Ionizable polymers, preparation of polymeric lipid hybrid nanoparticles and uses thereof

Abstract

The application discloses an ionizable polymer, a polymer-lipid hybrid nanoparticle and application, and belongs to the technical field of biological medicine. The ionizable polymer has a molecular structure as shown in formula (I): wherein n is an integer of 1-50. Through modular structure design, the charge state is regulated under different pH conditions, efficient loading and release of nucleic acids are completed. The polymer-lipid hybrid nanoparticle disclosed simultaneously can exhibit better in-vivo delivery capacity, tissue expression effect and local expression capacity, and thus has application potential in multiple drug delivery scenarios.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to an ionizable polymer, polymer-lipid hybrid nanoparticles, and their preparation and application. Background Technology

[0002] Nucleic acid drugs, especially messenger RNA (mRNA) therapy, have attracted widespread attention due to their enormous application potential in areas such as infectious disease vaccines, protein replacement therapy, immunotherapy, and gene editing. However, mRNA molecules themselves have inherent defects such as large molecular weight, susceptibility to degradation by nucleases, and difficulty in autonomously crossing cell membranes, requiring efficient and safe delivery systems to achieve their functional expression in target cells.

[0003] Currently, carrier materials used for nucleic acid delivery are mainly divided into two categories: viral carriers and non-viral carriers. Among non-viral carriers, cationic polymers and cationic lipids have become research hotspots due to their low immunogenicity and relatively flexible chemical designability. Cationic polymers can bind to negatively charged nucleic acid molecules through electrostatic interactions, compressing them into nanoscale composite particles, protecting nucleic acids from degradation and promoting cellular uptake. However, traditional cationic polymers often maintain a high density of positive charge on their surface under physiological pH conditions, leading to severe cytotoxicity, non-specific protein adsorption, and rapid in vivo clearance, significantly limiting their in vivo application efficacy.

[0004] To address these issues, the design concept of ionizable carrier materials has been proposed. Ideally, the delivery material should carry a positive charge under preparation conditions (such as a weakly acidic buffer) to achieve efficient binding and encapsulation of nucleic acids; however, under physiological pH conditions after entering the bloodstream, its positive charge should significantly decrease, and its surface charge should approach neutral, thereby reducing toxicity and non-specific interactions, and improving in vivo biocompatibility and circulation time. However, existing ionizable carrier materials still have significant room for improvement in terms of structural diversity, precise pH response tuning, and a comprehensive balance between in vivo delivery efficiency and safety.

[0005] Therefore, developing an ionizable carrier material that is charge-neutral under physiological conditions and is activated only in an acidic endosome environment is of great practical significance. Summary of the Invention

[0006] In view of this, the primary objective of this application is to provide an ionizable polymer that can be designed with diverse structures and possesses ionizable properties. Under acidic conditions, it can undergo protonation, enhancing its electrostatic interaction with mRNA and improving nucleic acid encapsulation capability; while under near-physiological conditions, its charge decreases, improving the biocompatibility of the system. In other words, it can regulate the charge state under different pH conditions, thereby achieving efficient encapsulation and release of nucleic acids.

[0007] To achieve the above objectives, this application adopts the following technical solution: One aspect of this application discloses a novel ionizable polymer having the molecular structure described in formula (I): , This ionizable polymer possesses diverse structural features, and its molecules exhibit pH-responsive charge-switching capabilities. Specifically, under acidic conditions, it undergoes protonation, enhancing the electrostatic interaction with mRNA and improving nucleic acid encapsulation capacity; while under near-physiological conditions, its charge decreases, improving the system's biocompatibility. This enables highly efficient encapsulation and in vivo delivery of nucleic acid molecules such as mRNA.

[0008] In equation (I), n is an integer from 1 to 50, for example, any integer from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a range between any two values. n represents the number of repeating units in the ionizable polymer, and its value directly affects the molecular weight and charge density distribution of the polymer. Those skilled in the art can design appropriate n values ​​within a defined range according to specific delivery requirements to achieve a balance between encapsulation efficiency, particle uniformity, and functionality of the ionizable polymer.

[0009] In formula (I), R1 acts as a linker or spacer in the polymer backbone. Choosing different structures for R1 can adjust the polymer's hydrophilicity / hydrophobicity, biodegradability, and chain flexibility. As an example, R1 is selected from one or more of the following: C2-C20 straight-chain or branched alkylene groups, polyether segments containing 1-10 oxygen atoms, C6-C18 aryl groups, C2-C20 alkylene groups containing thioether or disulfide bonds, C2-C20 alkylene groups containing ester bonds, and C2-C20 fluoroalkylene groups.

[0010] R2 is selected from one or more of the following: C1~C24 straight-chain or branched alkyl groups, C2~C24 alkenyl groups containing unsaturated bonds, C3~C12 cycloalkyl groups, C6~C30 steroidal groups, C1~C12 fluoroalkyl groups, and C6~C18 aryl groups. The R2 group mainly acts as a hydrophobic module, which provides hydrophobic driving force, promotes the assembly of hydrophobic cores during the formation of nanoparticles, enhances the structural stability of particles, and assists in hydrophobic membrane perturbation in the acidic environment of the endosome to promote the cytoplasmic release of nucleic acid molecules.

[0011] The R3 group is the core functional unit of the ionizable polymer, containing at least one tertiary amine group that can be protonated under acidic conditions, thereby endowing the polymer with pH-responsive charge-switching capabilities. Under the weakly acidic buffer conditions used to prepare nanoparticles, the tertiary amine group is protonated to form a positively charged ammonium cation, which interacts strongly with the negatively charged nucleic acid phosphate backbone, achieving efficient binding and tight compression. Under physiological pH conditions, the degree of protonation is significantly reduced, and the surface charge approaches neutral, thereby reducing toxicity and non-specific protein adsorption. As a specific example, R3 can be selected from one or more of the following: a C2-C12 alkylene group containing a tertiary amine, a polyether segment containing a tertiary amine structure, or a 5-12 member nitrogen-containing heterocyclic group containing 1-3 heteroatoms selected from N, O, and S; the nitrogen-containing heterocyclic group is preferably piperazine, morpholino, piperidinyl, N-alkylpiperazine, or their substituted derivatives. Preferably, R3 contains at least one protonable tertiary amine group.

[0012] The R4 group is located at the end of the polymer chain and can serve as a capping group for polymerization reactions. It can also be used to introduce specific functional end modifications, which can be designed according to actual needs. As an example, R4 is selected from one or more of the following: C1-C18 straight-chain or branched alkyl groups, C1-C8 hydroxy-substituted alkyl groups, C1-C8 amino-substituted alkyl groups, C1-C8 alkoxy-substituted alkyl groups, C3-C8 cycloalkyl groups, and 5-8 membered heterocyclic groups containing 1-3 heteroatoms selected from N, O, and S heteroatoms.

[0013] Furthermore, preferably, any one of the groups R1, R2, R3, and R4 may be optionally substituted with one or more substituents selected from halogens, hydroxyl groups, amino groups, mercapto groups, mercapto-substituents, carboxyl groups, ester groups, amide groups, ether bonds, thioether bonds, disulfide bonds, C1-C6 alkyl groups, C1-C6 alkoxy groups, C1-C6 fluoroalkyl groups, polyether segments, and heterocyclic groups. The physicochemical properties of the ionizable polymer can be finely tuned through substitution design, and the specific design can be tailored to actual needs without particular limitations.

[0014] Another aspect of this application discloses a method for preparing the aforementioned ionizable polymers. Specifically, through a modular design strategy, n, R1, R2, R3, and R4 can be independently selected and combined to rapidly construct a library of structurally diverse ionizable polymers. This structural extensibility facilitates systematic screening and optimization around key indicators such as delivery efficiency, tissue selectivity, and biosafety, and is beneficial for rapidly obtaining optimal polymer candidate molecules that meet the needs of specific therapeutic scenarios.

[0015] In this application, the synthetic route of the ionizable polymer is as follows: ; Its specific preparation includes the following steps: An ionizable polymer as shown in formula (I) was prepared from compounds A, B, C, D, and E via amidation, ring-opening, mercapto-Michael addition, and amine capping.

[0016] Specifically, in the synthesis process, compound B and compound C are first linked through an amidation reaction; then, under suitable conditions, compound B undergoes a ring-opening reaction with compound D, and undergoes a Michael addition reaction between a thiol group and an unsaturated carbonyl group with compound A, introducing compound D containing a tertiary amine group into the polymer backbone to form the R3 core functional unit; finally, through an amine end-capping reaction, compound E is introduced into the chain end, terminating the polymerization reaction and completing the modification of the terminal groups to obtain the target ionizable polymer.

[0017] As a preferred example, compounds A, B, C, D, and E are selected from the following library of ionizable polymers: .

[0018] The significant advantage of the modular synthesis method presented in this application lies in the fact that each structural unit can be independently replaced, thus allowing for flexible adjustment of the polymer's hydrophobic chain type, spacer group properties, tertiary amine structure, and terminal groups, enabling customized polymer synthesis and systematic screening. It should be understood that in practice, the temperature, solvent, catalyst, and purification method for each reaction step can be conventionally optimized based on the specific raw material type, and this application does not impose absolute limitations on these aspects.

[0019] Another aspect of this application discloses a composition for delivering mRNA, comprising an ionizable polymer as shown in formula (I) above and at least one auxiliary lipid. The efficient nucleic acid loading capacity of the ionizable polymer is organically combined with the in vivo stabilization function of the auxiliary lipid to form a synergistic delivery system.

[0020] In this application, assisting lipids refer to lipid molecules that, during the formation of nanoparticles, synergistically work with ionizable polymers to construct structurally complete and stable particulate formulations. As a typical example, assisting lipids are preferably selected from phospholipids, sterols, PEGylated lipids, or any combination thereof.

[0021] Phospholipids play a role in maintaining the structural integrity of particles. Their amphiphilic nature allows them to oriented and align at the aqueous-hydrophobic interface, forming a stable lipid layer framework. As a specific example, the phospholipid can be a conventional or self-developed phospholipid in the art, such as at least one of distearylphosphatidylcholine, dioleoylphosphatidylethanolamine, or similar compounds. In specific applications, a single type of phospholipid or a combination of two or more phospholipids can be selected as needed, and other phospholipid molecules with similar structures can also be chosen; this application does not impose specific limitations in this regard.

[0022] Sterols are used in particles to regulate the density and stability of the interfacial layer. Their rigid polycyclic skeletons are embedded between phospholipid molecules, filling gaps and enhancing interfacial density. As a specific example, sterols can be at least one of cholesterol or its derivatives, but are not limited thereto. It is understood that any sterols known in the art or independently developed can be used in this application, and no exhaustive list is made here.

[0023] PEGylated lipids are a class of amphiphilic molecules with polyethylene glycol chains covalently linked to lipid anchoring groups. They can extend from the particle surface via long hydrophilic PEG chains to form a sterically hindered hydration layer, reducing interparticle aggregation and non-specific adsorption of plasma proteins, prolonging in vivo circulation time, and improving the stability of the system in the physiological environment. As a specific example, the PEGylated lipid can be at least one of PEG-dimyristoylglycerol, PEG-distearatephosphatidylethanolamine, or their analogues, but is not limited to these. The number-average molecular weight of the PEG chain is typically between 500 and 5000 Daltons (Da), for example, any value from 500, 1000, 2000, 3000, 4000, 5000 Da or a range between two values. In a preferred embodiment, the PEG molecular weight is 2000 Da, at which point a better balance between shielding efficiency and cellular uptake can be achieved.

[0024] It should be understood that a suitable lipid ratio can impart better particle size uniformity, storage stability, and in vivo delivery efficiency to the particles. Those skilled in the art can adjust the ratio of each component in the auxiliary lipids according to actual research needs. In some specific examples, when phospholipids, sterols, and PEGylated lipids are present in the composition, the molar ratio of phospholipids, sterols, and PEGylated lipids is (5~25):(25~55):(0.1~5), preferably (10~20):(35~50):(1~3).

[0025] Another aspect of this application discloses polymer-lipid hybrid nanoparticles, comprising the composition described above and at least one mRNA.

[0026] This polymer-lipid hybrid nanoparticle employs a pre-polymerization followed by hybridization construction strategy: first, an ionizable polymer efficiently compresses mRNA molecules through electrostatic interactions, forming a dense polymer / mRNA nanocore; then, a hybrid lipid system is used to post-modify the surface of this core, forming a stable interfacial shell composed of phospholipids, sterols, and PEGylated lipids. This hybrid nanoparticle structure, with a polymer core and a lipid shell, balances the high loading capacity of the polymer system with the in vivo stability of the lipid system.

[0027] In this nanoparticle, the molar ratio (N / P ratio) between the protonable nitrogen atom in the ionizable polymer and the phosphate group of mRNA is from 1 to 50, for example, any value or a range between two values ​​from 1, 2, 3, 5, 8, 10, 15, 20, 25, 30, 40, 50. The tertiary amine nitrogen atom of the ionizable polymer becomes positively charged after protonation under weakly acidic conditions, and electrostatically binds to the negatively charged phosphodiester bond in the mRNA nucleotide unit. Therefore, the N / P ratio is a key parameter characterizing the degree of complexation between the ionizable polymer and mRNA. A suitable N / P ratio can be optimized and adjusted by those skilled in the art according to actual research needs. In some preferred embodiments, the N / P ratio is from 3 to 20, and more preferably, the N / P ratio is 20.

[0028] In this application, the polymer-lipid hybrid nanoparticles have an average particle size of 50 to 150 nanometers (nm) and a polydispersity index (PDI) of less than 0.3. The suitable particle size and polydispersity index indicate that the particles exhibit good batch-to-batch consistency and in vivo delivery reproducibility.

[0029] Furthermore, the polymer-lipid hybrid nanoparticles of this application exhibit a near-neutral surface potential (between -10mV and +10mV) under physiological conditions of pH 7.35 to 7.45. This near-neutral surface charge significantly reduces non-specific electrostatic adsorption between the particles and serum proteins, decreasing opsonin labeling and subsequent macrophage phagocytosis and clearance, thereby prolonging in vivo circulation time and increasing target tissue accumulation. The near-neutral physiological potential in this application is attributed to the unique molecular structure of the ionizable polymer, which is fully protonated under weakly acidic conditions and effectively deprotonated under physiological conditions.

[0030] Another aspect of this application discloses a method for preparing the polymer-lipid hybrid nanoparticles described above, comprising the following steps: (1) Dissolve or disperse mRNA and ionizable polymer in a weakly acidic buffer solution with a pH of 4.5 to 5.0 to form nucleic acid solution and polymer solution respectively.

[0031] The weakly acidic buffer solution includes, but is not limited to, acetate buffer, citrate buffer, or phosphate buffer. The aim is to ensure that the tertiary amine groups of the polymer are sufficiently protonated, carrying a sufficient positive charge, thus creating conditions for efficient electrostatic complexation with the negatively charged mRNA.

[0032] The concentration of the mRNA nucleic acid solution is typically set based on the target dosage and formulation size. In some specific embodiments, the concentration of mRNA in the weakly acidic buffer is 0.01 to 1.0 mg / mL, for example, any value or range between any two of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, and 1.0 mg / mL. Preferably, the mRNA concentration in the nucleic acid solution is 0.05 to 0.2 mg / mL. A suitable nucleic acid concentration range allows the nucleic acid molecules to be fully dispersed in the solution, which is beneficial for uniform complexation with polymers and avoids intermolecular entanglement or particle unevenness caused by excessive concentration. Those skilled in the art can choose according to their needs, and therefore there are no particular limitations.

[0033] The concentration of the ionizable polymer solution is prepared according to the target N / P ratio and the amount of nucleic acid used, as described above, and will not be elaborated here.

[0034] (2) At room temperature, the nucleic acid solution and polymer solution were mixed at a predetermined N / P ratio to ensure uniform contact between the mRNA and the ionizable polymer in the solution. Under electrostatic drive, the polymer spontaneously binds to the mRNA and compresses its conformation to form a polymer / mRNA nanocomplex. After mixing, an appropriate amount of weakly acidic buffer was added to adjust the total volume of the system to obtain an aqueous phase.

[0035] The specific mixing operation can be accomplished by simple vortex mixing, pipette blowing or microfluidic mixing, etc., and this application does not make specific limitations on this.

[0036] (3) Dissolve phospholipids, sterols and PEGylated lipids in an organic solvent in a predetermined ratio to form an organic phase of mixed lipids.

[0037] The organic solvent can be selected from commonly used solvents in the art, such as ethanol, isopropanol, methanol, or aqueous mixtures thereof. The purpose of selecting an organic solvent is to fully dissolve the lipid component and maintain its molecular-level dispersion so that it can be rapidly and uniformly deposited onto the polymer / mRNA core surface when subsequently mixed with the aqueous phase.

[0038] (4) The aqueous phase and the organic phase are thoroughly mixed so that the lipid components precipitate out when they come into contact with the aqueous phase due to the rapid change in solvent polarity, and are oriented and arranged on the core surface of the polymer / mRNA nanocomposite, self-assembling to form a stable interface layer composed of phospholipids, sterols and PEGylated lipids, thus obtaining polymer lipid hybrid nanoparticles.

[0039] The volume ratio of the aqueous phase to the organic phase is (1~10):1, for example, any ratio or a range between any two ratios from 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, to 10:1. Preferably, the volume ratio of the aqueous phase to the organic phase is 3:1 to 5:1. A suitable volume ratio range is beneficial for controlling the precipitation and self-assembly process of lipid components and promotes the formation of polymer-lipid hybrid nanoparticles with stable structure, uniform particle size, and good encapsulation properties. Those skilled in the art can determine the appropriate ratio according to actual needs.

[0040] In this application, the specific mixing operation can be accomplished using simple vortex mixing, pipette blowing, or microfluidic mixing. These are all conventional operating methods in the art, and the specific parameters and conditions can be adjusted as needed, which will not be described in detail here.

[0041] Understandably, the crude nanoparticles obtained after mixing typically require post-processing to obtain a purified formulation suitable for in vivo administration. In some specific embodiments, post-processing steps may include: solvent replacement and removal of residual organic solvents using a dialysis membrane, typically with a molecular weight cutoff of 100 kDa to 1000 kDa; or concentration and buffer replacement using ultrafiltration centrifuge tubes, ultimately transferring the nanoparticles to a pharmaceutical-grade buffer system, such as phosphate-buffered saline (PBS), Tris buffer, or HEPES buffer. If necessary, the purified nanoparticles can be lyophilized and reconstituted before use.

[0042] Another aspect of this application discloses the use of the polymer-lipid hybrid nanoparticles described above in the preparation of medicaments for delivering nucleic acids to subjects. Based on the differential in vivo expression characteristics exhibited by these nanoparticles under different routes of administration, they can be used for the prevention and treatment of various diseases.

[0043] In some specific embodiments, the drug is a vaccine or gene therapy drug for the prevention or treatment of infectious diseases, tumors, genetic diseases or autoimmune diseases.

[0044] In some examples, nanoparticles loaded with mRNA encoding viral antigen proteins can be administered via intramuscular injection or other methods, achieving efficient expression of the antigen proteins at the injection site. This induces humoral and cellular immune responses, generating protective immunity to prevent infectious diseases. Applicable pathogens include, but are not limited to, influenza viruses, novel coronaviruses, and respiratory syncytial viruses.

[0045] In other examples, the nanoparticles can carry mRNA encoding tumor-specific antigens, cytokines, or immune checkpoint regulators, enabling the activation of anti-tumor immune responses through efficient delivery, thereby achieving tumor therapy. In particular, they can also be used to in-situ engineer T cells or natural killer cells in vivo by delivering mRNA encoding chimeric antigen receptors.

[0046] In other examples, the nanoparticles are loaded with mRNA encoding the corresponding functional protein, and after intravenous injection, they can achieve continuous therapeutic protein expression supplementation in target organs such as the liver, so as to treat hereditary diseases, applicable to diseases caused by the absence or functional defects of specific proteins.

[0047] In addition, this system can also be used to deliver mRNA encoding immunomodulatory factors, regulate abnormally activated immune pathways, and restore immune homeostasis to achieve the treatment of autoimmune diseases.

[0048] It should be understood that the specific diseases and treatments listed above are merely illustrative examples, and any application based on the delivery of nucleic acid molecules by nanoparticles in this application to achieve the purpose of prevention, diagnosis or treatment does not depart from the protective essence of this application.

[0049] This application has at least the following beneficial effects: The ionizable polymer in this application introduces thiol groups through ring-opening of the thiolactone structure, followed by thiol-Michael addition with acrylate monomers, enabling rapid construction of the polymer backbone under mild conditions. Simultaneously, by introducing different types of amine-containing monomers and end-capping groups, the polymer can be endowed with ionizable properties, allowing for the regulation of hydrophobicity, charge density, and spatial structure. The polymer can undergo protonation under acidic conditions, thereby enhancing its electrostatic interaction with mRNA and improving nucleic acid encapsulation capabilities; while under near-physiological conditions, the charge decreases, contributing to improved biocompatibility. Furthermore, this system employs a modular design strategy, exhibiting strong structural scalability. Diverse polymer libraries can be rapidly constructed by altering alkyl chains, amine structures, and crosslinking units, facilitating systematic screening and optimization of delivery performance.

[0050] Furthermore, polymer-lipid hybrid particles constructed from ionizable polymers are created by post-modifying the polymer / mRNA complex with lipid components, thereby balancing the high loading capacity of the polymer system with the in vivo stability of the lipid system. Specifically, the polymer enables efficient mRNA compression and nanoparticle formation through electrostatic interactions, while the subsequently introduced phospholipids, cholesterol, and PEGylated lipids further form a stable interfacial layer on the particle surface. Phospholipids help maintain particle structural integrity, cholesterol regulates the density and stability of the interfacial layer, and PEGylated lipids reduce interparticle aggregation and non-specific adsorption of plasma proteins, thus improving the system's stability in physiological environments. Compared to nanocomposites formed from single polymers, these hybrid nanoparticles exhibit superior in vivo delivery and tissue expression under intravenous administration conditions; simultaneously, they maintain good local expression under intramuscular injection conditions, demonstrating the system's potential for application in various drug delivery scenarios. Attached Figure Description

[0051] Figure 1 For compound A1 1 H NMR spectrum (400MHz, CDCl3).

[0052] Figure 2 For compound BC1 1 H NMR spectrum (400MHz, CDCl3).

[0053] Figure 3 For compound A1BC1D1 1 H NMR spectrum (400MHz, CDCl3).

[0054] Figure 4 For compound A1BC1D1E1 1 H NMR spectrum (400MHz, CDCl3).

[0055] Figure 5 For compound A1BC1D1E2 1 1H NMR spectrum (400MHz, CDCl3).

[0056] Figure 6 For compound A1BC1D1E3 1 H NMR spectrum (400MHz, CDCl3).

[0057] Figure 7 The characterization results are for polymer-lipid hybrid nanoparticles, where, Figure 7 In the figure, A represents the particle size statistics. Figure 7 B represents the statistical result of the Polydispersity Index (PDI). Figure 7 C represents the statistical results of the Zeta potential.

[0058] Figure 8 The results of in vivo expression of polymer-lipid hybrid nanoparticles in mice after intravenous injection into the eye socket were presented. Figure 8 Image A in the image represents the luciferase signal in a mouse. Figure 8 Figure B shows a quantitative analysis of fluorescence intensity in mice.

[0059] Figure 9 The results of in vivo expression of polymer-lipid hybrid nanoparticles in mice after intramuscular injection were presented, among which... Figure 9 Image A in the image represents the luciferase signal in a mouse. Figure 9 Figure B shows a quantitative analysis of fluorescence intensity in mice. Detailed Implementation

[0060] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0062] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0063] Example 1: Preparation of ionizable polymer A1BC1D1E1 (1) Preparation of compound A1

[0064] Weigh 1,4-butanediol (0.901 g, 10 mmol, 1.0 equivalent) into a 50 mL round-bottom flask and dissolve it in dichloromethane (DCM, 10 mL). Under magnetic stirring, add triethylamine (TEA, 3.036 g, 30 mmol, 3.0 equivalent) to the system. Weigh acryloyl chloride (2.172 g, 24 mmol, 2.4 equivalent) into a 10 mL centrifuge tube and dilute with dichloromethane to a total volume of 10 mL to prepare a diluted acryloyl chloride solution. Stir the reaction system in an ice bath, and under this condition, slowly add the diluted acryloyl chloride solution dropwise to the reaction flask, controlling the addition time to be 10–20 min. After the addition is complete, continue the reaction in an ice bath for 30 min, then move to room temperature and stir overnight (12–16 h). After the reaction is complete, filter to remove the generated white solid (triethylamine hydrochloride). The resulting filtrate is concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by silica gel column chromatography using a petroleum ether / ethyl acetate system (volume ratio 20:1) as the eluent. The solvent was removed by concentration under reduced pressure to obtain the target product A1. 1 The H NMR characterization results are shown in the figure. Figure 1 It is a colorless and transparent liquid.

[0065] (2) Preparation of compound BC1

[0066] Compound B (DL-homocysteine ​​thiolactone hydrochloride, 1.536 g, 10 mmol, 1.0 equivalent) was weighed and placed in a 50 mL round-bottom flask. Dichloromethane (DCM, 10 mL) was added to dissolve it. Under magnetic stirring, triethylamine (3.036 g, 30 mmol, 3.0 equivalent) was added to the system as an acid-binding agent, and a large amount of white solid (triethylamine hydrochloride) was formed in the system. Compound C1 (octanoyl chloride, 1.952 g, 12 mmol, 1.2 equivalent) was weighed and placed in a 10 mL centrifuge tube. Diluted with dichloromethane to a total volume of 10 mL to prepare a diluted acyl chloride solution. The reaction system was stirred in an ice bath, and the diluted acyl chloride solution was slowly added dropwise to the reaction flask under the same conditions, with the addition time controlled at 10-20 min. After the addition was completed, the reaction was continued in an ice bath for 30 min, and then the mixture was moved to room temperature and stirred overnight (12-16 h). After the reaction was complete, the resulting white solid (triethylamine hydrochloride) was removed by filtration. The filtrate was concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by silica gel column chromatography using a petroleum ether / ethyl acetate system (volume ratio 5:1). The solvent was removed by concentration under reduced pressure to obtain the target product BC1. 1 The H NMR characterization results are shown in the figure. Figure 2 It is a white solid.

[0067] (3) Preparation of compound A1BC1D1

[0068] Compounds D1 (1,4-bis(3-aminopropyl)piperazine, 0.200 g, 1 mmol, 1.0 equivalent), BC1 (0.486 g, 2 mmol, 2.0 equivalent), and A1 (0.218 g, 1.1 mmol, 1.1 equivalent) were weighed separately and placed in 5 mL centrifuge tubes, respectively. Tetrahydrofuran (THF, 2 mL) was added to each tube to dissolve them. The solutions of compound BC1 and A1 were transferred to a 50 mL round-bottom flask and mixed thoroughly under magnetic stirring. Then, the solution of compound D1 was added, followed by the addition of 2 mL of THF. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the reaction solution was slowly added dropwise to pre-cooled anhydrous diethyl ether (100 mL) to precipitate the product. A white precipitate formed in the system. After standing, the supernatant was discarded, and the precipitate was dried overnight in a vacuum drying oven to obtain the target product A1BC1D1. 1 The H NMR characterization results are shown in the figure. Figure 3 According to the 1H NMR spectrum, the repeating unit n of this polymer is approximately 6, and the molecular weight is approximately 5500.

[0069] (4) Preparation of ionizable polymer A1BC1D1E1

[0070] Take 1 mL of A1BC1D1 polymer stock solution (100 mg / mL, dimethyl sulfoxide, DMSO) and place it in a 10 mL reaction flask. Add 400 μL of end-capping reagent E1 stock solution (0.5 M, N,N-diethylethylenediamine in DMSO solution), mix well, and allow the reaction system to react at room temperature for 24 h. After the reaction is complete, slowly add the reaction solution dropwise to pre-cooled anhydrous diethyl ether (30 mL) for precipitation. A white precipitate forms in the system. After standing, discard the supernatant and dry in a vacuum drying oven overnight to obtain the target product A1BC1D1E1. 1 The H NMR characterization results are shown in the figure. Figure 4 According to the 1H NMR spectrum, the repeating unit n of this polymer is approximately 6, and the molecular weight is approximately 5700.

[0071] Example 2: Preparation of ionizable polymer A1BC1D1E2

[0072] Take 1 mL of A1BC1D1 polymer stock solution (100 mg / mL, dimethyl sulfoxide, DMSO) and place it in a 10 mL reaction flask. Add 400 μL of end-capping reagent E2 stock solution (0.5 M, N,N-bis(2-hydroxyethyl)ethylenediamine in DMSO solution), mix well, and allow the reaction system to react at room temperature for 24 h. After the reaction is complete, slowly add the reaction solution dropwise to pre-cooled anhydrous diethyl ether (30 mL) for precipitation. A white precipitate forms in the system. After standing, discard the supernatant and dry in a vacuum drying oven overnight to obtain the target product A1BC1D1E2. 1 The H NMR characterization results are shown in the figure. Figure 5 According to the 1H NMR spectrum, the repeating unit n of this polymer is approximately 6, and the molecular weight is approximately 5800.

[0073] The preparation of the A1BC1D1 polymer is described in Example 1.

[0074] Example 3: Preparation of ionizable polymer A1BC1D1E3

[0075] Take 1 mL of A1BC1D1 polymer stock solution (100 mg / mL, dimethyl sulfoxide, DMSO) and place it in a 10 mL reaction flask. Add 400 μL of end-capping reagent E3 stock solution (0.5 M, DMSO solution of dodecyl primary amine), mix well, and allow the reaction system to react at room temperature for 24 h. After the reaction is complete, slowly add the reaction solution dropwise to pre-cooled anhydrous diethyl ether (30 mL) to precipitate. A white precipitate forms in the system. After standing, discard the supernatant and dry in a vacuum drying oven overnight to obtain the target product A1BC1D1E3. 1 The H NMR characterization results are shown in the figure. Figure 6 According to the 1H NMR spectrum, the repeating unit n of this polymer is approximately 6, and the molecular weight is approximately 5900.

[0076] The preparation of the A1BC1D1 polymer is described in Example 1.

[0077] Example 4: Polymer-Lipid Hybrid Nanoparticles 4.1 Preparation of polymer-lipid hybrid nanoparticles (1) Preparation of nucleic acid solution The mRNA encoding luciferase (Luc) was dissolved in 25 mM sodium acetate buffer (pH 4.5-5.0) to obtain 40 μL of nucleic acid solution, in which the mRNA concentration was 0.2 mg / mL.

[0078] (2) Preparation of polymer solution The ionizable polymers were dissolved or dispersed in 25 mM sodium acetate buffer (pH 4.5-5.0). A small amount of organic solvent, such as DMSO, could be added to aid dissolution, resulting in a 40 μL polymer solution with a polymer concentration of 5.375 mg / mL.

[0079] (3) Electrostatic recombination forms nanoparticles At room temperature, the above nucleic acid solution and polymer solution were mixed at a 1:1 volume ratio to control the molar ratio (N / P ratio) between protonable nitrogen atoms in the ionizable polymer and phosphate groups in mRNA at approximately 20:1. After mixing, rapid vortexing was used to promote uniform contact, thereby forming a polymer / mRNA nanocomposite.

[0080] (4) Construction of hybrid nanoparticles by post-lipid modification 70 μL of 25 mM sodium acetate buffer (pH 4.5-5.0) was added to the polymer / mRNA nanocomposite to adjust the system volume (as the aqueous phase). Then, 50 μL of a pre-prepared lipid organic phase (a mixture of DSPC, cholesterol, and DMG-PEG2000 dissolved in anhydrous ethanol at a molar ratio of 10:38.5:1.5, with a total lipid concentration of 5.149 mg / mL) was added, resulting in an aqueous-to-organic phase volume ratio of 3:1. At room temperature, vortexing caused the lipid components to adsorb and rearrange on the nanoparticle surface, forming a stable interfacial layer structure, thus yielding polymer-lipid hybrid nanoparticles.

[0081] (5) Buffer system adjustment By adding phosphate-buffered saline (PBS), the system is adjusted to near physiological pH and ionic strength conditions to obtain the final nanoparticle formulation for in vivo or in vitro experiments.

[0082] 4.2 Characterization of the preparation of polymer-lipid hybrid nanoparticles The particle size, polydispersity index (PDI), and zeta potential of the prepared polymer-lipid hybrid nanoparticles were determined using dynamic light scattering (DLS). The results are as follows: Figure 7 As shown, the obtained polymer lipid hybrid nanoparticles have a particle size distribution in the range of 50-150 nm, a polydispersity index of less than 0.3, and a surface potential close to neutral under physiological conditions.

[0083] Example 5: In vivo expression experiment 5.1 Intravenous injection in the eye Six- to eight-week-old Balb / c female mice (approximately 20g in weight) were acclimatized and then randomly divided into three groups, with one mouse in each group. Each mouse received an intravenous injection of 100 μL of the polymer-lipid hybrid nanoparticles from Example 4, with an additional 2 μg of mRNA injected into each mouse. The mice were fed normally during the injection period. Six hours after injection, each mouse received an intraperitoneal injection of 100 μL of Luciferase substrate (15 mg / mL), and was anesthetized for small animal imaging 5 minutes later.

[0084] The results are as follows Figure 8 As shown, after intravenous injection into the eye, the Total Flux of all three polymer-lipid hybrid nanoparticles was greater than 1 × 10⁻⁶. 7 (p / sec), and all were expressed in the liver and spleen. Among them, A1BC1D1E1 and A1BC1D1E3 had the highest total flux, reaching 7×10. 7 (p / sec).

[0085] 5.2 Intramuscular injection Six- to eight-week-old Balb / c female mice (approximately 20g in weight) were acclimatized and then randomly divided into three groups, with one mouse in each group. Each mouse received an intramuscular injection of 50 μL of the polymer-lipid hybrid nanoparticles from Example 4, with an additional 2 μg of mRNA injected into each mouse. The mice were fed normally during the injection period. Six hours after injection, each mouse received an intraperitoneal injection of 100 μL of Luciferase substrate (15 mg / mL), and was anesthetized for small animal imaging 5 minutes later.

[0086] The results are as follows Figure 9 As shown, after intramuscular injection, the Total Flux of all three polymer-lipid hybrid nanoparticles was greater than 1 × 10⁻⁶. 6 (p / sec), and all were expressed in situ in muscle. Among them, A1BC1D1E1 had the highest total flux, reaching 3×10. 6 (p / sec).

[0087] The above results indicate that, compared with nanocomposites formed by polymers alone, the polymer-lipid hybrid nanoparticles prepared in this application exhibit superior in vivo delivery capability and tissue expression effect under intravenous administration conditions; at the same time, they can still maintain good local expression capability under intramuscular injection conditions, indicating that the polymer-lipid hybrid delivery system in this application has the potential for application in multiple drug delivery scenarios.

[0088] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. An ionizable polymer characterized in that, It has the molecular structure described in formula (I): ， Where n is an integer from 1 to 50; R1 is selected from one or more of the following: C2-C20 straight-chain or branched alkylene groups, polyether segments containing 1-10 oxygen atoms, C6-C18 arylene groups, C2-C20 alkylene groups containing thioether bonds or disulfide bonds, C2-C20 alkylene groups containing ester bonds, and C2-C20 fluoroalkylene groups. R2 is selected from one or more of the following: C1~C24 straight-chain or branched alkyl groups, C2~C24 alkenyl groups containing unsaturated bonds, C3~C12 cycloalkyl groups, C6~C30 steroidal groups, C1~C12 fluoroalkyl groups, and C6~C18 aryl groups; R3 is selected from one or more of the following: C2-C12 alkylene groups containing tertiary amines, polyether segments containing tertiary amine structures, and 5-12 membered nitrogen-containing heterocyclic groups containing 1-3 heteroatoms selected from N, O, and S; the nitrogen-containing heterocyclic group is preferably piperazinyl, morpholinyl, piperidinyl, N-alkylpiperazinyl, or their substituted derivatives. Preferably, R3 contains at least one protonable tertiary amine group; R4 is selected from one or more of the following: C1-C18 straight-chain or branched alkyl groups, C1-C8 hydroxy-substituted alkyl groups, C1-C8 amino-substituted alkyl groups, C1-C8 alkoxy-substituted alkyl groups, C3-C8 cycloalkyl groups, and 5-8 membered heterocyclic groups containing 1-3 heteroatoms selected from N, O, and S. Preferably, any one of the groups R1, R2, R3 and R4 may be further substituted by one or more substituents selected from halogens, hydroxyl groups, amino groups, mercapto groups, mercapto substituents, carboxyl groups, ester groups, amide groups, ether bonds, thioether bonds, disulfide bonds, C1-C6 alkyl groups, C1-C6 alkoxy groups, C1-C6 fluoroalkyl groups, polyether segments and heterocyclic groups.

2. The method of claim 1, wherein the ionizable polymer is prepared by the process comprising: Its synthetic route is as follows: ； The preparation of the ionizable polymer includes the following steps: An ionizable polymer as shown in formula (I) was prepared from compounds A, B, C, D, and E via an amidation reaction, a ring-opening reaction, a mercapto-Michael addition reaction, and an amine capping reaction. Preferably, compounds A, B, C, D, and E are selected from the following library of ionizable polymers: 。 3. A composition for delivering mRNA, characterized in that, It comprises the ionizable polymer of claim 1 and at least one auxiliary lipid; Preferably, the auxiliary lipid is selected from phospholipids, sterols, PEGylated lipids, or any combination thereof.

4. The composition according to claim 3, characterized in that, The phospholipid is at least one of distearylphosphatidylcholine, dioleoylphosphatidylethanolamine, or an analogue thereof; And / or, the sterol is at least one of cholesterol or a derivative thereof; And / or, the PEGylated lipid is at least one of PEG-dimyristoylglycerol, PEG-distearatephosphatidylethanolamine or its analogues, with a PEG number-average molecular weight of 500-5000 Da.

5. The composition according to claim 3 or 4, characterized in that, When phospholipids, sterols and PEGylated lipids are present, the molar ratio of phospholipids, sterols and PEGylated lipids is (5~25):(25~55):(0.1~5).

6. Polymer-lipid hybrid nanoparticles, characterized in that, It includes the composition according to any one of claims 3-5 and at least one mRNA.

7. The polymer-lipid hybrid nanoparticles as described in claim 6, characterized in that, The molar ratio (N / P ratio) between protonable nitrogen atoms and mRNA phosphate groups in ionizable polymers is 1-50.

8. The polymer-lipid hybrid nanoparticles as described in claim 6, characterized in that, Its average particle size is 50~150nm, polydispersity index is less than 0.3, and surface potential is between -10mV and 10mV under physiological conditions of pH 7.35-7.

45.

9. The method for preparing polymer-lipid hybrid nanoparticles according to any one of claims 6-8, characterized in that, Includes the following steps: mRNA and ionizable polymers are dissolved or dispersed separately in a weakly acidic buffer solution with a pH of 4.5-5.0 to form nucleic acid solution and polymer solution, respectively. At room temperature, the nucleic acid solution and polymer solution are mixed in a predetermined ratio to allow the mRNA and ionizable polymer to come into uniform contact, forming a polymer / mRNA nanocomplex. Buffer solution is added to adjust the system volume to obtain an aqueous phase. Phospholipids, sterols, and PEGylated lipids are dissolved in an organic solvent to form an organic phase of mixed lipids; Polymer lipid hybrid nanoparticles were prepared by thoroughly mixing the aqueous phase and the organic phase.

10. Use of the polymer-lipid hybrid nanoparticles according to any one of claims 6-8 in the preparation of a medicament for delivering nucleic acids in a subject; Preferably, the drug is a vaccine or gene therapy drug for the prevention or treatment of infectious diseases, tumors, genetic diseases or autoimmune diseases.

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