WO2024149306A1 - 含疏水烷基链的高度支化聚(β-氨基酯)及其制备方法和mRNA递送应用 - Google Patents

含疏水烷基链的高度支化聚(β-氨基酯)及其制备方法和mRNA递送应用 Download PDF

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WO2024149306A1
WO2024149306A1 PCT/CN2024/071675 CN2024071675W WO2024149306A1 WO 2024149306 A1 WO2024149306 A1 WO 2024149306A1 CN 2024071675 W CN2024071675 W CN 2024071675W WO 2024149306 A1 WO2024149306 A1 WO 2024149306A1
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highly branched
amino ester
branched poly
alkyl chain
hydrophobic alkyl
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French (fr)
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雍海洋
李建忠
王凯旋
李智立
王晨飞
李明
潘超兰
周德重
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Xian Jiaotong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines

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  • the invention belongs to the technical field of biomedical materials, and specifically relates to a highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain, a preparation method thereof, and mRNA delivery application thereof.
  • mRNA drugs have great application potential in infectious disease vaccines, tumor diseases, protein replacement and gene therapy.
  • the lack of safe and efficient vectors limits the clinical application of mRNA gene therapy.
  • more than 70% of clinical trials still use viral vectors, but viral vectors have potential immunogenicity and teratogenicity, cumbersome preparation procedures and high prices.
  • the commercial lipid nanotechnology delivery (LNP) technology in non-viral vectors is controlled by Arbutus, a Canadian company.
  • inorganic nanoparticles can regulate the potential toxicity of inorganic materials by controlling the size and surface modification of nanoparticles, the difficulty of in vivo degradation is an important factor that needs to be considered in practical applications.
  • Polymers have become one of the non-viral vectors with potential for clinical transformation due to their low immune response, high safety, flexible design, easier production and chemical modification processes.
  • Poly( ⁇ -amino esters) have shown great application prospects in drug, protein and nucleic acid delivery due to their wide monomer sources, easy control of structural properties and in vivo degradability.
  • the application of poly( ⁇ -amino esters) in mRNA delivery has only explored polymers prepared with polyamines as branching units, and there has been no report on the use of tri(tetra)acrylate monomers as branching units to construct poly( ⁇ -amino esters) for efficient mRNA delivery.
  • the present invention aims to provide a highly branched poly ( ⁇ -amino ester) containing a hydrophobic alkyl chain and a preparation method and mRNA delivery application thereof, which solves the shortcomings of the prior art.
  • the application of poly( ⁇ -amino ester) in mRNA delivery has only explored polymers prepared with polyamines as branching units. There has been no report on the use of tri(tetra)acrylate monomers as branching units to construct poly( ⁇ -amino ester) for efficient mRNA delivery.
  • the present invention adopts the following technical scheme to achieve it: a highly branched poly ( ⁇ -amino ester) containing a hydrophobic alkyl chain, wherein the highly branched poly ( ⁇ -amino ester) has the following structural formula:
  • R 1 is one of the following structures:
  • R2 is one of the following structures:
  • the present invention also discloses a method for preparing a highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain, comprising the following steps:
  • Step 1 preparing highly branched poly( ⁇ -amino ester) by Michael addition reaction of acrylic acid ester monomers and small molecular amine monomers;
  • Step 2 Using an amino monomer as a capping agent to functionalize and cap the highly branched poly ( ⁇ -amino ester) to obtain a highly branched poly ( ⁇ -amino ester) containing a hydrophobic alkyl chain.
  • the acrylic acid ester monomers include diacrylate monomers and tri(tetra)acrylate monomers.
  • the Michael addition reaction in step 1 is carried out at 60-120° C. for 12-48 hours, and the concentration of the reaction material dissolved in dimethyl sulfoxide is 200-500 mg/mL.
  • the molar ratio of the end-capping agent to the acrylic ester monomer is 0.5-3:1.
  • the functionalization capping reaction temperature is 25° C. and the reaction time is 48 h.
  • the diacrylate monomer is one of the following structures:
  • the tri(tetra)acrylate monomer is one of the following structures:
  • the small molecule hydrophilic amine monomer is one of the following structures:
  • the small molecule hydrophobic amine monomer includes one of the following structures:
  • the amino-containing end-capping monomer R 2 —NH 2 is one of the following structures:
  • the invention also discloses the use of highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains in the preparation of mRNA drug delivery carriers.
  • the present invention discloses a highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain, and the poly( ⁇ -amino ester) is constructed by using tri(tetra)acrylate monomers as branching units.
  • a class of highly branched biodegradable polymers with adjustable structural components, structures, and properties are synthesized. Due to its special topological structure, it improves the ability to encapsulate and protect mRNA, and has higher mRNA delivery efficiency and cell activity.
  • the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains disclosed in the present invention has higher delivery efficiency, lower toxicity and greater clinical potential.
  • the present invention also discloses a method for preparing the highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain.
  • the highly branched poly( ⁇ -amino ester) is prepared by the Michael addition method using commercial acrylate monomer reagents and small molecule organic amines. The polymer molecular weight is monitored by gel permeation chromatography, and the molecular weight is controlled at 2000-40000Da.
  • the highly branched poly( ⁇ -amino ester) is capped using an amino monomer as an end-capping agent, and poly( ⁇ -amino ester) with different chemical compositions, branched structures and terminal groups is prepared in one pot, showing good mRNA transfection performance.
  • the above-mentioned preparation method disclosed in the present invention successfully prepares a highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain with the advantages of low cost, simple synthesis path, and easy control of structural properties.
  • the molar ratio of diacrylate monomer: tri(tetra)acrylate monomer is used to regulate the reaction speed and the obtained polymer has good mRNA delivery efficiency and safety.
  • reaction is well controlled within the reaction time and temperature range to obtain a product with a suitable molecular weight.
  • the molar ratio of the end-capping agent to the acrylate monomer ensures that the end-capping reaction is complete while no side reaction occurs.
  • the present invention also discloses the use of a highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain in the preparation of an mRNA drug delivery carrier. Due to its main chain peptide bond structure, it has good biodegradability; the ionizable or positively charged group can effectively compress mRNA, thereby achieving delivery. The effectiveness, accuracy and wide applicability of the invention have been verified in in vitro cell experiments, and it has good prospects for clinical transformation.
  • FIG1 is a schematic diagram of the synthesis of highly branched poly( ⁇ -amino ester);
  • FIG2 is a schematic diagram of the physical phase state of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in Example 1 at 25° C.;
  • FIG3 is a 1 H NMR spectrum of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in Example 1;
  • FIG4 is a gel permeation chromatography (GPC) curve of the purified highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain obtained in Example 1;
  • FIG5 is a TEM microscopic morphology of a complex formed by a highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain and mRNA prepared in Example 1;
  • FIG6 is a potential of a complex formed by a highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain prepared in Example 1 and mRNA;
  • FIG7a is a fluorescence photograph of HeLa cells transfected with mRNA encoding green fluorescent protein after the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in Example 1 was transfected with mRNA encoding green fluorescent protein;
  • Figure 7b is a fluorescent photograph of HeLa cells after the commercialized Lipo Messenger of Example 1 was transfected with mRNA encoding green fluorescent protein;
  • FIG8 shows the cell activity of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in Example 1 after transfection in HeLa cells;
  • FIG9 is a schematic diagram of the physical phase state of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in Example 2 at 25° C.;
  • FIG10 is a 1H NMR spectrum of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains obtained in Example 2;
  • FIG11 is a gel permeation chromatography (GPC) curve of the purified highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains obtained in Example 2;
  • FIG12 is a schematic diagram of the physical phase of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in Example 3 at 25° C.;
  • FIG13 is a 1 H NMR spectrum of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in Example 3;
  • FIG. 14 is a gel permeation chromatography (GPC) curve of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains obtained in Example 3 after purification.
  • GPC gel permeation chromatography
  • the preparation method of highly branched poly ( ⁇ -amino ester) containing hydrophobic alkyl chains, the synthesis route, see Figure 1, comprises the following steps:
  • Step 2 Add a certain amount of end-capping agent and dimethyl sulfoxide into a reaction bottle, react at 25° C. for 48 hours, and the molar ratio of the end-capping agent to the raw material acrylic ester monomer is 0.5-3:1; purify the product by precipitation method, vacuum dry, and obtain a highly branched poly ( ⁇ -amino ester) containing a hydrophobic alkyl chain.
  • the functional highly branched poly ( ⁇ -amino ester) prepared by the present invention is used for the delivery of mRNA, comprising the following steps:
  • HeLa cells were cultured under standard culture conditions, seeded in a 96-well plate at a density of 0.5*10 4 -2.0*10 4 cells/well, and cultured for 24 h;
  • FIG2 A schematic diagram of the physical phase state of the highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain obtained in this example at 25° C. is shown in FIG2 ; a 1 H NMR spectrum of the highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain prepared in this example is shown in FIG3 ; a GPC curve of the highly branched poly( ⁇ -amino ester) containing a hydrophobic alkyl chain prepared in this example is shown in FIG4 .
  • the highly branched poly ( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in this example was used for extracellular mRNA delivery experiments. After 10,000 HeLa cells were inoculated in a 96-well plate per well and cultured for 24 hours, the polymer and mRNA mass ratios were uniformly mixed at 10, 30, and 60:1, and the amount of mRNA per well was 25 ng.
  • the composite nanoparticles were incubated statically for 20 minutes to form composite nanoparticles, which were then transferred to a 96-well plate for transfection.
  • the microscopic morphology of the nanoparticles under the 30:1 condition is shown in Figure 5.
  • the nanoparticles are uniform in size and the potential is shown in Figure 6.
  • the positive charge increases accordingly, which can effectively compress mRNA;
  • the mRNA delivery efficiency of the polymer in HeLa cells is shown in Figures 7a and 7b, and the cell activity after transfection is shown in Figure 8.
  • the mRNA transfection efficiency and safety of the polymer are significantly better than those of commercial products.
  • the molar ratio of acrylate monomer bisphenol A glycerol diacrylate: pentaerythritol triacrylate is 3:1
  • organic small molecule amine monomer (3-amino-1,2-propylene glycol and dodecylamine are 6:1)
  • concentration of the system reaction material in dimethyl sulfoxide is 200 mg/mL.
  • the physical phase diagram of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains obtained in this example at 25° C. is shown in FIG9 ;
  • the 1 H NMR spectrum of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in this example is shown in FIG10 , indicating that the chemical composition of the product is basically consistent with the raw material input;
  • the GPC curve of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in this example is shown in FIG11 .
  • the physical phase diagram of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains obtained in this example at 25° C. is shown in FIG. 12 ; the 1 H NMR spectrum of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in this example is shown in FIG. 13 , indicating that the chemical composition of the product is consistent with the raw material input; the GPC curve of the highly branched poly( ⁇ -amino ester) containing hydrophobic alkyl chains prepared in this example is shown in FIG. 14 .
  • the present invention discloses a method for preparing a class of biodegradable highly branched poly( ⁇ -amino ester)s containing hydrophobic alkyl chains and their application in mRNA delivery, belonging to the technical field of biomedical materials.
  • Highly branched poly( ⁇ -amino ester)s are prepared by selecting different acrylate monomers and small molecule amine monomers, and efficient delivery of mRNA is achieved in typical HeLa cells.
  • jetPEI Lipofectamine
  • this type of polymer is cheap, easy to design and prepare, has better transfection performance, and has significant clinical application potential in mRNA drug delivery.
  • hydrophobic alkyl chains increases the hydrophobicity of the polymer and improves the affinity for mRNA, thereby achieving effective encapsulation of mRNA; in addition, due to the fusion of the hydrophobic alkyl chain with the endosomal membrane, the escape efficiency of the mRNA in the endosomal is effectively increased, thereby improving the transfection efficiency.
  • a multi-component synthesis strategy is proposed to quickly and effectively evaluate and screen branched poly ( ⁇ -amino esters) with better performance.

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Abstract

本发明属于生物医学材料技术领域，公开了一种含疏水烷基链的高度支化聚(β-氨基酯)及其制备方法和mRNA递送应用，将丙烯酸酯类单体和小分子胺单体通过迈克加成反应制备得到高度支化聚(β-氨基酯)；以氨基单体作为封端剂，对高度支化聚(β-氨基酯)进行功能化封端，制得含疏水烷基链的高度支化聚(β-氨基酯)，在HeLa等细胞中实现高效mRNA递送。本发明含疏水烷基链的高度支化聚(β-氨基酯)，价格便宜、易于设计制备、转染性能更优，在mRNA药物递送方面具有显著的临床应用潜力。

Description

含疏水烷基链的高度支化聚(β-氨基酯)及其制备方法和mRNA递送应用 技术领域
本发明属于生物医学材料技术领域，具体涉及一种含疏水烷基链的高度支化聚(β-氨基酯)及其制备方法和mRNA递送应用。
背景技术
mRNA药物在传染病疫苗、肿瘤疾病、蛋白质替代与基因治疗等方面具有巨大的应用潜力。然而缺乏安全高效的载体限制了mRNA基因治疗在临床中的应用。目前，超过70％的临床实验使用的仍然是病毒载体，但病毒载体具有潜在的免疫原性和致畸性、制备程序繁琐、价格高昂。非病毒载体中商用的脂质纳米技术递送(LNP)技术由加拿大Arbutus公司掌控，短时间突破LNPs的相关技术专利目前看来相对困难，而且其低温储存和稳定性差也极大限制了在商业化中的应用；无机纳米颗粒虽然可以通过控制纳米颗粒的尺寸和表面修饰来调节无机材料潜在毒性，但是体内难降解性是其在实际应用中的需要考虑的重要因素。
聚合物由于其低免疫反应、高安全性、灵活的设计、更容易的生产和化学修饰过程等特点而成为具有临床转化潜力的非病毒载体之一。聚(β-氨基酯)由于其单体来源广、结构性能易调控、体内可降解等优点已在药物、蛋白、核酸递送方面显示出巨大的应用前景。然而，目前聚(β-氨基酯)在mRNA递送中的应用只探究了以多元胺为支化单元制备的聚合物，以三(四)丙烯酸酯类单体为支化单元构建聚(β-氨基酯)用于mRNA高效递送尚未有任何报道。
发明内容
为了克服上述现有技术的缺点，本发明的目的在于提供一种含疏水烷基链的高度支化聚(β-氨基酯)及其制备方法和mRNA递送应用，解决了现有技术 中聚(β-氨基酯)在mRNA递送中的应用只探究了以多元胺为支化单元制备的聚合物，以三(四)丙烯酸酯类单体为支化单元构建聚(β-氨基酯)用于mRNA高效递送尚未有任何报道的问题。
为了达到上述目的，本发明采用以下技术方案予以实现：含疏水烷基链的高度支化聚(β-氨基酯)，所述高度支化聚(β-氨基酯)结构式如下：
其中，R1为下列结构中的一种：
R2是下列结构中的一种：
其中，n＝2-16，m＝3-50。
本发明还公开了一种含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，包括以下步骤：
步骤一、将丙烯酸酯类单体和小分子胺单体通过迈克加成反应制备得到高度支化聚(β-氨基酯)；
步骤二、以氨基单体作为封端剂，对高度支化聚(β-氨基酯)进行功能化封端，制得含疏水烷基链的高度支化聚(β-氨基酯)。
优选地，所述丙烯酸酯类单体包括二丙烯酸酯类单体和三(四)丙烯酸酯 类单体；小分子胺单体包括亲水胺和疏水胺；丙烯酸酯类单体和小分子胺单体的反应摩尔比为(1-2)：1；其中二丙烯酸酯单体：三(四)丙烯酸酯单体＝9：1-1：9；亲水胺：疏水胺＝9：1-1：9。
优选地，步骤一中迈克加成反应是在60-120℃下反应12-48h，反应物料溶解在二甲基亚砜中的浓度为200-500mg/mL。
优选地，步骤二中封端剂与丙烯酸酯类单体的摩尔比为0.5-3：1。
优选地，功能化封端反应温度为25℃，反应时间为48h。
优选地，所述二丙烯酸酯类单体为如下结构中的一种：
优选地，所述三(四)丙烯酸酯类单体为如下结构中的一种：
优选地，所述小分子亲水胺类单体为如下结构中的一种：
所述小分子疏水胺类单体包括如下结构中的一种：
所述含氨基的封端单体R2-NH2为如下结构中的一种：
本发明还公开了含疏水烷基链的高度支化聚(β-氨基酯)在制备mRNA药物递送载体中的应用。
与现有技术相比，本发明具有以下有益效果：本发明公开了含疏水烷基链的高度支化聚(β-氨基酯)，通过使用三(四)丙烯酸酯类单体为支化单元构建聚(β-氨基酯)。通过使用不同的单体组合、调控支化结构，使用不同的封端单体，合成一类结构组分、结构、性能可调控的的可生物降解的高度支化聚合物，由于其特殊的拓扑结构提高对mRNA的包裹和保护能力，具有较高的mRNA递送效率和细胞活性。与目前本领域技术中主要采用的商业化mRNA递送试剂和Lipofectamine MessengerMAXTM相比，本发明公开的含疏水烷基链的高度支化聚(β-氨基酯)具有较高的递送效率，毒性低，更具临床潜力。
本发明还公开了上述含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，通过商业化的丙烯酸酯类单体试剂和小分子有机胺，以迈克尔加成方法制备出高度支化聚(β-氨基酯)，通过凝胶渗透色谱监测聚合物分子量，分子量控制在2000-40000Da。以氨基单体作为封端剂，对高度支化聚(β-氨基酯)进行封端，一锅法制备出具有不同化学组成、支化结构以及末端基团的聚(β-氨基酯)，表现出良好的mRNA转染性能。本发明公开的上述制备方法以低成本、合成路径简单、结构性能易调控的优势成功制备含疏水烷基链的高度支化聚(β-氨基酯)。
进一步地，二丙烯酸酯单体：三(四)丙烯酸酯单体的摩尔比一是调控反应速度，二是所得到的聚合物具有良好的mRNA递送效率和安全性。
进一步地，反应时间和温度范围内反应控制得较好，得到具有合适分子量的产物。
进一步地，封端剂与丙烯酸酯类单体摩尔比确保封端反应进行完全，同时又不会发生副反应。
本发明还公开了含疏水烷基链的高度支化聚(β-氨基酯)在制备mRNA药物递送载体中的用途，由于其主链肽键结构，具有良好的生物降解性能；可离子化或带正电的基团能够有效的压缩mRNA，从而实现递送，已在体外细胞实验中验证了该发明的有效性、准确性和广泛的适用性，具有良好的临床转化前景。
附图说明
图1为高度支化聚(β-氨基酯)的合成示意图；
图2为实施例1制得的含疏水烷基链的高度支化聚(β-氨基酯)在25℃时的物理相态示意图；
图3为实施例1制得的含疏水烷基链的高度支化聚(β-氨基酯)的1H NMR谱图；
图4为实施例1制得的含疏水烷基链的高度支化聚(β-氨基酯)纯化后凝胶渗透色谱(GPC)曲线；
图5为实施例1制得的含疏水烷基链的高度支化聚(β-氨基酯)与mRNA所形成复合物的TEM微观形貌；
图6为实施例1制得的含疏水烷基链的高度支化聚(β-氨基酯)与mRNA所形成复合物的电位；
图7a为实施例1制得的含疏水烷基链的高度支化聚(β-氨基酯)在HeLa细胞中转染编码绿色荧光蛋白mRNA后细胞的荧光照片；
图7b为实施例1商业化Lipo Messenger在HeLa细胞中转染编码绿色荧光蛋白mRNA后细胞的荧光照片；
图8为实施例1制得的含疏水烷基链的高度支化聚(β-氨基酯)在HeLa细胞中转染后的细胞活性；
图9为实施例2制得的含疏水烷基链的高度支化聚(β-氨基酯)在25℃时的物理相态示意图；
图10为实施例2制得的含疏水烷基链的高度支化聚(β-氨基酯)的1H NMR谱图；
图11为实施例2制得的含疏水烷基链的高度支化聚(β-氨基酯)纯化后凝胶渗透色谱(GPC)曲线；
图12为实施例3制得的含疏水烷基链的高度支化聚(β-氨基酯)在25℃时的物理相态示意图；
图13为实施例3制得的含疏水烷基链的高度支化聚(β-氨基酯)的1H NMR谱图；
图14为实施例3制得的含疏水烷基链的高度支化聚(β-氨基酯)纯化后凝胶渗透色谱(GPC)曲线。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
需要说明的是，本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备 固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述：
含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，合成路线，参见图1，包括以下步骤：
步骤一、将一定量的丙烯酸酯类单体(二丙烯酸酯类单体、三(四)丙烯酸酯类单体等)和小分子有机胺(亲水胺、疏水胺)加入含二甲基亚砜的烧瓶中，丙烯酸酯类单体与小分子有机胺的投料摩尔比为(1-2)：1；其中二丙烯酸酯单体：三丙烯酸酯单体＝9：1-1：9；亲水胺：疏水胺＝9：1-1：9，在60-120℃下反应12-48h；在反应过程中使用凝胶渗透色谱监测聚合物分子量，当聚合物的分子量到达2000-40000Da，终止反应；
步骤二、将一定量的封端剂和二甲基亚砜加入到反应瓶中，在25℃下反应48h，封端剂与原料丙烯酸酯类单体投料摩尔比为0.5-3：1；使用沉淀法对产物进行提纯，真空干燥，得到含疏水烷基链的高度支化聚(β-氨基酯)。
将本发明制得的功能性高度支化聚(β-氨基酯)用于mRNA的递送，包括以下步骤：
1)细胞培养：HeLa细胞在标准培养条件下进行培养，按照0.5*104-2.0*104个细胞/孔的密度接种在96孔板中，培养24h；
2)将含疏水烷基链的高度支化聚(β-氨基酯)的溶液与含编码绿色荧光蛋白的mRNA溶液混合均匀，静置孵育15-30min，然后转移至96孔板中进行转染，其中聚合物：mRNA质量比＝10-200：1，每孔mRNA质量为25-250ng；
3)转染24-48h后，在荧光显微镜下对转染的绿色荧光蛋白的细胞进行观察和拍照。
5)转染24-48h后，移除细胞上清液后加入alamarBlue溶液，在培养箱中孵育30min，进行细胞活性测试。
实施例1
丙烯酸酯类单体(双酚A乙氧基化物二丙烯酸酯，EO/phenol＝1.5：三羟甲基丙烷三丙烯酸酯投料摩尔比为＝5：1，)和有机小分子胺类单体(5-氨基-1-戊醇与正辛胺投料摩尔比为8：1)的投料摩尔比为1.5：1，体系反应物料在二甲基亚砜中的浓度为400mg/mL。在80℃下反应12h；然后加入丙烯酸酯类单体摩尔用量0.6倍的1-(3-氨丙基)-4-甲基哌嗪，并在25℃下反应48h；提纯干燥后得到的分子量支化聚(β-氨基酯)分子量为6000Da，分子量分布为2.3，m等于10。结构式如下：
本实施例所得到的含疏水烷基链的高度支化聚(β-氨基酯)在25℃时的物理相态示意图如图2所示；本实施例制备得到的含疏水烷基链的高度支化聚(β-氨基酯)的1H NMR谱图如图3所示；本实施例制备得到的含疏水烷基链的高度支化聚(β-氨基酯)的GPC曲线如图4所示。
将本实施例制备的含疏水烷基链的高度支化聚(β-氨基酯)在细胞外进行mRNA递送实验。将HeLa细胞以每孔10000个接种在96孔板里培养24h后，以聚合物与mRNA质量比10、30、60：1均匀混合，每孔mRNA用量25ng，静止孵育20min形成复合纳米颗粒，然后转移至96孔板中进行转染。其中30：1条件下的纳米颗粒微观形貌如图5所示，纳米颗粒尺寸均一，电位如图6，随着聚合物的用量增加，正电荷相应增加，能够有效的压缩mRNA；聚合物在HeLa细胞中的mRNA递送效率如图7a和图7b，转染后细胞活性如图8，聚合物的mRNA转染效率和安全性明显优于商业化产品。
实施例2
丙烯酸酯类单体(双酚A甘油二丙烯酸：季戊四醇三丙烯酸酯投料摩尔比为＝3：1，)和有机小分子胺类单体(3-氨基-1,2-丙二醇与十二烷胺投料摩尔比为6：1)的投料摩尔比为1.1：1，体系反应物料在二甲基亚砜中的浓度为200mg/mL。在100℃下反应24h；然后加入丙烯酸酯类单体摩尔用量1倍的丙二胺，并在25℃下反应48h；提纯干燥后得到的分子量支化聚(β-氨基酯)分子量为15000Da，分子量分布为3.4，m等于20。结构式如下：
本实施例所得到的含疏水烷基链的高度支化聚(β-氨基酯)在25℃时的物理相态示意图如图9所示；本实施例制备得到的含疏水烷基链的高度支化聚(β-氨基酯)的1H NMR谱图如图10所示，表明产物的化学组成与原料投料基本一致；本实施例制备得到的含疏水烷基链的高度支化聚(β-氨基酯)的GPC曲线如图11所示。
实施例3
丙烯酸酯类单体(双酚A乙氧基化物二丙烯酸酯，EO/phenol＝2：季戊四醇四丙烯酸酯投料摩尔比为＝9：1，)和有机小分子胺类单体(2-氨乙基吗啉与十四烷胺投料摩尔比为4：1)的投料摩尔比为1.3：1，体系反应物在二甲基亚砜中的料浓度为350mg/mL。在80℃下反应36h；然后加入丙烯酸酯类单体摩尔用量1.3倍的1,11-二氨基-3,6,9-三氧杂十一烷，并在25℃下反应48h；提纯干燥后得到的分子量支化聚(β-氨基酯)分子量为22000Da，分子量分布为3.2， m等于25。结构式如下：
本实施例所得到的含疏水烷基链的高度支化聚(β-氨基酯)在25℃时的物理相态示意图如图12所示；本实施例制备得到的含疏水烷基链的高度支化聚(β-氨基酯)的1H NMR谱图如图13所示，表明产物的化学组成与原料投料一致；本实施例制备得到的含疏水烷基链的高度支化聚(β-氨基酯)的GPC曲线如图14所示。
本发明公开了一类可生物降解的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法和其在mRNA递送方面的应用，属于生物医用材料技术领域。通过选用不同的丙烯酸酯类单体和小分子胺类单体制备出高度支化聚(β-氨基酯)，在典型HeLa细胞中实现高效递送mRNA。该类聚合物与主流的商业转染试剂(jetPEI、Lipofectamine)相比，价格便宜、易于设计制备、转染性能更优，在mRNA药物递送方面具有显著的临床应用潜力。含疏水烷基链的高度支化聚(β-氨基酯)相较于以小分子胺做支化单元，以三(四)丙烯酸酯类单体为支化单元能够引入更多的酯键，得到的聚合物能够更快地降解，相应的细胞毒性更低，更具临床应用前景。mRNA有效封装保护其在翻译表达蛋白前免受体内核酸酶的降解。同时，目前已有研究表明mRNA在内涵体中的逃逸效率仅为1-4％， 提高内涵体逃逸效率是实现高效mRNA转染的关键步骤。引入疏水烷基链，增加聚合物的疏水性，提高对mRNA的亲和力，从而对mRNA实现有效封装；此外，由于该疏水烷基链与内涵体膜发生膜融合，有效地增加了该mRNA在内涵体中的逃逸效率，从而提高转染效率。相较于传统的合成方法，提出通过多组分合成策略快速有效评价和筛选出性能更优异的支化聚(β-氨基酯)。这些结果进一步表明含疏水烷基链的高度支化聚(β-氨基酯)能够有效地降低生物毒性，增加mRNA封装效率和内涵体逃逸效率，实现有效的胞内mRNA递送。
以上内容仅为说明本发明的技术思想，不能以此限定本发明的保护范围，凡是按照本发明提出的技术思想，在技术方案基础上所做的任何改动，均落入本发明权利要求书的保护范围之内。

Claims (10)

  1. 含疏水烷基链的高度支化聚(β-氨基酯)，其特征在于，所述高度支化聚(β-氨基酯)结构式如下：
    其中，R1为下列结构中的一种：
    R2是下列结构中的一种：
    其中，n＝2-16，m＝3-50。
  2. 根据权利要求1所述的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，其特征在于，包括以下步骤：
    步骤一、将丙烯酸酯类单体和小分子胺单体通过迈克加成反应制备得到高度支化聚(β-氨基酯)；
    步骤二、以氨基单体作为封端剂，对高度支化聚(β-氨基酯)进行功能化封端，制得含疏水烷基链的高度支化聚(β-氨基酯)。
  3. 根据权利要求2所述的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，其特征在于，所述丙烯酸酯类单体包括二丙烯酸酯类单体和三(四)丙烯酸酯类单体；小分子胺单体包括亲水胺和疏水胺；丙烯酸酯类单体和小分子胺单体的反应摩尔比为(1-2)：1；其中二丙烯酸酯单体：三(四)丙烯酸酯单体 ＝9：1-1：9；亲水胺：疏水胺＝9：1-1：9。
  4. 根据权利要求2所述的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，其特征在于，步骤一中迈克加成反应是在60-120℃下反应12-48h，反应物料溶解在二甲基亚砜中的浓度为200-500mg/mL。
  5. 根据权利要求2所述的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，其特征在于，步骤二中封端剂与丙烯酸酯类单体的摩尔比为0.5-3：1。
  6. 根据权利要求2所述的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，其特征在于，功能化封端反应温度为25℃，反应时间为48h。
  7. 根据权利要求2-6任意一项所述的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，其特征在于，所述二丙烯酸酯类单体为如下结构中的一种：
  8. 根据权利要求2-6任意一项所述的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，其特征在于，所述三(四)丙烯酸酯类单体为如下结构中的一种：
  9. 根据权利要求2-6中任意一项所述的含疏水烷基链的高度支化聚(β-氨基酯)的制备方法，其特征在于，所述小分子亲水胺类单体为如下结构中的一种：
    所述小分子疏水胺类单体为如下结构中的一种：
    所述含氨基的封端单体R2-NH2为如下结构中的一种：
  10. 权利要求1所述的含疏水烷基链的高度支化聚(β-氨基酯)在制备mRNA药物递送载体中的应用。
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