause systemic immunotoxicity. Meanwhile, MILP@mIL-12 significantly reshaped the tumor immune environment by inducing the infiltration and activation of effector T cells and DCs, the secretion of IFN-γ, and inhibiting the presence of immunosuppressive cells (Tregs, MDSCs), showing significant therapeutic potential against subcutaneous LLC and 4T1 tumor xenografts.

[0087] Furthermore, in mice bearing subcutaneous tumor xenografts, MILP@mIL-12 combined with αPD-1 provided 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-polypeptide, the chemical structure of which is as follows: ； in, m is 0 to 10, n is 5 to 100, z is 0 to 1 and does not include 0; Ny is a group containing a tertiary amine, Cx is an alkyl group, and z represents the molar fraction of the alkyl group.

2. The multivalent ionizable lipid-polypeptide according to claim 1, characterized in that: m is 1 to 8, n is 10 to 80, and z is 0.1 to 0.8; in the group containing tertiary amine, the tertiary amine is a linear or cyclic tertiary amine; and the alkyl group is a linear or cyclic alkyl group.

3. The method for preparing the multivalent ionizable lipid-polypeptide according to claim 1, comprising the following steps: using poly (β-benzyl-L-aspartic acid), an alkylamine compound, and an amine compound containing a tertiary amine group as raw materials to react and prepare the multivalent ionizable lipid-polypeptide.

4. The method for preparing a multivalent ionizable lipid-polypeptide according to claim 3, characterized in that: The reaction time is 5 to 50 hours; the reaction temperature is 0°C to room temperature; the amino compound and β-benzyl-L-aspartic acid are used as raw materials to prepare poly(β-benzyl-L-aspartic acid); the chemical structure of the poly(β-benzyl-L-aspartic acid) is as follows: 。 5. A multivalent ionizable lipid-polypeptide nucleic acid complex comprising the multivalent ionizable lipid-polypeptide according to claim 1 and a nucleic acid.

6. The method for preparing the multivalent ionizable lipid-polypeptide nucleic acid complex according to claim 5 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.

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

1.

8. A lyophilized powder obtained by lyophilizing the multivalent ionizable lipid-polypeptide nucleic acid complex according to claim 5.

9. A drug, the active ingredient of which comprises the multivalent ionizable lipid-polypeptide nucleic acid complex according to claim 5.

10. Use of the multivalent ionizable lipid-polypeptide according to claim 1 in the preparation of a drug or a nucleic acid carrier, or in the preparation of a drug.

Images