CN105985386B - 一类蔗糖酯型阳离子基因载体及其制备方法 - Google Patents
一类蔗糖酯型阳离子基因载体及其制备方法 Download PDFInfo
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- CN105985386B CN105985386B CN201510071065.XA CN201510071065A CN105985386B CN 105985386 B CN105985386 B CN 105985386B CN 201510071065 A CN201510071065 A CN 201510071065A CN 105985386 B CN105985386 B CN 105985386B
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- sucrose ester
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Abstract
本发明提供了一类蔗糖酯型阳离子类脂及其制备方法。利用该阳离子类脂制备的载体可用于核酸递送。本发明运用化学合成的方法制备了蔗糖酯型阳离子类脂，合成方法简单，产物收率较高。本发明的蔗糖酯型阳离子类脂化合物与类脂助剂混合，可制备包括悬浮液，乳浊液，微胶粒和脂质体等组合物。利用上述组合物可与核酸制备蔗糖酯型阳离子类脂复合物，其具有制备简单、低毒、转染效率高等优点，是一种新型的高效基因载体。
Description
技术领域
本发明属于生物医药技术领域，涉及一类蔗糖酯型阳离子类脂化合物及其制备方法，该类化合物可制备蔗糖酯型阳离子类脂组合物和复合物并用于核酸递送。
背景技术
近年来，人们发明了很多治疗疾病的新方法，但是对于癌症等疾病的治疗还没有有效的治疗方法。而基因治疗作为一种全新的和革命性的治疗手段，是治愈癌症、心血管疾病和先天性免疫缺陷等疑难疾病的潜在方法。然而，其核心技术—基因转运的难题一直限制着基因治疗的发展和临床试验，其中，获得高效、安全的基因转运载体无疑成为基因治疗成功与否的关键。
目前，全世界使用的基因转运载体主要为两大类：一类为病毒载体，另一类为非病毒载体。病毒基因载体是利用病毒侵染细胞的能力，以病毒为基础构建的一种运载基因的载体，与此相对应，不使用病毒进行基因转运的载体统称为非病毒基因载体。虽然病毒基因载体用于基因的转运具有效率高的优点，但存在免疫原性高、容量小、变异性和致癌性等缺点。而非病毒基因载体以其设计灵活、低毒性、低免疫原性、低致肿瘤性、易制备及可以实现细胞特异性和长期基因表达的优点，为基因治疗开辟出一条新的道路。到目前为止，在2000余例的基因治疗临床试验中，有接近三分之一是通过非病毒基因载体来实施的。然而，非病毒基因载体的转染效率与病毒载体相比还有一定的差距，成为制约其临床试验的瓶颈。开发高效、低毒的非病毒基因载体已成为基因治疗的重要研究内容之一。
阳离子脂质体作为非病毒基因载体中的一类因其制备简便，无免疫原性、可重复转染和易于商品化等优点，近年来得到了迅速的发展。1987年Felgner等人(PNAS1987,84:7413–7417)首次将阳离子类脂DOTMA制备成阳离子脂质体转运DNA，开创了阳离子脂质体在基因治疗领域的先河。由阳离子类脂所形成的脂质体的结构与生物膜相似，用它作为一种载体可包裹外源基因。阳离子脂质体在生理pH值下带有正电荷，能够借助静电作用与核酸分子中的带有负电性的磷酸基团自组装形成脂质体/基因复合物，再通过静电作用吸附于细胞表面，进而通过细胞内吞作用或其他作用将外源基因导入细胞，从而发挥基因的治疗作用。自1987年以来，人们已经设计合成了许多阳离子类脂并用于核酸递送(Focus 1993,15:73–83)。但是现有阳离子类脂毒性较高，且转染效率有待提高。
众所周知，糖类化合物在自然界中分布较为广泛，安全性高。蔗糖为原料制备成的蔗糖酯在水相中能形成囊泡状或双片层结构，具有良好的生物相容性和降解性。但是由于蔗糖中羟基基团很多，所以很难通过化学合成方法得到单一结构的蔗糖酯化合物(J.Am.Oil Chem.Soc.2014,91:1891-1901.)，到目前为止尚未有蔗糖酯型基因载体应用于基因载体的相关研究。因此，研究和开发生物相容性好、高效和低细胞毒性的蔗糖酯型基因载体，对于开发具有自主知识产权的基因载体具有重要的科学意义和经济价值。
发明内容
本发明的目的在于提供一类安全高效的蔗糖酯型阳离子基因载体及其制备方法，其主要包括：一类蔗糖酯型阳离子类脂化合物及其制备方法、一类蔗糖酯型阳离子组合物及其制备方法及一类蔗糖酯型阳离子类脂复合物及其制备方法。
除了另有说明外，本文中使用的术语具有以下含义。
本文中使用的术语“烃基”包括烷基、烯基和炔基。
本文中使用的术语“烷基”包括直链烷基和支链烷基。例如“C1-4烷基”包括甲基、乙基、正丙基、异丙基、正丁基和叔丁基。类似的规则也适用于本说明书中使用的其他基团。
本发明中使用的术语“卤素”包括氟、氯、溴和碘。
一、本发明的蔗糖酯型阳离子类脂化合物，具有通式I的结构：
其中：
R选自C10-20烃基酯、胆汁酸基和胆固醇酯基；
其中所述胆固醇酯基(B1)结构如下：
在优选的技术方案中，R优选选自C12-18烷基酯、十八烯基酯(C17H34COO-)、胆汁酸基和胆固醇酯基。更优选R选自C12烷基酯、C14烷基酯、十八烯基酯(C17H34COO-)和胆固醇酯基。
Y选自–NRaRb和–N+RaRbRcXˉ，其中Ra、Rb和Rc相同或不同，并且选自氢、C1-6烃基、C1-6羟烷基、半乳糖基、甘露糖基和/或叶酸酯基；Xˉ选自Fˉ、Clˉ、Brˉ、Iˉ。在优选的技术方案中，Y优选选自NRaRb，其中Ra和Rb优选选自C1-6烷基、C1-6羟烷基、C6H11O6-(半乳糖基)、C6H11O6-(甘露糖基)和叶酸酯基(C18H18N7O4COOCH2CH2-)；更优选Ra和Rb选自甲基、乙基、羟甲基、羟乙基、半乳糖基和叶酸酯基；其中所述半乳糖基(B2)、甘露糖基(B3)、叶酸酯基(B4)的结构如下：
二、上述一类蔗糖酯型阳离子类脂化合物的制备方法，包括如下步骤：
(1)三氯蔗糖和式ⅰ的化合物按照摩尔比1:1～50反应，制备化合物A：
反应温度为10～100℃，反应时间为10～50h，反应溶剂为如N,N-二甲基甲酰胺、吡啶、乙腈、哌啶、三乙胺等含氮溶剂，反应物与溶剂的质量体积比为1:1～50；
在一个优选的实施方案中，反应温度为10～70℃，反应时间为10～35h，反应溶剂为N,N-二甲基甲酰胺和吡啶，三氯蔗糖和式ⅰ的化合物的摩尔比1:1～1:20；在一个更优选的实施方式中，反应温度为30～60℃,反应时间为20～30h，反应溶剂为N,N-二甲基甲酰胺和吡啶，三氯蔗糖和式ⅰ的化合物的摩尔比1:1～1:10。
(2)化合物A和式ⅱ的化合物按照摩尔比1:1～8反应，制备化合物B：
具体操作如下：
a分别在容器中加入三氯蔗糖酯，再取一定量式ⅱ化合物加入容器中，反应温度为10～120℃，反应时间为10～40h，反应压力为1.0～5.0atm，溶剂为N,N-二甲基甲酰胺(DMF)、甲醇、乙醇、异丙醇或异丁醇，反应物与溶剂的质量体积比为1:1～50；
b反应结束后，减压蒸馏除去未反应的原料，所得即为反应粗产物。
c将粗产物经柱色谱分离提纯得到产物后以溶剂溶解结晶数次，得到淡黄色粉末状固体，即为化合物B。
在一个优选的实施方式中，反应温度为50～110℃,反应时间为10～35h,反应压力为1.0～3.0atm，反应溶剂为DMF、乙醇或异丙醇，化合物A和式ⅱ的化合物的摩尔比为1:1～1:6；在一个更优选的实施方式中，反应温度为80～105℃,反应时间为15～25h,反应压力为1.0～2.0atm，反应溶剂为乙醇或异丙醇，化合物A和式ⅱ的化合物的摩尔比为1:1～1:3。
当所合成的产物为季铵盐时，平衡阴离子Fˉ、Brˉ、Iˉ可以通过离子交换柱替换Cl-获得。
上述对本发明的一类蔗糖酯型阳离子类脂化合物的制备方法的描述中，各个取代基的定义，均与上述对化合物的描述中的定义相同。
三、本发明的蔗糖酯型阳离子类脂组合物，该组合物是由上述的蔗糖酯型阳离子类脂化合物和类脂助剂组成，所述的蔗糖酯型阳离子类脂化合物与类脂助剂的质量比为10:1～1:10。在优选的技术方案中，蔗糖酯型阳离子类脂化合物与类脂助剂的质量比为5:1～1:5,更优选的质量比为3:1～1:3。
所述类脂助剂选自卵磷脂、磷脂酰乙醇胺、糖脂、二油酰磷脂酰氯(DOPC)、棕榈酰油酰磷脂酰乙醇胺(POPE)、二油酰磷脂酰乙醇胺(DOPE)、胆固醇(Chol)、壳聚糖、蔗糖酯中的一种或两种以上的混合物。在优选的技术方案中，类脂助剂选自卵磷脂、DOPC、DOPE、Chol、壳聚糖、蔗糖酯，更优选选自DOPE、Chol、壳聚糖、蔗糖酯。
上述蔗糖酯型阳离子类脂组合物外观为悬浮液、乳浊液、微胶粒或脂质体。
四、本发明的阳离子类脂复合物，其是由蔗糖酯型阳离子类脂组合物与所述核酸形成的阳离子类脂复合物，两者的质量比20:1～1:20，在优选的技术方案中，蔗糖酯型阳离子类脂化合物与核酸的质量比为5:1～1:15,更优选的质量比为1:1～1:10。
上述复合物中所述核酸为pDNA、microRNA、siRNA中的一种或两种以上的混合物。
本发明与现有技术相比具有如下优点：
1、本发明的蔗糖酯型阳离子类脂化合物在制备上利用三氯蔗糖代替蔗糖作为原料，与蔗糖相比减少了三个羟基，只有一个伯羟基，可以与酸类化合物通过酯化反应，获得单一结构的蔗糖酯型类脂化合物。该制备方法简单、环保、安全，适于实验室制备以及工业化生产。
2、本发明的蔗糖酯型阳离子类脂组合物(如脂质体)，具有良好的生物相容性和降解性，细胞毒性低于目前常用的转染试剂Lipofectamine 2000和DOTAP。
3、本发明的蔗糖酯型阳离子类脂组合物(如脂质体)，用于核算递送，其转染效率优于目前常用的转染试剂Lipofectamine 2000和DOTAP。
综合上述结果可知，本发明制备的蔗糖酯型阳离子载体，具有制备简单、毒性低、转染效率高的特点，作为基因载体有着良好的应用前景。
附图说明
图1是本发明的一类蔗糖酯型阳离子类脂化合物的结构通式I。
图2是实施例1的核磁氢谱图。
图3是实施例1的核磁碳谱图。
图4是实施例5的阳离子脂质体的粒径图。
图5是实施例5的阳离子脂质体zeta电位图。
图6是实施例7的阳离子脂质体-DNA复合物的电泳图谱。
图7是实施例8的阳离子脂质体-DNA复合物对Hep-2细胞转染后绿色荧光蛋白检测结果图。
图8是实施例8的阳离子脂质体-DNA复合物对Hep-2细胞转染后荧光素酶检测结果图。
图9是实施例9用MTT法测定的Hep-2细胞存活率图。
具体实施方式
本发明采用上述方法合成的蔗糖酯型阳离子类脂化合物，采用核磁共振谱(1HNMR和13C NMR)或质谱来确认其结构。
实施例1三氯蔗糖月桂酸酯季铵盐(TSI12)的制备
(1)三氯蔗糖月桂酸酯的合成
在装有温度计和回流管的250mL容器中加入3.9g的三氯蔗糖、3g无水碳酸钾、5mL吡啶和N,N-二甲基甲酰胺(DMF，10mL)，油浴恒温75℃，磁力搅拌下充分溶解后滴加5mL月桂酸，反应24h后得到淡黄色滤液，将其减压旋转蒸干溶剂，得到琥珀色粘稠油状液体。用柱层析纯化后，即得白色无定形状的酯化产物，其结构表征如下：1H NMR(400MHz,CDCl3)δ:5.44(t,J＝124.9,2H),6.37–4.56(m,6H),6.37–4.28(m,10H),6.37–3.89(m,20H),6.37–3.59(m,26H),6.37–2.37(m,28H),2.32(t,J＝14.7,4H),2.07(s,2H),1.99(s,2H),1.77(s,2H),1.74–1.58(m,6H),1.42–1.15(m,33H),1.52–0.58(m,39H),1.43–0.58(m,39H),1.11–0.53(m,6H).13C NMR(400MHz,CDCl3)δ:174.44(s),107.88(s),94.90(s),82.14(s),80.40(s),74.63(s),74.02(s),73.37(s),71.64(s),65.88(s),64.52(s),44.29(s),42.93(s),33.92(s),31.73(s),29.07(t,J＝7.9),25.42(s),23.16(s),14.00(s).ESI-MS,m/z:Found[M+3H]3+,581.10,[M+2H-Cl]+,545.1,C24H41Cl3O9calcd for[M]＝578.1816,[M+3H]＝581.1894,[M+2H-Cl]＝545.2127.
(2)三氯蔗糖月桂酸酯季铵盐的合成
在装有具N2保护、温度计、回流管和滴液漏斗的250mL容器中加入10g三氯蔗糖酯和30mL N,N-二甲基甲酰胺(DMF)，量取30mL的三甲胺溶液，水浴恒温100℃，压力为1atm，反应24h后，减压蒸馏除去溶剂。所得即为反应粗产物，将其经柱色谱分离提纯得到产物后以溶剂溶解结晶数次，得到淡黄色粉末状固体。红外快速干燥箱中干燥2h，得三氯蔗糖月桂酸酯季铵盐，其结构表征如下：1H NMR(400MHz,CDCl3)δ:5.40–5.53(d,1H),4.54–4.56(t,1H),4.43(d,1H),[4.35(d,1H),4.23(d,1H)],4.28–4.30(d,1H),4.30–4.33(d,1H),4.10(s,1H),4.07(s,1H),3.95–3.99(m,1H),[3.84(d,1H),3.65(d,1H)],3.61–3.78(d,2H),3.22–3.30(s,9H),2.36–2.41(t,J＝7.6Hz,2H),1.60～1.66(dd,J＝14.6,8.2Hz,2H),1.28–1.30(m,J＝8.9Hz,16H),0.88–0.89(t,J＝6.4Hz,3H).13C NMR(400MHz,CDCl3)δ:172.97(s),103.57(s),92.22(s),75.12(s),74.94(s),74.44(s),71.14(s),68.03(s),67.43(s),66.91(s),63.53(s),63.06(s),52.94(s),42.93(s),32.95(s),31.05(s),28.19–28.72(m),23.99(s),21.71(s),12.43(s).Q-TOF-MS,m/z:Found[M-Cl]+,602.2856，C27H50Cl3NO9calcd for[M]＝637.2551,[M-Cl]＝602.2863.
实施例2胆汁酸-蔗糖酯型阳离子类脂(TSB-2I)的制备
(1)三氯蔗糖胆汁酸酯的制备
在装有温度计和回流管的250mL容器中加入3.9g的三氯蔗糖、3g无水碳酸钾、5mL乙腈和20mL哌啶，油浴恒温75℃，磁力搅拌下充分溶解后滴加10mmol胆汁酸，反应24h后得到淡黄色滤液，将其减压旋转蒸干溶剂，得到琥珀色粘稠油状液体。用柱层析纯化后，即得白色无定形状单酯化产物。1H NMR(400MHz,CDCl3)δ:4.86–4.29(m,4H),4.86–3.92(m,8H),3.89–3.68(m,3H),3.68–3.41(m,2H),3.37–3.18(m,2H),2.38(t,J＝16.1,2H),2.38(t,J＝16.1,2H),3.08–1.78(m,5H),3.08–0.98(m,29H),3.08–0.91(m,35H),3.08–0.44(m,39H).13C NMR(400MHz,CDCl3)δ:174.51(s),107.88(s),94.90(s),82.14(s),80.40(s),74.63(s),74.09(d),73.56(s),73.37(s),71.64(s),70.69(s),67.02(s),65.88(s),64.52(s),50.15(s),47.48(s),45.82(s),44.29(s),42.93(s),41.84(s),40.62(s),40.36(s),38.98(s),35.64(s),34.24(s),31.84(d,J＝16.2),30.78(s),29.76(s),29.33(s),28.14(s),24.87(s),18.78(s),17.69(s),11.94(s).Q-TOF-MS,m/z:Found[M+Na]+,825.2860,C36H57Cl3O13calcd for[M]＝802.2865,[M+Na]＝825.2865.
(2)胆汁酸-蔗糖酯型阳离子类脂的合成
在装有N2保护、温度计、回流管和滴液漏斗的250mL容器中加入5mmol的三氯蔗糖胆汁酸酯的异丙醇(20mL)溶液、0.7g无水碳酸钾。水浴恒温60℃，磁力搅拌下滴加10mmol二乙醇胺的乙醇(10mL)溶液，滴加时间约1h，滴完后，水浴温度升至100℃，反应6h。过滤反应混合物，得到滤液，将其减压旋转蒸干溶剂，得到混合物，最后通过制备色谱纯化可获得纯度较高的胆汁酸-蔗糖酯型阳离子类脂。1H NMR(400MHz,CDCl3)δ:5.57(d,J＝14.8,2H),4.77(d,J＝14.2,2H),4.56–4.35(m,7H),4.30–4.05(m,6H),3.81–3.60(m,4H),3.59–3.37(m,10H),3.37–3.18(m,4H),2.92–2.40(m,12H),2.35(t,J＝15.7,4H),2.15–1.82(m,10H),1.82–1.28(m,43H),1.24(s,6H),1.18–1.03(m,2H),0.96(s,12H),0.88(d,J＝12.8,6H),0.75–0.44(m,2H).13C NMR(400MHz,CDCl3)δ:174.51(s),107.88(s),94.90(s),80.40(s),78.39(s),78.09(s),74.09(d),73.56(s),73.37(s),71.64(s),70.69(s),67.02(s),65.88(s),64.52(s),59.18(s),58.37(s),57.48(s),50.15(s),47.48(s),45.82(s),44.29(s),41.84(s),40.62(s),40.36(s),38.98(s),35.64(s),34.24(s),31.84(d),30.78(s),29.76(s),29.33(s),28.14(s),24.87(s),18.78(s),17.69(s),11.94(s).Q-TOF-MS,m/z:Found[M+Na]+,894.3891,C40H67Cl2NO15calcd for[M]＝871.3888,[M+Na]＝994.3888.
实施例3胆固醇-蔗糖酯型阳离子类脂(TSD-2D)的制备
(1)三氯蔗糖胆固醇酯的制备
在装有温度计和回流管的250mL容器中加入3.9g的三氯蔗糖、3g无水碳酸钾、5mL三甲胺和20mL N,N-二甲基甲酰胺(DMF)，油浴恒温75℃，磁力搅拌下充分溶解后滴加10mmol胆固醇顺丁烯二酸，反应24h后得到淡黄色滤液，将其减压蒸干溶剂，得到琥珀色粘稠油状液体。用柱层析纯化后，即得淡黄色无定形状的酯化产物。1H NMR(400MHz,CDCl3)δ:6.31(s,4H),4.73–4.56(m,6H),4.55–4.37(m,6H),4.26(ddd,J＝23.6,19.1,12.2,4H),3.79–3.57(m,5H),2.66–0.61(m,87H),1.82–0.61(m,71H),1.73–0.88(m,65H),0.82(s,6H).13C NMR(400MHz,CDCl3)δ:168.13(s),167.08(s),140.62(s),132.99(s),131.80(s),121.95(s),107.88(s),94.90(s),82.14(s),80.40(s),74.89(s),74.63(s),74.02(s),73.37(s),71.64(s),66.85(s),64.52(s),55.26(s),51.39(s),50.92(s),44.29(s),42.93(s),42.59(s),39.59(s),39.43(s),39.19(s),37.80(s),37.22(s),32.76(d),32.25(s),30.45(s),28.55-28.27(m),27.55(s),24.28(s),22.74(s),21.49(s),18.73(s),12.92(s).Q-TOF-MS,m/z:Found[M+Na]+,971.3435,C42H63Cl3O11calcd for[M]＝848.3436,[M+Na]＝971.3436.
(2)胆固醇-蔗糖酯型阳离子类脂的合成
在装有N2保护、温度计、回流管和滴液漏斗的250mL容器中加入5.2mmol的三氯蔗糖胆固醇酯的异丁醇(20mL)溶液、1.0g无水碳酸钾。水浴恒温60℃，磁力搅拌下滴加16.5mmol二乙胺的甲醇(20mL)溶液，滴加时间约1h，滴完后，水浴温度升至100℃，加压到2atm下反应6h。过滤反应混合物，得到滤液，将其减压蒸干溶剂异丁醇，得到混合物，最后通过制备色谱纯化可获得纯度较高的胆固醇-蔗糖酯型阳离子类脂。
1H NMR(400MHz,CDCl3)δ:6.31(s,4H),5.01–4.24(m,10H),5.01–3.73(m,24H),2.85(ddd,J＝23.3,12.5,6.7,6H),2.39(q,J＝6.3,4H),2.24–2.14(m,4H),2.00–1.83(m,8H),2.68–0.75(m,107H),2.46–0.75(m,105H),1.72–1.45(m,20H),1.44–1.35(m,6H),1.34–0.88(m,53H),0.82(s,6H).13C NMR(400MHz,CDCl3)δ:168.13(s),167.08(s),140.62(s),132.99(s),131.80(s),121.95(s),107.88(s),94.90(s),80.40(s),78.39(s),78.09(s),74.89(s),74.02(s),73.37(s),71.64(s),66.85(s),64.52(s),58.25(s),55.26(s),51.39(s),50.92(s),48.12(s),44.29(s),42.59(s),39.59(s),39.43(s),39.19(s),37.80(s),37.22(s),32.76(d),32.25(s),30.45(s),28.55-28.27(m),27.55(s),24.28(s),22.74(s),21.49(s),18.73(s),12.92(s),12.32(s).Q-TOF-MS,m/z:Found[M+Na]+,908.4565,C46H73Cl2NO11calcd for[M]＝885.4561,[M+Na]＝908.4561.
实施例4阳离子脂质体的制备
上述实施例1、2和3得到的蔗糖酯型阳离子类脂TSI12、TSB-2I、TSD-2D、TSI14(三氯蔗糖肉豆蔻酸酯季铵盐)和TSI16(三氯蔗糖棕榈酸酯季铵盐)在紫外灯下照1h，称取1mg的阳离子类脂与一定量的DOPE(阳离子类脂与DOPE的摩尔比为2:1、1:1和1:2)或胆固醇Chol(阳离子类脂与Chol的摩尔比为5:1、4:1、3:1、2:1和1:1)加入到1mL氯仿中充分溶解，均匀氮气流吹干，形成薄膜，真空干燥10h，除净有机溶剂。加入1mL无菌去离子水，恒温55℃下超声1-3h，至透明澄清，制备成终浓度为1.00mM的阳离子脂质体。
实施例5阳离子脂质体的粒径以及Zeta电位的测定
取约20μL实施例4制备的阳离子脂质体加入约1mL水中，利用Zeta-sizer-1000型激光粒径分析仪测定阳离子脂质体的粒径大小及分布(日本，HORIBA Scientific公司，激光波长630nm，散射角90°～173°)，结果如图4所示。利用Zetaplusζ-电位分析仪(日本，HORIBA Scientific公司)测定阳离子脂质体的Zeta电位，结果如图5所示。图4和图5中Lipofectamine 2000(美国Invitrogen公司)和DOTAP(N-(2,3-二油酰氧基-1-丙基)三甲基甲磺酸铵，瑞士Roche公司)是目前本领域中常使用的阳离子脂质体试剂。图4的结果表明，本发明的脂质体的粒径在100～350nm之间，粒径范围在有效转染粒径范围(<1μm)内。图5的结果表明，本发明的脂质体的Zeta电位在25～90mV之间，商品试剂DOTAP的Zeta电位为29.8mV和Lipofectamine 2000的Zeta电位为46.4mV，具有结合负电性离子的能力(包括核酸等分子)。
实施例6脂质体-DNA复合物的制备
首先将一定量的质粒DNA(pGFP-N2或pGL-3)稀释于DMEM中，制成25μL的体系；再取一定体积的浓度为1mg/mL实施例4制备的阳离子脂质体稀释于DMEM中，制成25μL的体系，然后将25μL脂质体稀释液滴加到25μL质粒DNA的稀释液中，使得脂质体和DNA的质量比(μg/μg)分别为1:1、2:1、3:1、4:1、6:1、8:1和10:1，用旋涡振荡器充分混匀，室温温育20min,使其形成50μL脂质体-DNA复合物。
实施例7阳离子脂质体与DNA的结合能力检测
取20μL实施例6制备的复合物溶液与1.6μL的上样缓冲液(loading buffer，日本Takara公司)混匀，加入到1.2％琼脂糖凝胶中，90V电泳1h。在凝胶成像系统中观察DNA电泳图谱并照相，如图6所示。图6a为单独的TSI14脂质体的结合DNA的能力测试结果；图6b，图6c和图6d分别为TSI14与DOPE分别以2:1、1:1和1:2混合制备的脂质体的结合DNA的能力测试结果。
图6a、图6b、图6c和图6d：第1泳道为2kb DNA marker(λDNA/EcoR I+Hind IIIMarkers，购自SABC)，第2泳道为裸DNA，第3-10泳道为脂质体与DNA比例分别是1:1、2:1、3:1、4:1、6:1、8:1和10:1的脂质体-DNA复合物。在未加入脂质体时(裸DNA)，出现典型的质粒条带。在加入脂质体后，DNA条带明显减弱，随着脂质体的加入量的增加，DNA的延滞能力明显增强，说明本发明所制备的脂质体具有与DNA结合的能力。
实施例8转染效率的测定
取实施例6制备的脂质体-DNA复合物在细胞系Hep-2(人喉癌上皮细胞)中进行条件实验，考察不同条件下的转染效率，优化试剂的使用条件。
将细胞种植于96孔细胞培养板中，每孔接种适量的细胞，细胞培养液(含血清和抗生素)总体积100μL。将细胞放置在37℃，5％CO2培养箱中孵24h使在转染日细胞密度达到1～1.5×104。
移去生长培养基，用等量(100μL，无血清和抗生素)培养基替换。直接将50μL实施例6的脂质体-DNA复合物样品加入板孔中，每个样品设3个平行孔，摇动培养板，轻轻混匀。在37℃、5％CO2培养箱中培养5h，更换含血清和抗生素的培养基，培育48h后进行基因表达分析。
基因表达分析：
(1)绿色荧光蛋白检测：用倒置荧光显微镜观察绿色荧光蛋白信号。阳性细胞发出明亮的绿色荧光，而阴性细胞则无，结果如图7所示。
(2)萤光素酶检测：先用PBS洗3次，然后每孔加入100μL裂解缓冲液，收集溶胞产物，在12000rpm、4℃离心5min，取20μL上清液用于荧光素酶活力的检测，用荧光素酶检测试剂，在Luminometer上进行检测相对光单元(RUL)值，测量发光时间为10s，结果如图8所示。
实验结果如图7和8所示，本发明制备的脂质体均具有转运DNA的能力，其中脂质体TSI14+C3/1与DNA比例为6/1和8/1时以及脂质体TSI14+D 1/1与DNA比例为10/1时的转染效率要高于商品试剂Lipofectamine2000和DOTAP。
实施例9MTT毒性测定
在96孔细胞培养板上种植Hep-2(人喉癌上皮细胞)，平行3孔，取平均值。每孔种植1.5×104个细胞/100μL培养液，在37℃，5％CO2环境下培养至90％汇合度。移去培养基，用D-Hank’s洗1次。质粒DNA(pGFP-N2)0.5μg和转染试剂分别稀释于DMEM 25μL中,然后将实施例6所制备的脂质体-DNA复合物加入到96孔细胞培养板里；同样Lipofectamine2000(Lipo)(与质粒的质量比为3∶1)和DOTAP(与质粒的质量比为6∶1)的复合物作为阳性对照加入到96孔细胞培养板里；采用不含阳离子脂质体的100μL培养基(无血清和无抗生素)作为阴性对照。在37℃，5％二氧化碳培养箱中继续培养，24h后向每孔加入20μL 3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐(MTT)溶液(5mg/mL inPBS,pH 7.4)，继续培养5h，移除培养基。生成的甲臜结晶用150μL二甲基亚砜(DMSO)溶解，剧烈混合使甲臜溶解。在酶标仪上进行读数，吸收波长570nm，酶标仪用无细胞的培养基调零。阳离子脂质体相对于对照细胞的相对存活率按下式计算：
[A]样品/[A]对照×100
[A]样品为测试孔的吸光值，[A]对照为阴性对照孔的吸光值。
实验结果如图9所示，本发明所制备的蔗糖酯型阳离子脂质体在Hep-2细胞中的细胞存活率普遍较高，均高于商品试剂Lipofectamine2000和DOTAP。
实施例10阳离子微胶粒的制备
将1mg的TSD-2D与一定量的蔗糖酯(阳离子类脂与蔗糖酯的摩尔比为2:1、1:1和1:2)、壳聚糖(阳离子类脂与壳聚糖的摩尔比为2:1、1:1和1:2)或棕榈酰油酰磷脂酰乙醇胺(阳离子类脂与棕榈酰油酰磷脂酰乙醇胺的摩尔比为5:1、4:1、3:1、2:1和1:1)加入溶于DMF中,制成浓度为1mg/mL的溶液。在剧烈搅拌的条件下逐滴加入去离子水,直至溶液轻度变浑,得到稳定的胶束溶液。将其转移至透析袋中,置于去离子水中透析,透析过程中多次更换去离子水3d后,配成一定浓度为1mg/mL水溶液。
实施例11微胶粒-siRNA复合物的制备
首先将一定量的siRNA稀释于DMEM中，制成25μL的体系；再取一定体积的浓度为1mg/mL实施例10制备的阳离子微胶粒稀释于DMEM中，制成25μL的体系，然后将25μL微胶粒稀释液滴加到25μL质粒DNA的稀释液中，使得微胶粒和siRNA的质量比(μg/μg)分别为1:1、2:1、3:1、4:1、6:1、8:1和10:1，用旋涡振荡器充分混匀，室温温育20min,使其形成50μL微胶粒-siRNA复合物。
实施例12阳离子微胶粒与siRNA的结合能力检测
取20μL实施例11制备的微胶粒-siRNA复合物溶液与1.6μL的上样缓冲液(loadingbuffer，日本Takara公司)混匀，加入到1.2％琼脂糖凝胶中，90V电泳1h。在凝胶成像系统中观察siRNA电泳图谱并照相。
Claims (8)
1.一类蔗糖酯型阳离子类脂化合物，其特征在于：具有通式I的结构：
其中：
R选自C10-20烃基、胆汁酸基和胆固醇丙烯酸酯基；
其中胆汁酸基的结构为：
其中胆固醇丙烯酸酯基的结构为：
Y选自-NRaRb和-N+RaRbRcX-，其中Ra、Rb和Rc相同或不同，并且选自氢、C1-6烃基、C1-6羟烷基、半乳糖基、甘露糖基和/或叶酸乙酯基；X-选自F-、Cl-、Br-、I-，
其中半乳糖基的结构为：
其中甘露糖基的结构为：
其中叶酸乙酯基的结构为：
2.权利要求1的蔗糖酯型阳离子类脂化合物的制备方法，其特征在于：包括如下步骤：
(1)三氯蔗糖和式i的化合物按照摩尔比1∶1～50反应，制备化合物A：
反应温度为10～100℃，反应时间为10～50h，反应溶剂为N，N-二甲基甲酰胺、吡啶、乙腈、哌啶或三乙胺，反应物与溶剂的质量体积比为1∶1～50；
式i的化合物选自C10-20烃基酸、胆汁酸或胆固醇丁烯二酸单酯；
(2)化合物A和式ii的化合物按照摩尔比1∶1～8反应，制备化合物B：
反应温度为10～120℃，反应时间为10～40h，压力为1.0～5.0atm，反应溶剂为N，N-二甲基甲酰胺、甲醇、乙醇、异丙醇或异丁醇，反应物与溶剂的质量体积比为1∶1～50；
式ii的化合物中Ra、Rb和Rc相同或不同，并且选自氢、C1-6烃基、C1-6羟烷基、半乳糖基、甘露糖基和/或叶酸乙酯基，化合物B中Y为-NRaRb或-N+RaRbRcX-，X-为F-、Cl-、Br-、I-。
3.根据权利要求2所述的蔗糖酯型阳离子类脂化合物的制备方法，其特征在于：当所合成的产物为季铵盐时，平衡阴离子F-、Br-、I-可以通过离子交换柱替换Cl-获得。
4.一种阳离子类脂组合物，其特征在于：该组合物是由权利要求1中的蔗糖酯型阳离子类脂化合物和类脂助剂组成，所述的蔗糖酯型阳离子类脂化合物与类脂助剂的质量比为10∶1～1∶10。
5.根据权利要求4所述的一种阳离子类脂组合物，其特征在于：所述类脂助剂选自卵磷脂、磷脂酰乙醇胺、糖脂、二油酰磷脂酰氯、棕榈酰油酰磷脂酰乙醇胺、二油酰磷脂酰乙醇胺、胆固醇、壳聚糖、蔗糖酯中的一种或两种以上的混合物。
6.根据权利要求4所述的阳离子类脂组合物，其特征在于：所述组合物为悬浮液，乳浊液，微胶粒或脂质体中的一种或两种以上的混合物。
7.一种阳离子类脂复合物，其特征在于：其是由权利要求4-6任意一项中的阳离子类脂组合物与核酸结合组成的物质，并且阳离子类脂组合物与核酸的质量比为20∶1～1∶20。
8.根据权利要求7所述的阳离子类脂复合物，其特征在于：核酸为pDNA、microRNA、siRNA中的一种或两种以上的混合物。
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