CN115197272A - 一种具有活性氧响应性的脂质体、其制备方法及应用 - Google Patents
一种具有活性氧响应性的脂质体、其制备方法及应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及纳米递送载体技术领域,特别涉及一种具有活性氧响应性的脂质体、其制备方法及应用。
背景技术
基因治疗(gene therapy)是指将外源性基因通过分子生物学方法导入靶细胞内,以取代、补偿或纠正异常的基因,从而实现治疗或者改善某种疾病的目的。这一概论在1972年首次提出以后得到了飞速发展,被应用于多种疾病的治疗。最早开发的基因治疗递送系统是基于病毒载体的,然而由于其免疫原性强、细胞毒性大、成本高及缺乏特异性和靶向性等问题在临床上的应用受到限制。相比较而言,非病毒载体具有成本低、制备简单、安全性高等优点,一直是基因递送领域的研究热点。其中,由脂质体介导的基因转染技术是基因治疗研究最早、也是应用最为广泛的非病毒转染策略之一。
目前,批准上市的脂质体基因载体较少,其面临以下几个关键问题:首先,面临的最主要问题是转染效率低,达不到临床要求,如何进一步提高转染效率是基因治疗的关键因素。其次,脂质体的化学合成及可修饰问题,临床上常用的脂质体以天然的卵磷脂和人工合成为主,其由磷脂酰胆碱两性离子头部和长链烷烃尾部组成,磷脂酰胆碱头部缺乏有效的反应位点,很难通过化学反应实现进一步修饰,导致其在结构和应用上比较单一且合成方法也不够简洁、高效。最后,脂质体的控制释放问题,临床上使用的脂质体不具备环境的刺激响应性,没有明确的释放机制,只能依靠胞内物质竞争缓慢释放,这种机制大多是缓慢且被动的,不具有可控的释放性。
发明内容
有鉴于此,本发明提供了一种具有活性氧响应性的脂质体、其制备方法及应用。本发明提供的脂质体在合成方法上具有成本低、制备简单及合成结构多样化等优点,同时脂质体具有活性氧响应性能够进入靶向部位,实现可控的释放,提高转染效率。
为实现上述目的,本发明提供了如下方案:
一种具有活性氧响应性的脂质体,具有式(I)所示结构:
其中,R1选自以下结构:
n=1~5;
R2选自H或者Me;
R3选自硼酸酯、硼酸、NO2或者OAc;
R4选自取代或者非取代C2~C22的饱和烷基醇、取代或者非取代C2~C22的饱和烷基醇;
R5与R4相同。
所述具有活性氧响应性的脂质体的制备方法,合成路线如下:
所述具有活性氧响应性的脂质体的制备方法,具体包括以下步骤:
将1~2当量亚胺中间体与1~2当量亚磷酸酯置于反应器中,在氮气保护下,于50~120℃反应1~3h,薄层色谱法(TLC)监测反应进度,反应完成后通过快速柱层析分离纯化,得到具有活性氧响应性的脂质体。
其中,R1选自以下结构:
n=1~5;
R2选自H或者Me;
R3选自硼酸酯、硼酸、NO2或者OAc。
进一步地,所述亚胺中间体的制备方法,合成路线如下:
所述亚胺中间体的制备方法,具体包括以下步骤:
在氮气保护下,将1~2当量化合物胺加入到1~2当量醛或者酮的甲醇(0.3~0.6M)溶液中,加热至50~100℃,反应1~5h,反应结束后温度降至室温,旋转蒸发掉溶剂,得到所述亚胺中间体。
进一步地,所述化合物胺的结构式如下:
所述醛或者酮的结构式如下:
其中,R1选自以下结构:
n=1~5;
R2选自H或者Me;
R3选自硼酸酯、硼酸、NO2或者OAc。
一种包括所述具有活性氧响应性的脂质体组装体。所述组装体为胶束、囊泡、脂质双层膜或多层膜。组装体的脂质体来源于上述方法制备的脂质体。组装方式包括但不限于亲疏水组装、静电相互作用组装、氢键相互作用组装。组装方式并不会影响本发明脂质体的转染效率。
一种包括负载物和所述具有活性氧响应性的载物脂质体。
进一步地,所述负载物包括核酸、药物分子、蛋白质中的一种或者几种。
进一步地,所述核酸包括DNA、质粒DNA、siRNA、microRNA及CRISPR/Cas。
所述具有活性氧响应性的脂质体在制备靶向性药物中的应用。
所述具有活性氧响应性的脂质体在基因递送领域的应用。
本发明公开了以下技术效果:
本发明提供了一种具有上述式(I)结构的阳离子脂质体,芳基官能团以左为含有仲胺的亲水性阳离子头部,以右的部分为疏水性的长链饱和及不饱和烷烃结构,芳基官能团部分则带有硼酸酯、硼酸等具有刺激响应性官能团。三者通过两步连续的亚胺缩合及Kabachnik-Fields反应,连接为上述式(I)的结构。与现有技术相比本发明具有如下优点:首先,本发明脂质体具有优异的转染效率。含有仲胺基的阳离子脂质体,表面带有的正电荷能稳定包裹的带负电性的基因,而其与带负电的细胞膜也可以通过静电相互作用而结合,这一过程有利于细胞内吞,从而提高载体的基因转染效率。而适宜长度的脂肪链能够增加脂质膜的流动性,有利于其在细胞膜间转运,也能提高转染效率。实验表明,本发明制备的脂质体在DNA质粒的转染效果上优于市售的脂质体转染试剂Lipofectamine 2000。其次,合成脂质体的原料便宜、易得且合成方法简便,不需要特殊的仪器设备。同时,具有不同结构的原料都可以利用上述合成路线,得到结构多样化的产物,产物也能继续进行修饰,拓展了该脂质体在不同体系下的适用性。最后,该脂质体具有刺激响应性,可以实现可控的释放过程。含有仲胺的阳离子头部能缓冲内涵体的酸化程度,从而保护递送的基因免受降解,促进内涵体逃逸。而硼酸酯、硼酸基团的存在,实现主动靶向具有活性氧的部位实现脂质体的解体,破坏脂质体的双层膜结构,从而释放出内包的基因。上述过程都在一定程度上提高了转染的效率。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1的核磁共振1H NMR(400MHz,CDCl3)图。
图2是实施例1的核磁共振13C NMR(101MHz,CDCl3)图。
图3是实施例1的核磁共振31P NMR(162MHz,CDCl3)图。
图4是部分实施例的转染荧光效果图;
图5是部分实施例的细胞流式统计图。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
本发明实施例中薄层色谱法(TLC)监测反应进度为本领域常规方法,且并非本发明重点,在此不做赘述。
本发明实施例中的室温指的是25±1℃。
实施例1
在氮气保护下,将N,N-二甲基乙二胺(55.1μL,0.5mmol)加入到4-甲酰基苯硼酸频哪醇酯(0.12g,0.5mmol,1.0equiv)的甲醇(1mL)溶液中,加热至70℃,反应3h。反应结束后温度降至室温,旋转蒸发掉溶剂,所得亚胺中间体粗产品直接用于下一步反应。
将上述亚胺中间体(0.5mmol,1.0equiv)与14C亚磷酸酯(0.24g,0.5mmol,1.0equiv)置于反应器中,在氮气保护下,于100℃反应1.5h。薄层色谱法(TLC)监测反应进度,反应完成后以甲醇、二氯甲烷及氨水的混合溶液为淋洗剂(甲醇:二氯甲烷:氨水=1:40:0.005,体积比),通过快速柱层析分离纯化,得到目标产物0.16g为无色液体,产率为40%。实施例1的核磁共振1H NMR(400MHz,CDCl3)图见图1,核磁共振13C NMR(101MHz,CDCl3)图见图2,核磁共振31P NMR(162MHz,CDCl3)图见图3。1H NMR(400MHz,CDCl3)δ7.94–7.78(m,2H),7.48–7.37(m,2H),4.10–4.02(m,1H),4.01–3.64(m,4H),2.90–2.54(m,4H),2.31(s,6H),1.67–1.58(m,2H),1.56–1.44(m,2H),1.32–1.18(m,56H),0.87(t,J=6.7Hz,6H);13CNMR(101MHz,CDCl3)δ139.60,134.83,128.00,127.94,83.81,66.98,66.91,66.84,62.26,60.74,58.78,45.72,45.55,45.44,31.98,30.64,30.59,30.53,30.48,29.75,29.72,29.65,29.60,29.42,29.25,29.20,25.51,25.44,24.93,22.74,14.17;31P NMR(162MHz,CDCl3)δ23.30.
实施例2
实施例2的制备过程与实施例1相同,不同的是,以N,N-二乙基乙二胺(54.6μL,0.5mmol)替换N,N-二甲基乙二胺(55.1μL,0.5mmol),得到目标产物0.15g为无色液体,产率为37%。1H NMR(400MHz,CDCl3)δ8.11–7.79(m,2H),7.48–7.32(m,2H),4.11–4.01(m,1H),4.03–3.85(m,4H),3.80–3.66(m,1H),2.59–2.44(m,8H),1.66–1.56(m,2H),1.56–1.46(m,2H),1.28–1.21(m,56H),0.99(t,J=7.0Hz,6H),0.87(t,J=6.7Hz,6H);31P NMR(162MHz,CDCl3)δ23.60.
实施例3
实施例3的制备过程与实施例1相同,不同的是,以1-(3-氨基丙基)吡咯烷(64.1mg,0.5mmol)替换N,N-二甲基乙二胺(55.1μL,0.5mmol),得到目标产物0.13g为无色液体,产率为33%。1H NMR(400MHz,CDCl3)δ7.78–7.70(m,2H),7.42–7.34(m,2H),4.05–3.79(m,4H),3.73–3.55(m,1H),3.12–3.02(m,1H),2.83–2.68(m,2H),2.63–2.38(m,4H),2.28–1.93(m,2H),1.87–1.75(m,4H),1.72–1.64(m,1H),1.63–1.52(m,2H),1.49–1.41(m,2H),1.23(s,56H),0.85(t,J=6.7Hz,6H);31P NMR(162MHz,CDCl3)δ23.01.
实施例4
实施例4的制备过程与实施例1相同,不同的是,以1-(2-氨乙基)哌啶(64.1mg,0.5mmol)替换N,N-二甲基乙二胺(55.1μL,0.5mmol),得到目标产物0.16g为无色液体,产率为38%。1H NMR(400MHz,CDCl3)δ8.13–7.83(m,2H),7.49–7.32(m,2H),4.14–4.02(m,1H),4.04–3.84(m,4H),3.80–3.65(m,1H),2.74–2.38(m,8H),1.72–1.49(m,10H),1.36–1.19(m,56H),0.90(t,J=6.7Hz,6H);31P NMR(162MHz,CDCl3)δ23.45.
实施例5
实施例5的制备过程与实施例1相同,不同的是,以3-氮杂环庚烷-1-丙胺(78.1mg,0.5mmol)替换N,N-二甲基乙二胺(55.1μL,0.5mmol),得到目标产物0.11g为无色液体,产率为27%。1H NMR(400MHz,CDCl3)δ8.17–7.77(m,2H),7.46–7.28(m,2H),4.08–3.99(m,1H),3.99–3.81(m,4H),3.66–3.27(m,1H),3.05–2.44(m,10H),1.77–1.53(m,12H),1.43–1.13(m,56H),0.87(t,J=6.7Hz,6H);31P NMR(162MHz,CDCl3)δ27.30.
实施例6
实施例6的制备过程与实施例1相同,不同的是,以1-(3-氨基丙基)咪唑(59.6μL,0.5mmol)替换N,N-二甲基乙二胺(55.1μL,0.5mmol),得到目标产物0.11g为无色液体,产率为26%。1H NMR(400MHz,CDCl3)δ8.02–7.89(m,2H),7.64–7.49(m,1H),7.41–7.31(m,2H),7.03(s,1H),6.86–6.70(m,1H),4.09–3.95(m,4H),3.92–3.78(m,2H),3.67–3.55(m,1H),2.57–2.43(m,2H),1.92–1.75(m,2H),1.69–1.57(m,2H),1.52–1.39(m,2H),1.34–1.11(m,56H),0.86(t,J=6.6Hz,6H);31P NMR(162MHz,CDCl3)δ23.44.
实施例7
实施例7的制备过程与实施例1相同,不同的是,以4-(2-氨乙基)吡啶(59.9μL,0.5mmol)替换N,N-二甲基乙二胺(55.1μL,0.5mmol),得到目标产物0.13g为无色液体,产率为32%。1H NMR(400MHz,CDCl3)δ8.55(s,2H),7.96(d,J=7.4Hz,2H),7.33(d,J=7.6Hz,2H),7.13(s,2H),4.07–3.81(m,4H),3.70–3.60(m,1H),2.85–2.68(m,4H),1.65–1.55(m,2H),1.50–1.40(m,2H),1.34–1.15(m,56H),0.86(t,J=7.0Hz,6H);31P NMR(162MHz,CDCl3)δ23.03.
实施例8
实施例8的制备过程与实施例1相同,不同的是,以4-(2-氨基乙基)吗啉(65.7μL,0.5mmol)替换N,N-二甲基乙二胺(55.1μL,0.5mmol),得到目标产物0.16g为无色液体,产率为32%。1H NMR(400MHz,CDCl3)δ7.94(d,J=7.5Hz,2H),7.42(d,J=8.0Hz,2H),4.08–3.88(m,4H),3.70–3.65(m,4H),3.64–3.59(m,1H),2.60–2.45(m,4H),2.39–2.31(m,4H),1.68–1.60(m,2H),1.55–1.48(m,2H),1.34–1.20(m,56H),0.87(t,J=6.6Hz,6H);31P NMR(162MHz,CDCl3)δ23.49.
实施例9
实施例9的制备过程与实施例1相同,不同的是,以6C亚磷酸酯(0.14g,0.5mmol,1.0equiv)替换14C亚磷酸酯(0.24g,0.5mmol,1.0equiv),得到目标产物0.10g为无色液体,产率为35%。1H NMR(400MHz,CDCl3)δ7.85–7.75(m,2H),7.48–7.30(m,2H),4.06–3.92(m,4H),3.89–3.79(m,1H),3.09–2.98(m,2H),2.95–2.80(m,2H),2.72(s,6H),1.65–1.56(m,2H),1.46–1.38(m,2H),1.30–1.21(m,24H),0.90–0.82(t,J=6.7Hz,6H);31P NMR(162MHz,CDCl3)δ22.68.
实施例10
实施例10的制备过程与实施例1相同,不同的是,以9C亚磷酸酯(0.17g,0.5mmol,1.0equiv)替换14C亚磷酸酯(0.24g,0.5mmol,1.0equiv),得到目标产物92.3mg为无色液体,产率为29%。1H NMR(400MHz,CDCl3)δ7.91(d,J=7.5Hz,2H),7.41(d,J=7.6Hz,2H),4.10–3.82(m,4H),3.78–3.65(m,1H),2.84–2.42(m,4H),2.21(s,6H),1.67–1.57(m,2H),1.54–1.45(m,2H),1.35–1.11(m,30H),0.86(t,J=6.3Hz,6H);13C NMR(101MHz,CDCl3)δ137.18,134.50,134.23,127.65,67.11,66.96,66.89,66.84,62.09,60.57,58.47,45.09,44.93,44.61,31.88,31.86,30.65,30.60,30.54,30.48,29.71,29.52,29.48,29.26,29.23,29.22,29.16,25.49,25.41,22.68,14.13;31P NMR(162MHz,CDCl3)δ23.43.
实施例11
实施例11的制备过程与实施例2相同,不同的是,以9C亚磷酸酯(0.17g,0.5mmol,1.0equiv)替换14C亚磷酸酯(0.24g,0.5mmol,1.0equiv),得到目标产物0.11g为无色液体,产率为32%。1H NMR(400MHz,CDCl3)δ8.12–7.78(m,2H),7.45–7.30(m,2H),4.10–3.86(m,4H),3.79–3.64(m,1H),2.62–2.49(m,8H),1.64–1.58(m,2H),1.54–1.47(m,2H),1.31–1.19(m,36H),0.99(t,J=6.8Hz,6H),0.86(t,J=7.2Hz,6H);31P NMR(162MHz,CDCl3)δ23.40.
实施例12
实施例12的制备过程与实施例1相同,不同的是,以18C亚磷酸酯(0.29g,0.5mmol,1.0equiv)替换14C亚磷酸酯(0.24g,0.5mmol,1.0equiv),得到目标产物0.16g为无色液体,产率为35%。1H NMR(400MHz,CDCl3)δ7.90(d,J=7.6Hz,2H),7.40(d,J=7.7Hz,2H),4.11–3.85(m,4H),3.76–3.62(m,1H),2.85–2.33(m,4H),2.20(s,6H),1.66–1.56(m,2H),1.54–1.44(m,2H),1.37–1.07(m,72H),0.86(t,J=6.7Hz,6H);13C NMR(101MHz,CDCl3)δ137.31,134.60,134.30,127.67,67.01,66.93,66.84,62.18,60.66,60.39,58.58,45.12,44.93,44.68,31.97,30.73,30.68,30.61,30.55,29.77,29.71,29.68,29.66,29.64,29.61,29.41,29.29,29.23,25.56,25.47,22.72,14.13;31P NMR(162MHz,CDCl3)δ23.44.
实施例13
实施例13的制备过程与实施例2相同,不同的是,以18C亚磷酸酯(0.29g,0.5mmol,1.0equiv)替换14C亚磷酸酯(0.24g,0.5mmol,1.0equiv),得到目标产物0.17g为无色液体,产率为38%。1H NMR(400MHz,CDCl3)δ8.11–7.76(m,2H),7.46–7.30(m,2H),4.06–3.87(m,4H),3.78–3.62(m,1H),2.72–2.51(m,8H),1.65–1.57(m,2H),1.55–1.47(m,2H),1.32–1.20(m,72H),1.05–0.98(t,J=7.2Hz,6H),0.87(t,J=6.6Hz,6H);31P NMR(162MHz,CDCl3)δ23.38.
实施例1~13的中间体产物收率、终产物收率和终产物结构见表1。
表1
脂质体对质粒的转染效果研究
倒置荧光显微镜(定性分析)
将HEK 293T细胞接种于2个6孔板中,每孔5~8×105个/孔,设空白对照组(控制组)1孔、Lipo2000组1孔和材料组,共9孔。将培养板置于37℃、5%CO2,培养箱培养过夜。待细胞长至70%-80%时,弃去原有培养基,加入2mL新鲜无血清培养基,并加入配制好的材料,材料配制:Lipo2000组按照试剂盒配制质量比为5mg/mL的溶液,并加入质粒(1mg/mL)孵育15min备用。材料组按照脂质体:质粒为1:3的质量比加入各实施例制备的脂质体和质粒,孵育15min。转染时,将制备好的脂质体/质粒复合物加入到相应的孔中,转染12h后更换新鲜含血清培养基,48h后弃去培养基,每孔加入D-hanks溶液2mL。倒置荧光显微镜下观察,结果如图4所示。通过倒置荧光显微镜拍摄转染后细胞表达的荧光蛋白图片,可以通过荧光强度的强弱判断转染的效率,荧光强度越强转染效果越好。由图4可以看出,控制组在倒置荧光显微镜下为黑色未发现荧光点,而其它组有不同强度的荧光点。其中,实施例1与对照组Lipo2000的荧光强度最大,表明其转染效率最好。
流式细胞仪测定(定量分析)
将HEK 293T细胞接种于2个6孔板中,每孔5-8×105个/孔,设空白对照组(控制组)1孔、Lipo2000组1孔和材料组,共9孔。将培养板置于37℃、5%CO2,培养箱培养过夜。待细胞长至70%-80%时,弃去原有培养基,加入2mL新鲜无血清培养基,并加入配制好的材料,材料配制:Lipo2000组按照试剂盒要求配制质量比为5mg/mL的溶液,并加入质粒(1mg/mL)孵育15min备用。材料组按照脂质体:质粒为1:3的质量比加入稀释好的材料和质粒,孵育15min。转染时,将制备好的脂质体/质粒复合物加入到相应的孔中,转染12h后更换新鲜含血清培养基,48h后弃去培养基。胰酶消化并离心(1000rpm,3min),利用流式细胞仪测定细胞摄取的EGFP的荧光强度(激发波长为488nm,发射波长为507nm),细胞流式统计结果如图5所示。通过流式细胞仪定量测定表达的荧光蛋白,计算得到转染效率,流式细胞仪定量测定的结果表明实施例1的转染效率高于商品化的Lipo2000,达到99.9%。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (10)
2.一种权利要求1所述具有活性氧响应性的脂质体的制备方法,其特征在于,包括以下步骤:
将1~2当量亚胺中间体与1~2当量亚磷酸酯置于反应器中,在氮气保护下,于50~120℃反应1~3h,反应完成后分离纯化,得到具有活性氧响应性的脂质体。
4.根据权利要求3所述制备方法,其特征在于,所述亚胺中间体的制备方法,包括以下步骤:
在氮气保护下,将1~2当量化合物胺加入到1~2当量醛或者酮的甲醇溶液中,加热至50~100℃,反应1~5h,反应结束后温度降至室温,蒸发掉溶剂,得到所述亚胺中间体。
6.一种脂质体组装体,其特征在于,包括权利要求1所述具有活性氧响应性的脂质体。
7.一种载物脂质体,其特征在于,包括负载物和权利要求1所述具有活性氧响应性的脂质体。
8.根据权利要求7所述一种载物脂质体,其特征在于,所述负载物包括核酸、药物分子、蛋白质中的一种或者几种。
9.权利要求1所述具有活性氧响应性的脂质体在基因递送领域的应用。
10.权利要求1所述具有活性氧响应性的脂质体在制备靶向性药物中的应用。
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