CN109432443B - CT/荧光双模态成像荷载siRNA的纳米粒及制备方法和应用 - Google Patents
CT/荧光双模态成像荷载siRNA的纳米粒及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种CT/荧光双模态成像荷载siRNA的纳米粒及制备方法和应用，该纳米粒包括载体成分、药物成分、CT成像成分和荧光成像成分。载体成分是脂质，药物成分是siRNA，CT成像成分是粒径5nm左右的金纳米颗粒，荧光成像成分是Cy5（碳氰类染料）。利用乙醇稀释法制备以脂质分子包裹siRNA、金纳米颗粒与Cy5而形成的纳米颗粒，可以有效的克服药物成分siRNA在体内的递送障碍，还能赋予金纳米颗粒本身不具有的肿瘤靶向特性和较长的CT成像时间窗。
Description
技术领域
本发明涉及多模态成像和siRNA递送技术领域，尤其是一种CT/荧光双模态成像荷载siRNA的纳米粒及制备方法和应用，是同时实现CT成像、荧光成像监控siRNA在体内的释放位点与时间的有效方法。
背景技术
siRNA因其显著的基因沉默效果带来的应用前景而备受关注。然而裸露的siRNA存在易被核酶降解、细胞内吞效果差和细胞靶向能力差等缺点，建立合适的基因递送系统尤为关键。纳米递送系统因其安全性、低毒性、低免疫原性、靶向性及易于组装等优点而受到广泛研究。纳米粒表面修饰亲水聚合物如聚乙二醇PEG可增加体内循环时间，从而易于通过EPR效应(高渗透与滞留效应)被动蓄积在肿瘤内部。传统的基因药物纳米递送系统主要包括脂质体和阳离子聚合物两大类。近年发展起来的阳离子脂质纳米粒是融合了阳离子聚合物和脂质体的载药优势而设计的递送系统。
siRNA在体内必须通过细胞内Dicer酶切才能发挥RNA干扰效果，而这一过程的发生位点与发生时间无法被实时监控，目前只能通过处死动物取出靶器官做PCR实验考察基因表达量来评价siRNA的体内疗效。如果能用成像技术实时监测基因药物在体内释放及发挥基因沉默效果的部位与时间，无疑将对基因治疗领域起到关键性指导作用。
在众多成像技术中，CT成像是应用最广泛的诊断成像技术，其缺点是软组织对比度差，无法显示各组织器官。目前临床上一般通过引入CT造影剂来增强病变组织与正常组织的对比度，从而提高CT对病灶组织的分辨能力。目前应用广泛的造影剂基本是含碘元素的有机化合物，但其分子量较小，难以在血液中长循环，导致成像时间窗窄、缺乏组织特异性、难以对病灶组织造影。因此，许多原子序数较大的金属元素化合物如金、铋、镱等被开发作为良好的CT造影剂。
荧光成像一般灵敏度高，低浓度的荧光探针就可进行明显的荧光成像，但其应用受限于组织穿透能力低。任何成像模式均有其固有缺陷，仅靠一种成像模式难以提供全面信息。将CT成像与荧光成像结合开发出CT/荧光双模态成像对比剂，可以结合两种成像的优势、弥补各自的缺陷，实现更灵活更多样的功能。
发明内容
本发明的目的是提供一种CT/荧光双模态成像荷载siRNA的纳米粒，该纳米粒可作为基因药物递送系统将siRNA递送至靶部位。
本发明的另一目的是提供所述CT/荧光双模态成像荷载siRNA的纳米粒的制备方法。
本发明的另一目的是提供该纳米粒在CT成像、荧光成像中的应用。
实现本发明目的的具体技术方案是：
一种CT/荧光双模态成像荷载siRNA的纳米粒，特点是：该纳米粒包括载体成分、药物成分、CT成像成分和刺激响应性荧光成像成分，所述载体成分为具有微正电特性的阳离子脂质1，2-二羟基-3-二甲基氨基丙烷DlinDMA、PEG修饰的脂质聚乙二醇二甲基丙烯酸酯PEG-C-DMA、二硬脂酸磷脂酰胆碱DSPC及胆固醇Cholesterol，其摩尔比为30-40∶5-10∶5-10∶30-40；药物成分为抑制Survivin基因表达的siRNA；CT成像成分为金纳米颗粒；刺激响应性荧光成像成分为碳氰类近红外染料Cy5；碳氰类近红外染料Cy5通过化学修饰位于siRNA反义链的3’端，而正义链的5’端修饰有巯基，siRNA通过正义链5’端的巯基与CT成像成分纳米金相连；然后包裹上载体成分构成所述的纳米粒；其中：载体成分、药物成分、CT成像成分和刺激响应性荧光成像成分的摩尔比为1∶7-8∶210-240∶7-8。
一种上述的CT/荧光双模态成像荷载siRNA的纳米粒的制备方法，该方法包括以下具体步骤：
步骤1：配制溶液
1)Au-siRNA水相溶液
ⅰ)制备金纳米颗粒
将氯金酸溶液加入水中，磁力搅拌后，加入柠檬酸钠溶液，搅拌，逐滴加入新配置的预冷硼氢化钠，此时溶液逐渐变为酒红色，继续搅拌即得金纳米颗粒，加入焦碳酸二乙酯(DEPC)搅拌过夜后高温高压灭菌除去可降解siRNA的核酶，得到纯化金纳米颗粒；制备过程中，氯金酸、柠檬酸钠、硼氢化钠的摩尔比为1∶7∶0.4；
ⅱ)将siRNA与纯化纳米金相连
siRNA正义链加5％-10％体积的100nM浓度的三(2-羧乙基)膦盐酸盐激活1h-3h，加入与siRNA正义链等摩尔量的已被DEPC水溶解了的siRNA反义链，75℃-90℃水浴10至30min，室温冷却后加入30倍摩尔量的纯化金纳米颗粒，1％-2％体积的饱和氯化钠溶液，每超声一段时间后加入1％-2％体积的饱和氯化钠溶液，共超声12h至36h；加20％-40％体积的异丙醇后高速离心；沉淀物即为金纳米颗粒与siRNA的偶联物Au-siRNA；
ⅲ)配制Au-siRNA水相溶液
配制柠檬酸溶液、用氢氧化钠溶液调至pH＝5，加入氯化钠溶解，混合后得水相溶液；
水相溶液中加入Au-siRNA溶解，即得Au-siRNA水相溶液；
2)脂类油相溶液
8-9体积的无水乙醇与1-2体积的超纯水混合摇匀得油相溶剂，脂类溶质为阳离子脂质1，2-二羟基-3-二甲基氨基丙烷DlinDMA、PEG修饰的脂质聚乙二醇二甲基丙烯酸酯PEG-C-DMA、二硬脂酸磷脂酰胆碱DSPC及胆固醇Cholesterol，将其按照摩尔比为1∶7-8∶210-240∶7-8溶解于所述油相溶剂中，即得脂类油相溶液；
步骤2：搭建制备装置
用两个T形三孔接头连接两个注射器和一个超滤离心管，构成制备装置；
步骤3：纳米粒制备
将搭建好的制备装置置于恒温条件下，两个注射器中分别吸入所述的水相溶液和油相溶液；两个注射器同时挤出，超滤离心管接收制备出的纳米粒，并经超滤离心管离心除去溶剂、稀释液及断裂的siRNA；在超滤离心管内加入磷酸盐缓冲溶液获得纳米粒子溶液，过微孔滤膜除菌后保存。
一种上述CT/荧光双模态成像荷载siRNA的纳米粒在CT成像中的应用。
一种上述CT/荧光双模态成像荷载siRNA的纳米粒在荧光成像中的应用。
本发明的有益效果
本发明的纳米粒Au-siRNA@LP的成像成分包括金纳米颗粒与Cy5荧光成像分子。其中金纳米粒对X射线可产生较强的吸收，较大的分子量使其不容易被肾小球滤过、血液循环时间更长，可显著延长CT成像时间窗，用来作为CT成像对比剂增加软组织之间对比度；由于金和巯基基团有很强的配位作用，易于修饰带有巯基和Cy5成像分子的siRNA；其次纳米金在540纳米左右对光有很强的吸收，Cy5荧光分子靠近纳米金会发生FRET效应(荧光共振能量转移作用)，使荧光处于猝灭状态，而当siRNA被细胞内的Dicer酶剪切脱落时，Cy5离开纳米金表面从而使荧光恢复。载体成分包括阳离子脂质、聚乙二醇-脂质、膜融合脂质和胆固醇，这些成分在递送功能中分别发挥着不同的作用。其中阳离子脂质能够使得脂质双层和带负电荷的核酸产生静电吸引作用，从而增加核酸药物包封率；聚乙二醇-脂质通过聚乙二醇的水化作用，进入体内可以避免网状内皮系统的吞噬，使纳米粒在血液中长循环，从而延长CT成像时间窗且获得良好的血池造影效果，也有利于纳米粒更多的被动靶向至肿瘤区域，从而获得良好的肿瘤造影效果；膜融合脂质可参与体内膜融合，有利于纳米粒被细胞吞噬，从而释放出基因药物；胆固醇能够提高脂质纳米粒整体结构的稳定性。该纳米粒不仅可以有效的实现基因药物在体内的传递，解决其递送障碍发挥治疗效果，而且可以进行CT成像和荧光成像，其中CT成像可为纳米粒的体内分布提供影像学指导，荧光成像可以实时监控药物成分在体内的释放时间与位点。
附图说明
图1为用动态光散射仪测量本发明纳米粒的粒径图；
图2为用透射电镜测量本发明纳米粒的形貌图；
图3为未包裹载体材料的纳米粒的荧光淬灭图；
图4为未包裹载体材料的纳米粒在Dicer酶作用下的荧光恢复图；
图5为向4T1小鼠皮下乳腺癌模型注射本发明纳米粒Au-siRNA@LP后的活体荧光成像图；
图6为向4T1小鼠皮下乳腺癌模型注射本发明纳米粒Au-siRNA@LP后3小时的CT成像图。
具体实施方式
以下结合附图及试验实例对本发明进行详细描述。
本发明纳米粒的制备
1)配制溶液
ⅰ)Au-siRNA水相溶液
首先制备金纳米颗粒：用王水浸泡实验器具过夜，用去离子水充分洗涤，将1体积的1％的氯金酸溶液加入80体积的水中，磁力搅拌5分钟后，一次性加入7体积的1％柠檬酸钠溶液，搅拌5分钟后，逐滴缓慢加入8体积的新配置的4℃0.05％硼氢化钠，溶液逐渐变为酒红色，继续搅拌30分钟即得金纳米颗粒，按溶液最后体积加入1％体积的纯焦碳酸二乙酯(DEPC)溶液搅拌过夜后高温高压灭菌(120℃，15min)除去可降解siRNA的核酶，用ICP-MS定量纳米金溶液中金元素含量。其次将siRNA与纳米金相连：每10D的正义链加250ul DEPC水和2ul 100nM TCEP激活1h，加入10D已被250ulDEPC水溶解了的反义链，75℃水浴10min，室温冷却后加入按siRNA比金元素摩尔比1∶30的量加入金纳米颗粒，10ul的饱和氯化钠溶液，每超声4h后加入10ul的饱和氯化钠溶液，共计3次，共超声12h。加500ul异丙醇后14000rpm/min离心20min，DEPC水洗涤三次。沉淀物即为Au-siRNA。最后配置Au-siRNA水相溶液：配制200mM的柠檬酸溶液、2M的氢氧化钠溶液调至pH5，加入150mM的氯化钠，混合溶解后得水相溶液，每1mL水相溶液中加入40nmol siRNA量的Au-siRNA，即得到Au-siRNA水相溶液，其中siRNA浓度约为40nmol/ml；
ⅱ)脂类油相溶液
按无水乙醇与超纯水的体积比为9︰1配制，摇匀得油相溶液，脂类溶质包括阳离子脂质DLinDMA、PEG修饰的脂质PEG-C-DMA、磷脂DSPC及胆固醇Cholesterol，将分别称量的脂类溶质溶解于同一个油相溶剂中，使其溶解在油相溶液中的浓度分别为1.32nmol/ml，0.16nmol/ml，0.33nmol/ml，1.48nmol/ml，溶解后即得到脂类的油相溶液；各脂质的化学结构式：
阳离子脂质DlinDMA
脂质DSPC
PEG修饰的脂质PEG-C-DMA
胆固醇
2)搭建制备装置
用两个T形三孔接头连接两个注射器和一个截留分子量为的100K的超滤离心管用于接收纳米粒，构成制备装置；
3)纳米粒的制备与表征
将搭建好的制备装置置于37℃恒温条件下，两个注射器中分别吸入Au-siRNA水相溶液、脂类油相溶液，体积比为1∶1。两个注射器同时挤出，脂类油相溶液中的阳离子脂类与负电性的siRNA结合并与其它脂类通过静电吸引力和疏水作用力自发形成油包水型的纳米结构；超滤离心管端接收制备出的纳米粒，并经100K截留分子量的超滤离心管以4500转/分钟的转速离心812分钟，除去溶剂及断裂的siRNA，在超滤离心管内加入1体积的等渗溶液(磷酸盐缓冲液或注射用生理盐水)，通过0.22um微孔滤膜除菌，将得到的纳米粒溶液置于4℃冰箱保存。吸取500ul纳米粒溶液用马尔文动态光散射仪测定粒径大小，结果如图1所示，粒径在70nm左右。吸取10ul纳米粒Au-siRNA@LP溶液滴于铜网上在烘干机内彻夜烘干，用透射电镜拍照观察纳米粒形貌，结果如图2所示，形貌均一。
2.荧光猝灭-恢复效率的检测
将1/50D的siRNA-Cy5溶解于1mL的水中测定荧光强度，将1/50DsiRNA量的Au-siRNA溶解于1mL的水中测定荧光强度。考察金纳米颗粒对siRNA-Cy5的荧光淬灭能力，结果如图3所示，金纳米颗粒加入后荧光值明显降低。向1/50D siRNA量的Au-siRNA中加入Dicer酶与ATP，5min后测量荧光值，考察Au-siRNA的荧光恢复效率，结果如图4所示，荧光接近100％的恢复。
4.肿瘤动物模型的建立及双模态纳米粒的成像功能检测
1)肿瘤动物模型的建立与活体荧光成像效果检测
5周大babl/c裸鼠皮下注射200ul 106/100ul 4T1小鼠乳腺癌细胞，一周后测量肿瘤大小大于200mm3认证肿瘤模型构建成功。尾静脉注射200ul的纳米粒Au-siRNA@LP，剂量按siRNA剂量10D/20g体重注射。异氟烷气体麻醉各小鼠后放置于小动物活体成像仪内，激发光波长610，收集660纳米的红光成像。结果如图5所示，纳米粒Au-siRNA@LP在肿瘤部位有明显的荧光。
2)CT成像效果检测
尾静脉注射200ul金浓度为10mM的纳米粒Au-siRNA@LP。异氟烷气体麻醉各小鼠后放置于小动物活体CT成像仪内，100kev强度的X射线照射小鼠身体中部，第3h观测肿瘤CT造影效果。结果如图6所示，成像时间宽、肿瘤部位有明显的造影。
Claims (2)
1.一种CT/荧光双模态成像荷载siRNA的纳米粒，其特征在于，该纳米粒包括载体成分、药物成分、CT成像成分和刺激响应性荧光成像成分，所述载体成分为具有微正电特性的阳离子脂质1,2-二羟基-3-二甲基氨基丙烷DlinDMA、PEG修饰的脂质聚乙二醇二甲基丙烯酸酯PEG-C-DMA、二硬脂酸磷脂酰胆碱DSPC及胆固醇Cholesterol，其摩尔比为30-40∶5-10∶5-10∶30-40；药物成分为抑制Survivin基因表达的siRNA；CT成像成分为金纳米颗粒；刺激响应性荧光成像成分为碳氰类近红外染料Cy5；碳氰类近红外染料Cy5通过化学修饰位于siRNA反义链的3’端，而正义链的5’端修饰有巯基，siRNA通过正义链5’端的巯基与CT成像成分纳米金相连；然后包裹上载体成分构成所述的纳米粒；其中：载体成分、药物成分、CT成像成分和刺激响应性荧光成像成分的摩尔比为1∶7-8∶210-240∶7-8。
2.一种权利要求 1 所述的CT/荧光双模态成像荷载siRNA的纳米粒的制备方法，其特征在于，该方法包括以下具体步骤：
步骤1：配制溶液
1）Au-siRNA-Cy5水相溶液
ⅰ）制备金纳米颗粒
将氯金酸溶液加入水中，磁力搅拌后，加入柠檬酸钠溶液，搅拌，逐滴加入新配置的预冷硼氢化钠，此时溶液逐渐变为酒红色，继续搅拌即得金纳米颗粒，加入焦碳酸二乙酯（DEPC）搅拌过夜后高温高压灭菌除去可降解siRNA的核酶，得到纯化金纳米颗粒；制备过程中，氯金酸、柠檬酸钠、硼氢化钠的摩尔比为1∶7∶0.4；
ⅱ）将siRNA-Cy5与纯化纳米金相连
siRNA-Cy5正义链加5%-10%体积的100nM浓度的三（2- 羧乙基）膦盐酸盐激活1h-3h，加入与siRNA-Cy5正义链等摩尔量的已被DEPC水溶解了的siRNA-Cy5反义链，75℃-90℃水浴10至30min，室温冷却后加入30倍摩尔量的纯化金纳米颗粒，1%-2%体积的饱和氯化钠溶液，每超声一段时间后加入1%-2%体积的饱和氯化钠溶液，共超声12h至36h；加20%-40%体积的异丙醇后高速离心；沉淀物即为金纳米颗粒与siRNA-Cy5的偶联物Au-siRNA-Cy5；
ⅲ）配制Au-siRNA-Cy5水相溶液
配制柠檬酸溶液、用氢氧化钠溶液调至pH=5，加入氯化钠溶解，混合后得水相溶液；
水相溶液中加入Au-siRNA-Cy5溶解，即得Au-siRNA-Cy5水相溶液；
2）脂类油相溶液
8-9体积的无水乙醇与1-2体积的超纯水混合摇匀得油相溶剂，脂类溶质为阳离子脂质1,2-二羟基-3-二甲基氨基丙烷DlinDMA、PEG修饰的脂质聚乙二醇二甲基丙烯酸酯PEG-C-DMA、二硬脂酸磷脂酰胆碱DSPC及胆固醇Cholesterol，将其按照摩尔比为1∶7-8∶210-240∶7-8溶解于所述油相溶剂中，即得脂类油相溶液；
步骤2：搭建制备装置
用两个T形三孔接头连接两个注射器和一个超滤离心管，构成制备装置；
步骤3：纳米粒制备
将搭建好的制备装置置于恒温条件下，两个注射器中分别吸入所述的水相溶液和油相溶液；两个注射器同时挤出，超滤离心管接收制备出的纳米粒，并经超滤离心管离心除去溶剂、稀释液及断裂的siRNA-Cy5；在超滤离心管内加入磷酸盐缓冲溶液获得纳米粒子溶液，过微孔滤膜除菌后保存。
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