CN111265669A - 一种核酸转运用载体组合物及其在制备小干扰rna药物中的应用 - Google Patents
一种核酸转运用载体组合物及其在制备小干扰rna药物中的应用 Download PDFInfo
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- CN111265669A CN111265669A CN202010246551.1A CN202010246551A CN111265669A CN 111265669 A CN111265669 A CN 111265669A CN 202010246551 A CN202010246551 A CN 202010246551A CN 111265669 A CN111265669 A CN 111265669A
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Abstract
本发明公开了一种核酸转运用载体组合物及其在制备小干扰RNA药物中的应用。该组合物是由聚乙二醇‑聚乳酸及(聚酰胺‑胺)‑2‑丙酸‑3‑甲基马来酸酐‑聚己内酯两者混合均匀得到的。本发明将siRNA加入所述载体组合物溶液中,通过双乳化的方法得到siRNA与纳米颗粒的复合体,得到小干扰RNA药物。本发明不但可以制备出能够高效结合siRNA的纳米颗粒,降低siRNA与纳米颗粒完全结合所需最低NP比。而且该小干扰RNA药物能有效进入细胞,有效沉默致病靶基因的表达,并在体内抑制乳腺癌的生长。因而此给药体系在siRNA和类似小核酸药物的给药用于疾病治疗中具有良好的前景。
Description
技术领域
本发明属于小干扰RNA药物领域,具体涉及一种核酸转运用载体组合物及其在制备小干扰RNA药物中的应用。
背景技术
小干扰RNA能够简单高效地沉默靶基因的表达,因此成为研究基因功能与疾病治疗的重要工具。和小分子药物相比,siRNA能够特异性下调致病基因的表达,并不影响细胞中正常基因表达,这与传统的小分子药物相比更具有治疗潜力。2018年8月,Alnylam公司的Onpattro(patisiran)获美国和欧盟批准,用于遗传性ATTR(hATTR)淀粉样变性多发性神经病的治疗,成为RNAi现象被发现整整20年以来获准上市的首款RNAi药物,这标志着基因药物开发的一个重大里程碑。2019年11月,Alnylam公司的另一款RNAi药物Givlaari(givosiran)成为获得FDA批准的全球第二款RNAi药物,该药通过皮下注射给药,治疗急性肝卟啉症(AHP)成人患者,鼓舞了RNAi相关药物研发领域的热情。
然而,siRNA在人体内的给药面临着巨大挑战。由于siRNA分子在血液中不稳定,容易被机体清除,穿透细胞膜的能力极差等缺点,如何设计siRNA的给药***是RNAi疗法相关研究的焦点。RNAi传递的细胞内障碍。siRNA的载体设计要求可分为三个部分:1)有效荷载并保护siRNA;2)将荷载siRNA的***递送到靶点细胞;3)siRNA在细胞质中的有效释放。
目前用于siRNA给药***构建的材料主要包括阳离子胶束、阳离子脂质体和阳离子高分子聚合物等大类。这些载体与天然电负性的siRNA通过电荷相互作用形成复合物,从而实现荷载siRNA的目的。然而为实现高效的siRNA荷载率,一般研究中的氮磷比(NP比),即阳离子材料和siRNA的电荷比会比较高。而高NP比会增加阳离子材料消耗,升高给药***表面正电性而降低体内稳定性,使载体更容易被免疫***清除,并导致一定的毒性作用。如何使载体***能够高效荷载siRNA且降低阳离子材料与siRNA的比例,是相关研究的一个重难点。例如由Calando制药公司开发的siRNA药物CALAA-01,是第一个用于RNAi疗法肿瘤临床试验的聚合物类纳米载体。在CALAA-01的几个主要成分中,CDP能提供正电荷与siRNA通过电荷作用形成颗粒内核;金刚烷-PEG(AD-PEG)与金刚烷-PEG-转铁蛋白(AD-PEG-Tf)形成颗粒主要结构,其中PEG能够提高颗粒在体内循环中的稳定性,转铁蛋白Tf能够与肿瘤表面高表达的转铁蛋白受体CD71特异性结合,增强递送体系的肿瘤靶向能力。但是,由于少数患者的毒性反应,该体系已于2014年终止临床实验(KanastyR,Dorkin J R,Vegas A,etal.Delivery materials for siRNA therapeutics[J].Nature Materials,2013,12(11):967-977)。
发明内容
为了克服现有技术存在的上述不足,本发明的目的是提供一种核酸转运用载体组合物及其在制备小干扰RNA药物中的应用。
本发明的目的至少通过如下技术方案之一实现。
本发明的目的是提供一种核酸药物给药载体,具体涉及一种利用两种两亲性高分子聚合物材料共同制备的核酸药物的给药载体、载体制备方法和药物组合物。本发明提供的给药载体组合物具有很高的siRNA结合效率。
本发明提供的核酸转运用载体组合物是由两种两亲性高分子聚合物PEG-PLGA(聚乙二醇-聚乳酸)和PAMAM-CDM-PCL((聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯)共同制备的小干扰RNA(siRNA)的给药***和制剂。
本发明将siRNA加入到两种两亲性高分子材料PEG-PLGA和PAMAM-CDM-PCL的溶液中,通过双乳化的方法得到siRNA与纳米颗粒的复合体。这种方法不但可以制备高效结合siRNA纳米颗粒,而且纳米颗粒能有效进入细胞,有效沉默致病靶基因的表达,并在体内抑制乳腺癌的生长。
本发明提供的核酸转运用载体组合物是一种利用两亲性聚合物通过双乳化制备得到能够包载siRNA的给药***。
本发明提供的一种核酸转运用载体组合物,按重量份数计,包括:
聚乙二醇-聚乳酸 100份;
(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯 11-55份。
进一步地,所述聚乙二醇-聚乳酸中,聚乙二醇的嵌段分子量为3000-7000,聚乳酸的嵌段分子量为9000-13000。
进一步地,所述(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯中,聚酰胺-胺的代数为四代,聚酰胺-胺的分子量为14215,聚己内酯的分子量为3000-5000。
进一步地,其粒径为90-140纳米。所述核酸转运用载体组合物为纳米颗粒,可以在其纳米颗粒表明进行化学修饰、抗体修饰或配体修饰。
本发明提供的小干扰RNA药物,包含所述的核酸转运用载体组合物和小干扰RNA(siRNA)。
本发明提供的一种制备所述小干扰RNA药物的方法,包括如下步骤:
(1)将聚乙二醇-聚乳酸和(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯加入油相中,混合均匀,得到所述核酸转运用载体组合物的溶液,然后加入siRNA水溶液,超声处理,得到初始乳液;
(2)将步骤(1)初始乳液加入水相中,超声乳化处理,然后减压去除有机溶剂,得到所述药物组合物的水溶液。
进一步地,步骤(1)所述聚乙二醇-聚乳酸和(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯的质量比为100:11-55;所述油相为氯仿;所述聚乙二醇-聚乳酸与油相的质量体积比为3-5:1mg/mL。
进一步地,步骤(1)所述siRNA水溶液的浓度为2-3mg/mL;所述油相与siRNA水溶液的体积比为15-25:1。
进一步地,步骤(1)所述超声处理的时间为0.5-1.5分钟,超声处理的功率为60-100W。
进一步地,步骤(2)所述超声乳化处理的时间为0.5-1.5分钟,超声乳化处理的功率为60-100W。
优选地,步骤(2)所述减压去除有机溶剂的压力为1000帕。
优选地,步骤(2)所述初始乳液与水相的体积比为1:8
本发明提供的药物组合物,可以通过与细胞接触,使核酸导入到细胞内。
所述核酸为小干扰核酸(siRNA)。
本发明提供的药物组合物(两种两亲性高分子聚合物的组合)可以应用在制备核酸转运用载体组合物中。
所述核酸转运用载体组合物可以应用在制备抗肿瘤药物中的应用。所述肿瘤优选为乳腺肿瘤。
本发明提供的核酸药物给药载体由两种两亲性高分子聚合物材料共同制备而成。所述两亲性高分子聚合物是指在一个大分子链上同时含有亲水性和疏水性链段。其中一种两亲性共聚物为PEG-PLGA,其中PEG(聚乙二醇)的数均分子量为5000g/mol,PLGA(聚乳酸)数均分子量为11000g/mol。这种嵌段共聚物能够在水介质中自组装成胶束或纳米粒,相对疏水性的PLGA聚集成疏水性的核,PEG嵌段组装成亲水性的壳,具有稳定胶束、有效躲避生物体内质网***的捕捉和蛋白质吸附的作用。另一种两亲性聚合物为PAMAM-CDM-PCL,PAMAM(聚酰胺-胺)的代数为四代,PCL(聚己内酯)的数均分子量为4000g/mol,PAMAM和PCL通过一个小分子CDM相结合。PAMAM的表面具有许多氨基,在水溶液中能够质子化从而带正电,与带负电的siRNA通过静电相互作用形成复合物,在此给药***中的主要作用是通过提高核酸药物与载体的结合效率,降低siRNA与纳米颗粒完全结合所需最低NP比。
由上述两种两亲性高分子材料共同制备的给药载体能够形成纳米颗粒,所述纳米颗粒的直径约为130nm。
本发明的核酸转运用载体组合物适用的核酸,对其种类或结构没有特殊的限定。作为该核酸的具体例,可以为siRNA、mRNA、tRNA、rRNA、cDNA、miRNA(微RNA)、核酶、反义寡核普酸、质粒DNA、肽核酸、三链形成型寡核普酸(Triplex Forming Oligonucleotide,TFO)、基因等。其中,本发明的核酸转运用载体组合物在将siRNA向细胞内转运方面特别有效。本发明的核酸转运用载体适用的核酸,可以是来自人、动物、植物、细菌、病毒等的核酸,另外,也可以是通过化学合成制备的核酸。进而,上述核酸可以是单链、双链、三链中的任一种,并且对其分子量也没有特殊的限定。另外,本发明中,核酸可以为被化学、酶或肽修饰的核酸。
本发明中,核酸可以单独使用1种,也可以2种以上适当地组合使用。在一较佳的实施方式中,本发明的核酸转运用载体组合物优选转运小干扰核酸(siRNA)或其类似物。
本发明还提供了一种药物组合物,该组合物包含上述的核酸转运用载体组合物及核酸。药物组合物中适用的核酸,对其种类或结构没有特殊的限定。作为该核酸的具体例,可以为siRNA、mRNA、tRNA、rRNA、cDNA、miRNA(微RNA)、核酶、反义寡核普酸、质粒DNA、肽核酸、三链形成型寡核普酸(Triplex Forming Oligonucleotide,TFO)、基因等。在一较佳的实施方式中,优选为小干扰核酸(siRNA)。
本发明还提供了上述药物组合物的制备方法,所述方法包括如下步骤:将两种两亲性高分子材料溶于油相(氯仿等)中,加入siRNA水溶液后超声(80瓦,1分钟)形成初始乳液,将初始乳液加入到水相中并再次超声(80瓦,1分钟)乳化,将乳液减压(1000帕)除去有机溶剂,得到纳米颗粒水溶液。
本发明还提供了一种核酸导入方法,通过使上述药物组合物与细胞接触,将核酸导入到细胞内。所述细胞优选为哺乳动物细胞,更优选为病理状态下或非正常生理状态下的哺乳动物细胞,所述核酸优选为小干扰核酸(siRNA)。
与现有技术相比,本发明具有如下优点和有益效果:
(1)本发明提供的核酸转运用载体组合物,其具有良好的稳定性,制备方法简单,siRNA结合效率高,能保护siRNA免于降解,并能将核酸药物高效率地转运至细胞内,沉默靶点基因,达到相应治疗效果;
(2)本发明提供的核酸转运用载体组合物,能够输送特异性siRNA,在细胞和动物水平证明了其沉默靶基因表达的功效,以及沉默癌基因Plk1表达并抑制乳腺癌生长的效果。
附图说明
图1为实施例1中用双乳化方法和纳米沉淀法制备得到的核酸转运用载体组合物的电泳图;
图2为实施例1的核酸转运用载体组合物粒径图;
图3为荷载Cy5-siRNA的核酸转运用载体组合物与MDA-MB-231细胞培养5小时后在细胞内分布的激光共聚焦显微镜照片;
图4为荷载siPlk1的核酸转运用载体组合物进入MDA-MB-231细胞后下调Plk1的mRNA水平的效果图;
图5为尾静脉注射包载siPlk1的的靶向给药***抑制小鼠原位植入乳腺癌生长的效果图;
图6为治疗实验结束后降低肿瘤组织中Plk1的mRNA水平的效果图。
具体实施方式
以下结合实例对本发明的具体实施作进一步说明,但本发明的实施和保护不限于此。需指出的是,以下若有未特别详细说明之过程,均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者,视为可以通过市售购买得到的常规产品。
本发明所述的核酸转运用载体组合物由两种两亲性高分子聚合物材料共同制备而成。所述两亲性高分子聚合物是指在一个大分子链上同时含有亲水性和疏水性链段。其中一种两亲性共聚物为PEG-PLGA,其中PEG(聚乙二醇)的数均分子量为5000g/mol,PLGA(聚乳酸)数均分子量为11000g/mol。这种嵌段共聚物能够在水介质中自组装成胶束或纳米粒,相对疏水性的PLGA聚集成疏水性的核,PEG嵌段组装成亲水性的壳,具有稳定胶束、有效躲避生物体内质网***的捕捉和蛋白质吸附的作用。另一种两亲性聚合物为PAMAM-CDM-PCL,PAMAM(聚酰胺-胺)的代数为四代,PCL(聚己内酯)的数均分子量为4000g/mol,PAMAM和PCL通过一个小分子CDM(2-丙酸-3-甲基马来酸酐)相结合。PAMAM的表面具有许多氨基,在水溶液中能够质子化从而带正电,与带负电的siRNA通过静电相互作用形成复合物,在此给药***中的主要作用是通过提高核酸药物与载体的结合效率,降低siRNA与纳米颗粒完全结合所需最低NP比(PAMAM表面正电荷与siRNA负电荷之比)。
本发明的核酸转运用载体组合物适用的核酸,对其种类或结构没有特殊的限定。作为该核酸的具体例,可以为siRNA、mRNA、tRNA、rRNA、cDNA、miRNA(微RNA)、核酶、反义寡核普酸、质粒DNA、肽核酸、三链形成型寡核酸(Triplex Forming Oligonucleotide,TFO)、基因等。其中,本发明的核酸转运用载体组合物在将siRNA向细胞内转运方面特别有用。本发明的核酸转运用载体组合物适用的核酸,可以是来自人、动物、植物、细菌、病毒等的核酸,另外,也可以是通过化学合成制备的核酸。进而,上述核酸可以是单链、双链、三链中的任一种,并且对其分子量也没有特殊的限定。另外,本发明中,核酸可以为被化学、酶或肽修饰的核酸。本发明中,核酸可以单独使用1种,也可以2种以上适当地组合使用。
通过双乳化方法制备负载siRNA的纳米颗粒(即所述小干扰RNA药物),具体方法包括:将聚合物PEG5000-PLGA(2.25mg)和不同质量(参考表1)的PAMAM-CDM-PCL溶于0.5mL氯仿中,加入FAM-siRNA(0.025mL,0.055mg)溶液后超声下(超声功率80瓦,每超声五秒停止两秒,共超声一分钟)形成初始乳液后,将初始乳液加入到5mL DEPC水溶液中并再次超声乳化(声功率80瓦,每超声十秒停止两秒,共超声一分钟),减压下(1000帕)挥发有机溶剂,收集颗粒溶液。
实施例中所用原料来源及处理方法:
丙交酯(D,L-Lactide;CAS:4511-42-6),购自济南岱罡生物工程有限公司,使用前用升华瓶在真空下加热至90℃升华纯化,40℃下抽真空干燥12小时;
乙交酯(Glycolide;CAS:02-97-6)购自济南岱罡生物工程有限公司,使用前使用乙酸乙酯重结晶3次,溶剂质量比依次为70%,90%,130%,40℃下抽真空干燥12小时;
异辛酸亚锡(Tin(II)2-ethylhexanoate;CAS:301-10-0)购自Sigma-Aldrich,于手套箱中保存和取用。
单甲醚的聚乙二醇5000(mPEG5K;CAS:9004-74-4)购买自百灵威,使用前经甲苯135℃常压共沸除水(1g/10mL),随后降温至70℃抽真空搅拌干燥12h;
二氯甲烷(DCM;CAS:75-09-2),购自国药试剂,HPLC级别DCM经MB-SPS溶剂净化***纯化后接入手套箱内直接取用PEG5K-PLGA11K为本发明合成,在手套箱内称取单体和引发剂固体于干燥圆底烧瓶中,丙交酯和乙交酯物质的量之比为3:1,由目标聚合物的分子量计算投料比,考虑单体活性及反应损失,相对引发剂丙交酯投入1.2倍过量,乙交酯投入1.1倍过量。将烧瓶置于油浴中125℃加热,并缓慢搅拌约20min后引发剂和单体完全熔化混匀,搅拌过程中避免混合液飞溅到瓶壁上;确认无固态反应物后,用玻璃滴管取一滴(5-10mg)异辛酸亚锡,于烧瓶口正上方小心滴入,塞上瓶塞。反应持续约30min,在搅拌过程中体系粘稠度逐渐增大,可以适当减小转速以维持体系搅拌,待搅拌子无法正常搅拌后,取下反应体系,迅速取出手套箱并置于液氮中淬冷以终止反应。向冷却的体系中加入适量二氯甲烷,震荡溶解,旋蒸浓缩体系至20mL左右,将粘稠的浓缩液滴入0℃,5%甲醇的无水***中搅拌沉淀,过滤,干燥,最终得到白色固体聚合物。
羟基末端聚己内酯(PCL-OH)均聚物为本发明合成,三异丙醇铝(0.15g,1.0eqv)溶解于2mL无水甲苯中,加入到己内酯(10.0g,125.0eqv)中并在室温下反应1h,然后加入1mL冰醋酸并继续搅拌12小时。将得到的产物用二氯甲烷溶解并沉淀至冰***中,真空下干燥12h后得到白色固体产物,
PCL-CDM为本发明合成。合成方法包括:将CDM(2-丙酸-3-甲基马来酸酐,60mg,1.0eqv)溶解于4mL无水二氯甲烷中,再依次加入草酰氯(52mg,1.25eqv)、N,N-二甲基甲酰胺(DMF,80μL)。上述反应先置于冰水浴中反应10min,然后转移至室温反应2h。在真空条件下除去二氯甲烷、N,N-二甲基甲酰胺和过量的草酰氯,得到酰氯化CDM中间产物。将酰氯化CDM溶解于4mL无水二氯甲烷中,将预先通过甲苯共沸除水干燥后的PCL-OH(0.20g,0.33eqv)溶解于3mL无水二氯甲烷,置于干燥的恒压滴液漏斗于冰水浴中缓慢加入,滴加完毕后转移至室温继续反应2h。加入饱和氯化铵水溶液(20mL)消耗过量的酰氯化CDM后,用氯仿萃取三次(20mL×3),干燥浓缩有机相后,沉淀至冰***中,真空下干燥12h后得到浅棕色固体产物。
***聚酰胺-胺树枝状高分子(PAMAM,G4,Mn=14,215g/mol)购自美国Dendritech公司。
PCL-CDM-PAMAM通过PCL-CDM中酸酐与PAMAM/Pt表面氨基的开环反应合成。PAMAM(142mg,1.0eqv)与PCL-CDM(40mg,1.0eqv)溶解于5mL二甲亚砜中,并在避光条件下于室温下搅拌2h。于搅拌条件下加入20mL超纯水使产物组装成纳米颗粒,进而通过超滤(MWCO=100,000)进行纯化,收集上层溶液并冻干,得到白色固体。
Alexa 488-phalloidin、LipofectamineRNAiMAX购于Invitrogen公司。
RNAiso Plus、PrimeScriptTM RT reagent Kit(Perfect Real Time)购自TaKaRa公司。
FastStart Universal SYBR Green Master(ROX)购自Roche公司。
siRNA是一种由二十多个核苷酸组成的双链小分子RNA,带有负电荷。
以下实验使用中的siPlk1,对应反义链序列为:
UAAGGAGGGUGAUCUUCUUCAdTdT。
对照阴性siRNA(siN.C.),对应反义链序列为:
AACCACUCAACUUUUUCCCAAdTdT。
Cy5-siRNA为荧光染料Cy5标记的siN.C.。FAM-siRNA为荧光染料FAM标记的siN.C.。
以上siRNA均由苏州瑞博制药技术有限公司合成。
未经说明的其它试剂直接使用。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
实施例一.siRNA给药***的制备与表征
使用两亲性嵌段共聚物PEG-PLGA和两亲性聚合物PAMAM-CDM-PCL,利用超声双乳化的方法制备负载siRNA的纳米颗粒。
1.不同NP比下颗粒与siRNA结合能力
制备负载siRNA的纳米颗粒,具体方法为:将聚合物PEG-PLGA(2.25mg)和不同质量(参考表1)的PAMAM-CDM-PCL分别溶于0.5mL氯仿中,加入siRNA(0.025mL,0.055mg)溶液后超声下(超声功率80瓦,每超声五秒停止两秒,共超声一分钟)形成初始乳液后,将初始乳液加入到5mL DEPC水中并再次超声乳化(声功率80瓦,每超声十秒停止两秒,共超声一分钟),减压下(1000帕)挥发有机溶剂,并将颗粒溶液浓缩至1ml。
表1.不同NP比的纳米颗粒制备中各组分的投料量
得到不同NP比的颗粒后,通过凝胶电泳检测siRNA的结合。每种颗粒取20μL加入4μL6×loading buffer(擎科,TSJ010),混匀后将含20pmol siRNA的上述溶液于2%琼脂糖凝胶、100V下和三羟甲基氨基甲烷-硼酸-乙二胺四乙酸缓冲液中电泳15分钟。siRNA条带在凝胶成像仪下检测。
从图1可见,纳米颗粒(即所述核酸转运用载体组合物)可以在NP比等于4的时候完全结合siRNA。
2.纳米颗粒表征
以NP比为4的纳米颗粒(即所述核酸转运用载体组合物)为实例说明其性质。利用马尔文Malvern Zetasizer Nano ZSE纳米粒度电位仪测量颗粒的粒径。
从图2可见,得到的纳米颗粒粒径强度统计在130nm左右。
实施例二.此给药***在细胞水平的效应评价
NP比为4时,纳米颗粒(即所述核酸转运用载体组合物)可以完全结合siRNA,并且此NP比下使用的材料最少,故将NP比为4的纳米颗粒命名为NCN(Nano-confinedNanoparticle),并以此实例比来说明此给药***的生物学效应。
1.纳米颗粒进入细胞的能力
用实施例1中所述方法制备包载Cy5-siRNA的NCN来研究此给药***的细胞摄取。
将包载FAM-siRNA纳米颗粒(Cy5-siRNA终浓度为100nM)与人乳腺癌MDA-MB-231细胞(24孔板,5×104细胞/孔)在37℃共同培养5小时后,用冰PBS洗涤细胞3次,使用4wt%的多聚甲醛固定细胞15分钟。去除4wt%多聚甲醛,PBS洗涤细胞3次。随后用0.1%的TritonX-100(聚乙二醇辛基苯基醚)穿膜5分钟,PBS缓冲液洗涤细胞3次。加入含1wt%BSA的PBS进行封闭,室温避光放置20分钟。以1:100倍稀释的Alexa Fluor 488 phalloidin(Invitrogen,A12379)标记细胞骨架,室温标记20分钟,PBS缓冲液洗3次。用含1μg/mL 4',6-二脒基-2-苯基吲哚(DAPI)的PBS缓冲液染色5分钟,PBS缓冲液洗3次。最后用抗荧光淬灭剂进行封片,封片后使用型号为Zeiss LSM880的激光共聚焦显微镜进行观察。结果见图3。
图3为荷载Cy5-siRNA的核酸转运用载体组合物(包载Cy5-siRNA的NCN)与MDA-MB-231细胞培养5小时后在细胞内分布的激光共聚焦显微镜照片;图3中,细胞内绿色荧光来源于Alexa 488-phalloidin标记的细胞骨架;红色荧光来源于Cy5-siRNA;蓝色荧光来源于DAPI标记的细胞核。通过三种颜色叠加的结果可知5小时培养后,纳米颗粒能有效进入细胞胞浆中,呈现出颗粒状分布。
2.细胞水平包载Plk1 siRNA的纳米颗粒对靶基因的沉默效果。
用实施例1中所述方法制备包载siRNA的NCN来研究此给药***的体外沉默水平。使用的siRNA分别为siPlk1和siN.C.,Plk1有助于促进和加速哺乳动物细胞有丝***,并在多种肿瘤细胞中高表达。通过沉默其表达,可以抑制肿瘤生长。
将MDA-MB-231细胞以1×105细胞/孔的密度接种于12孔板,37℃培养12小时后,分别进行如下处理:
处理一(对照组):加入等体积PBS溶液。
处理二(裸siPlk1组):加入等体积siPlk1溶液,siPlk1终浓度为100nM。
处理三(RNAiMAX组):用载siPlk1的RNAiMAX溶液处理细胞,siPlk1终浓度为20nM。
处理四(NCN组):用携载siPlk1的纳米颗粒(包载siPlk1的NCN)处理细胞,siPlk1的终浓度为100nM。
在转染培养24h后,用RNAiso Plus(TaKaRa)提取细胞中的总RNA,用Nanodrop测定提取的RNA样品浓度,然后用PrimeScriptTM RT reagent Kit(Perfect Real Time)合成cDNA,每个样品使用2μg总mRNA。在合成cDNA后,按照FastStart Universal SYBR GreenMaster(ROX)试剂盒进行实时荧光定量PCR反应。其中Plk1和甘油三磷酸脱氢酶GAPDH基因的PCR引物如下:
Plk1-上游引物5'-AGCCTGAGGCCCGATACTACCTAC-3',
Plk1-下游引物5'-ATTAGGAGTCCCACACAGGGTCTTC-3';
GAPDH-上游引物5'-TTCACCACCATGGAGAAGGC-3',
GAPDH-下游引物5'-GGCATGGACTGTGGTCATGA-3'。
PCR反应如下:
1)95℃预热600秒。
2)95℃加热15秒,57℃加热30秒,72℃加热30秒。此步循环45次。
3)37℃加热30秒。
利用2-ΔΔCT对不同实验组中Plk1基因表达差异进行了分析,其中以GAPDH为内参,分析不同实验组中Plk1基因表达水平。以PBS实验组为100%,其他实验组表达表示为相对于PBS组表达量,实验结果见图4。
图4中,不经过处理的PBS组细胞内Plk1表达量高,包载siN.C.的实验对照组对细胞中Plk1基因表达没有明显影响,而包载siPlk1的实验组(处理四组)可以有效抑制Plk1基因的表达。
实施例三.此给药***在动物水平的生物效应评价
1.携载Plk1 siRNA的纳米颗粒对肿瘤生长的抑制作用
在裸鼠第二个乳腺的脂肪垫下原位接种MDA-MB-231细胞(0.5×107),9天左右形成可见肿瘤,肿瘤体积约50mm3,随机分成三组,进行尾静脉注射治疗,每两天注射一次。肿瘤的体积按公式计算:V=0.5×a×b2计算,其中a指肿瘤长径,b指肿瘤短径。
处理一(PBS对照组):每只裸鼠注射200μL PBS。
处理二(裸siPlk1组):每只裸鼠注射200μL裸siPlk1,siPlk1剂量为40μg。
处理三(NCN/siN.C.组):每只裸鼠注射包载siN.C.的纳米颗粒NCN,聚合物质量为2.2mg,siN.C.剂量为40μg。
处理四(NCN/siPlk1组):每只裸鼠注射包载siPlk1的纳米颗粒NCN,聚合物质量为2.2mg,siPlk1剂量为40μg。
在治疗开始后,每隔一天对肿瘤体积进行测量。实施例结果如图5所示,所有阴性对照组中肿瘤生长速度均较快,而在使用包载siPlk1纳米颗粒治疗组中,肿瘤生长速度与阴性对照组相比受到明显抑制。说明本发明提供的包载siRNA纳米颗粒(所述小干扰RNA药物)在体内能够有效沉默致癌基因,从而抑制肿瘤生长。
2.携载Plk1 siRNA的纳米颗粒对肿瘤部位Plk1基因的沉默效果
收集治疗结束后各组荷瘤小鼠的肿瘤组织。取部分肿瘤组织,用RNAiso Plus(TaKaRa)提取细胞中的总RNA,用Nanodrop测定提取的RNA样品浓度,然后用PrimeScriptTMRT reagent Kit(Perfect Real Time)合成cDNA,每个样品使用2μg总mRNA。在合成cDNA后,按照FastStart Universal SYBR Green Master(ROX)试剂盒进行实时荧光定量PCR反应。相关引物序列与实时荧光定量PCR反应具体步骤与细胞水平实验相同。结果如图6所示,阴性对照组中肿瘤部位Plk1基因的表达水平与PBS组相比无明显区别,而使用包载siPlk1纳米颗粒治疗组中,肿瘤部位Plk1基因的表达受到明显抑制。
由上述结果可知,此siRNA给药***在动物水平能够有效抑制肿瘤生长,并且这种抑制效果是由于沉默肿瘤部位靶基因的表达导致的。
以上实施例仅为本发明较优的实施方式,仅用于解释本发明,而非限制本发明,本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。
Claims (10)
1.一种核酸转运用载体组合物,其特征在于,按重量份数计,包括:
聚乙二醇-聚乳酸 100份;
(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯 11-55份。
2.根据权利要求1所述的核酸转运用载体组合物,其特征在于,所述聚乙二醇-聚乳酸中,聚乙二醇的嵌段分子量为3000-7000,聚乳酸的嵌段分子量为9000-13000。
3.根据权利要求1所述的核酸转运用载体组合物,其特征在于,所述(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯中,聚酰胺-胺的代数为四代,聚酰胺-胺的分子量为14215,聚己内酯的分子量为3000-5000。
4.根据权利要求1所述的核酸转运用载体组合物,其特征在于,其粒径为90-140纳米。
5.一种小干扰RNA药物,其特征在于,包含权利要求1-3任一项所述的核酸转运用载体组合物和小干扰RNA。
6.一种制备权利要求5所述的小干扰RNA药物的方法,其特征在于,包括如下步骤:
(1)将聚乙二醇-聚乳酸、(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯加入油相中,然后加入siRNA水溶液,超声处理,得到初始乳液;
(2)将步骤(1)所述初始乳液加入水相中,超声乳化处理,然后减压去除有机溶剂,得到所述小干扰RNA药物的水溶液。
7.根据权利要求6所述的小干扰RNA药物的制备方法,其特征在于,步骤(1)所述聚乙二醇-聚乳酸和(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯的质量比为100:11-55;所述油相为氯仿;所述聚乙二醇-聚乳酸与油相的质量体积比为3-5 :1mg/mL。
8.根据权利要求6所述的小干扰RNA药物的制备方法,其特征在于,步骤(1)所述siRNA水溶液的浓度为2-3 mg/mL;所述油相与siRNA水溶液的体积比为15-25:1。
9.根据权利要求6所述的小干扰RNA药物的制备方法,其特征在于,步骤(1)所述超声处理的时间为0.5-1.5分钟,超声处理的功率为60-100 W。
10.根据权利要求6所述的小干扰RNA药物的制备方法,其特征在于,步骤(2)所述超声乳化处理的时间为0.5-1.5分钟,超声乳化处理的功率为60-100 W。
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