CN114209848A - 一种具有运输siRNA功能的铂肽共聚物的制备方法及其应用 - Google Patents
一种具有运输siRNA功能的铂肽共聚物的制备方法及其应用 Download PDFInfo
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- CN114209848A CN114209848A CN202111610856.7A CN202111610856A CN114209848A CN 114209848 A CN114209848 A CN 114209848A CN 202111610856 A CN202111610856 A CN 202111610856A CN 114209848 A CN114209848 A CN 114209848A
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Abstract
本发明公开了一种具有运输siRNA功能的铂肽共聚物的制备方法与应用，是以具有抗肿瘤作用的羧基铂为交联分子，将含有精氨酸与赖氨酸的多肽交联形成铂肽共聚物，再利用静电相互作用与具有诱导细胞凋亡的siRNA混合组装，最后用负电荷脂质体包裹，形成稳定的纳米复合物，最终制成化学/基因协同治疗的纳米传递系统。该纳米传递系统解决了单一疗法治疗效果不佳，siRNA到达肿瘤途中易被降解或清除的问题，具有水溶性，分散性好，稳定性高和生物相容性好等优点。
Description
技术领域
本发明属于生物医学应用领域，具体涉及一种具有运输siRNA功能的铂肽共聚物的制备方法及其应用。
背景技术
在当今社会，癌症是一种严重危害人类健康和生命的疾病。化疗是目前癌症治疗最常用和最有效的方法之一。在各类化疗药物中，铂类药物因其抗癌谱广，治疗效果好而在临床治疗中应用广泛，尤其是顺铂及其衍生物。然而Pt(II)类药物在细胞质环境中易被灭活，从而影响其治疗效果，用预氧化的Pt(IV)配合物被认为是解决这个问题的有效方法，而且可以在其轴向位置引入新基团，有更多化学修饰的可能。
RNA干扰(RNAi)具有优越的能力，可以通过敲除靶基因来诱导广泛的遗传靶点的有效、持久和特异性沉默，从而达到治疗癌症的效果。siRNA与其他癌症治疗药物相比具有一定的优势，siRNA可以在极低的细胞内浓度下以高度有序特异性的方式提供高效的基因沉默，并且几乎没有任何毒副作用。但因siRNA是一种带负电荷的大分子，容易被血清中的核酸酶所降解，也易被肾脏迅速清除，同时由于其阴离子电荷且亲水性高而导致不易穿过细胞膜，细胞摄取量低。因此如何将siRNA运输到目标癌组织中仍然是一个不小的挑战。
化疗和基因治疗的联合通过使用选择性化疗药物和功能性基因而展现出巨大的癌症治疗潜力，不仅可以克服多种药物的耐药性，还能达到良好的协同治疗效果。目前已有各种化疗药物和功能性基因的共传递系统被报道，证明了此类药物系统具有明显增强的肿瘤治疗效果，尤其是有通透性增强和保留效应的纳米材料，更适合开发成靶向肿瘤的传递系统。但是由于化疗药物和功能性基因不能独立地负载进纳米颗粒中，从而影响载体结构大小及其负载量，同时由于分子之间的干扰，基因和药物分子也有可能发生非特异性泄漏，这可能会在体循环过程中造成不必要的副作用。
综上所述，如何开发一个化疗药物和功能性基因互不干扰，尺寸合适，稳定性高，能够安全高效地共传递化疗药物和功能性基因到目标癌组织的系统成为本领域科研人员亟待解决的问题。
发明内容
本发明旨在提供一种具有运输siRNA功能的铂肽共聚物的制备方法及其应用，以避免化疗药物和功能性基因(RNA)的互相干扰，减少RNA在运输过程中的损失，能够安全高效地共传递化疗药物和功能性基因到目标癌组织，实现化疗和基因治疗的协同效果。
本发明具有运输siRNA功能的铂肽共聚物的制备方法，其特征是将羧基铂与多肽进行交联形成铂肽共聚物，再利用静电相互作用与siRNA混合组装，最后用负电荷脂质体包裹，形成稳定的纳米复合物。
本发明所采用的多肽具有2-4个赖氨酸残基用于羧基铂的交联反应、具有2-4个精氨酸使多肽在赖氨酸反应后仍具有正电荷，例如KRKRKR或KRFKRFRF。
本发明所采用的多肽单体的电荷较少、不足以有效负载核酸，而在羧基铂的多肽交联具有较多的负电荷，可有效负载核酸。当铂药物从多肽中解离后，多肽复合物解聚，每个单体分子不能有效负载核酸，使核酸释放。
所述siRNA具有抑制细胞增殖的功能，例如干扰Bcl-2的siRNA。
所述负电荷脂质体包括生育酚聚乙二醇琥珀酸酯脂质体或双十六烷基磷酸酯脂质体。
本发明具有运输siRNA功能的铂肽共聚物的制备方法，包括如下步骤：
步骤1：用Rink Amide MBHA树脂固相合成多肽。
所述多肽包含2-4个赖氨酸残基和2-4个精氨酸残基，例如KRKRKR或KRFKRFRF。
步骤2：四价羧基铂的合成
2a、称取顺铂加入圆底烧瓶中，再加入H2O2溶液，60℃～75℃避光搅拌反应3～8h，结束反应，旋蒸除去溶剂，冷却静置，收集沉淀，分别用丙酮和乙醚洗涤，冻干，得到羟基铂；
2b、称取羟基铂和丁二酸酐加入圆底烧瓶中，加入N,N-二甲基甲酰胺(DMF)，40～60℃避光搅拌12～48h，结束反应，冻干除去溶剂，用丙酮溶解，滴加乙醚析出沉淀，收集沉淀，沉淀用乙醚洗涤，冻干沉淀得到四价羧基铂产物Pt(IV)-COOH；
步骤3：铂肽交联
将多肽溶于吡啶水溶液或pH＝7.4的PBS溶液中，再将Pt(IV)-COOH、N-羟基丁二酰亚胺(NHS)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)溶于水中反应10～60min，然后将两种溶液混合均匀，室温避光搅拌10～24h，冻干除去溶剂后，分别用氯仿和丙酮提纯除去有机小分子杂质，再通过凝胶色谱柱除去杂质，得到铂肽交联产物Pt-AA；
在上述实施方案中，所述的Pt(IV)-COOH添加的摩尔量是多肽摩尔量的0.5～4倍，N-羟基丁二酰亚胺(NHS)添加的摩尔量是多肽摩尔量的4～8倍，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCI)添加的摩尔量是多肽摩尔量的10～20倍。
步骤4：Pt-AA-RNA
将铂肽交联产物Pt-AA和siRNA(市购获得)在常温下混合均匀后在超声中组装；AA-RNA以同样的方法制备，用以对比。
在上述实施方案中，所述的siRNA具有抑制细胞增殖的功能，例如干扰Bcl-2的siRNA和干扰survivin的siRNA。
在上述实施方案中，所述的铂肽交联产物Pt-AA和siRNA的质量比为50:1～200:1。
步骤5：Liposome-Pt-AA-RNA(LPR)
5a、阴离子脂质体(LP)：称取大豆卵磷脂、生育酚聚乙二醇琥珀酸酯和胆固醇，并将其溶解在氯仿中，通过旋转蒸发除去溶剂，形成均匀的脂质膜，然后加水，并通过超声处理和过滤膜得到阴离子脂质体(LP)；
在上述实施方案中，所述的脂质体的阴性成分可以是生育酚聚乙二醇琥珀酸酯(TPGS)、磷脂酸(PA)、磷脂酰丝氨酸(PS)和双十六烷基磷酸酯(DCP)。
5b、将LP水化，与Pt-AA-RNA混合后进行超声得到LPR。
在上述实施方案中，所述的Liposome、Pt-AA-RNA的质量比为2:1～16:1。
本发明具有运输siRNA功能的铂肽共聚物的应用，是在癌症靶向治疗过程中作为化疗和基因协同治疗的纳米传递系统使用。
本发明将抗癌广谱、治疗效果好的铂类药物与阳离子聚合物交联，通过静电相互作用加载能够高效诱导基因沉默的治疗siRNA，再包裹脂质体来稳定，最终制成化学/基因协同治疗的纳米传递系统。
与现有技术相比，本发明的有益效果体现在：
1、本发明以具有抗肿瘤功能的铂药物作为多肽的交联剂，在制备siRNA载药体系的同时实现化疗药物的负载，使铂配合物具有化学交联和抑制肿瘤的双重功能；
2、本发明利用铂药交联获得多聚肽分子，使其具有正电荷富集的作用，有利于核酸的负载，并在铂药释放后使肽解聚，促进核酸的释放；
3、本发明所制备的多肽-铂交联物具有可在药物释放的同时将聚合物解聚，每个多肽分子单体不能有效负载核酸，同时释放所负载的核酸。
4、本发明将siRNA装载在稳定的纳米颗粒里，解决了siRNA到达肿瘤途中易被降解或清除的问题，有效减少siRNA在运输到癌细胞过程中的损失。
5、本发明中基因和药物装载互不影响，且四价铂前药在癌细胞中才被还原成二价铂，从而实现了药物和siRNA的同时释放，减少了药物和基因的相互干扰，避免可能发生的非特异性泄露，减小副作用，提供了一种独立、可控地结合和释放的装载策略。
6、本发明中包裹脂质体后，纳米结构更加稳定，形成的纳米颗粒尺寸合适，水溶性好，生物相容性好。
7、本发明达到化疗药物和功能性基因的协同作用，有效增强肿瘤治疗的效果。
附图说明
图1为本发明具有运输siRNA功能的铂肽共聚物的制备路线示意图。
图2为本发明Liposome-PtAA-RNA的尺寸大小和电势。
图3为Liposome-PtAA-RNA的透射电镜图。
图4为Liposome-PtAA-RNA在H2O和含FBS的DMEM中的稳定性测试图。
图5为Liposome-PtAA-RNA和RNA的细胞摄取荧光图。
图6为Liposome-PtAA-RNA、Cisplatin和Pt(IV)-COOH的Pt的摄取图。
图7为Liposome-PtAA-RNA、Cisplatin和Pt(IV)-COOH的Pt的DNA铂化图。
具体实施方式
下面结合具体实施方案对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：固相合成多肽KRKRKR(Lys-Arg-Lys-Arg-Lys-Arg)
1a、称取500mg Rink Amide MBHA树脂，加入DMF(N，N-二甲基甲酰胺)溶胀1h；加入5ml 20％哌啶/DMF溶液反应10min，再用DMF溶液洗涤4次；加入700mgFomc-Lys(Boc)-OH，180mg1-羟基苯并三唑(HOBT)，520mg苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)和450ul N,N-二异丙基乙胺(DIEA)，DMF溶解反应12h，用DMF洗涤；然后进行茚三酮检测氨基，显示未变蓝，表明连接成功；加入8ml 20％哌啶/DMF溶液反应10min，将此步骤(加入8ml20％哌啶/DMF溶液反应10min)重复一次，用DMF洗涤，然后进行茚三酮检测氨基，显示变蓝，表明脱保护成功。
1b、加入750mg Fmoc-Arg(pbf)-OH，193mg HOBT，540mg HBTU和470ul DIEA，用DMF溶解反应12h；用DMF洗涤，然后进行茚三酮检测氨基，显示未变蓝，表明连接成功；加入8ml20％哌啶/DMF溶液反应10min，将此步骤(加入8ml 20％哌啶/DMF溶液反应10min)重复一次；再进行一次茚三酮检测氨基，显示变蓝，表明脱保护成功。
1c、将以上步骤(从加入Fomc-Lys(Boc)-OH开始，后面每接一个相同的氨基酸，方法相同)重复两次。
1d、切割多肽：用异丙醇和正己烷洗涤数次，晾干；取出树脂，加入10ml 95％TFA/H2O，室温搅拌2.5h，分离出液体，树脂用TFA和50％TFA/CH2Cl2洗涤，旋干溶液，加入少量二氯甲烷溶解，滴加乙醚析出固体，再用甲醇溶解，乙醚沉淀，将用甲醇溶解，乙醚沉淀重复2-3次，吹干，冻干得产物KRKRKR多肽。
实施例2：四价羧基铂的合成
2a、称取100mg顺铂加入50ml圆底烧瓶中，再加入7-8ml浓度为30％的H2O2溶液，75℃避光搅拌反应4h；结束反应，通过旋转蒸发除去大部分溶剂，再冷却过夜，收集沉淀，分别用丙酮和乙醚洗涤1-2次，冻干，得到羟基铂；
2b、称取100mg羟基铂和120mg丁二酸酐加入50ml圆底烧瓶中，加入3-5mlDMF，50℃避光搅拌24h；结束反应，通过冻干除去溶剂，用少许丙酮溶解，滴加乙醚析出沉淀，收集沉淀，沉淀用乙醚洗涤3次，冻干得到四价羧基铂产物Pt(IV)-COOH。
实施例3：铂肽交联产物Pt-AA的合成
称取74mg的多肽KRKRKR溶于15ml100mM pH＝7.4的PBS中，再将2倍摩尔量(以多肽计)Pt(IV)-COOH，8倍摩尔量(以多肽计)NHS和20倍摩尔量(以多肽计)EDCI溶于5ml水中反应30min，然后将两种溶液混合均匀，室温避光搅拌12h，冻干后除去溶剂，先分别用氯仿和丙酮提纯除去有机小分子杂质，再通过凝胶色谱柱除去杂质，得到铂肽交联产物Pt-AA。
实施例4：铂肽交联产物Pt-AA的合成
称取74mg的多肽KRKRKR溶于15ml吡啶水溶液中，再将1.5倍摩尔量(以多肽计)Pt(IV)-COOH，6倍摩尔量(以多肽计)NHS和15倍摩尔量(以多肽计)EDCI溶于5ml水中反应30min，然后将两种溶液混合均匀，室温避光搅拌12h，冻干后除去溶剂，先分别用氯仿和丙酮提纯除去有机小分子杂质，再通过凝胶色谱柱除去杂质，得到铂肽交联产物Pt-AA。本实施例中的吡啶水溶液中吡啶的量和EDCI同摩尔量。
实施例5：铂肽交联产物Pt-AA的合成
称取100mg的多肽KRFKRFRF溶于15ml吡啶水溶液中，再将1.5倍摩尔量(以多肽计)Pt(IV)-COOH，6倍摩尔量(以多肽计)NHS和15倍摩尔量(以多肽计)EDCI溶于5ml水中反应30min，然后将两种溶液混合均匀，室温避光搅拌12h，冻干后除去溶剂，先分别用氯仿和丙酮提纯除去有机小分子杂质，再通过凝胶色谱柱除去杂质，得到铂肽交联产物Pt-AA。本实施例中的吡啶水溶液中吡啶的量和EDCI同摩尔量。
实施例6：Pt-AA-RNA的合成
称取质量比为100:1的铂肽交联产物Pt-KRKRKR和Bcl-2siRNA，常温下混合均匀后在超声中组装；KRKRKR-Bcl-2siRNA以同样的方法制备。
实施例7：Pt-AA-RNA的合成
称取质量比为150:1的铂肽交联产物Pt-KRFKRFRF和survivin siRNA，常温下混合均匀后在超声中组装；KRFKRFRF-survivin siRNA以同样的方法制备
实施例8：Liposome-Pt-AA-RNA(LPR)的合成
称取100mg大豆卵磷脂、4mg生育酚聚乙二醇琥珀酸酯，和6mg胆固醇，并将其溶解在5mL氯仿中，通过旋转蒸发除去溶剂，形成均匀的脂质膜，然后加入8ml水，并通过超声处理和过220nm滤膜得到阴离子脂质体(LP)。
将阴离子脂质体(LP)水化，将LP与Pt-AA-RNA以8:1的质量比混合后进行超声得到LPR。
实施例9：Liposome-Pt-AA-RNA(LPR)的合成
称取100mg大豆卵磷脂、4mg双十六烷基磷酸酯，和6mg胆固醇，并将其溶解在5mL氯仿中，通过旋转蒸发除去溶剂，形成均匀的脂质膜，然后加入8ml水，并通过超声处理和过220nm滤膜得到阴离子脂质体(LP)。
将阴离子脂质体(LP)水化，将LP与Pt-AA-RNA以4：1的质量比混合后进行超声得到LPR。
实施例10：本发明制备的具有运输siRNA功能的铂肽共聚物的粒径、电位和形貌
采用高灵敏度Zeta电位及粒度分散仪测定实施例8制备的具有运输siRNA功能的铂肽共聚物在含血清(FBS)的培养基(DMEM)粒径和电位如图2所示。结果显示制备的纳米颗粒粒径约124nm，电位在-22.5V左右。除此之外，我们还采用了透射电镜测定了纳米颗粒的粒径和外观形貌，如图3所示，纳米颗粒的直径为直径100-200nm的球形颗粒，分散性比较好，结果基本与高灵敏度Zeta电位及粒度分散仪测定的结果相吻合。
实施例11：本发明制备的具有运输siRNA功能的铂肽共聚物的稳定性检测
采用动态光散射测定了实施例8制备的具有运输siRNA功能的铂肽共聚物的稳定性。如图4所示该纳米颗粒可以在水中和含FBS的培养基(DMEM)中至少96h都能保持稳定，具有较高的稳定性。
实施例12：本发明制备的具有运输siRNA功能的铂肽共聚物的细胞摄取
选用处于增殖旺盛的对数生长期的细胞，按照5×105个细胞/孔的密度将HeLa细胞接种于6孔板中，在细胞培养箱中培养12h。将含有FAM-RNA制成的LPR和FAM-RNA与HeLa的1ml的新鲜培养基替代旧培养基，37℃下孵育7h后，去除培养基并用PBS洗涤，然后在荧光显微镜下观察。如图5所示，LPR的FAM荧光图中有明显的绿色荧光，而RNA的FAM图则没有，证明该纳米运输系统可以安全高效将功能性基因运输到癌细胞里，避免在运输途中被降解或清除。
实施例13：本发明制备的具有运输siRNA功能的铂肽共聚物的细胞摄取
选用处于增殖旺盛的对数生长期的细胞，按照5×105个细胞/孔的密度将HeLa细胞接种于6孔板中，在细胞培养箱中培养12h。然后用含40μM铂的顺铂、四价羧基铂和LPR的1mL新鲜培养基替代原有培养基，37℃下孵育6h。随后弃去培养基，用PBS洗涤细胞三次，用胰酶消化细胞并对细胞计数，最后用王水消化细胞，ICP-MS测定细胞内铂的积累量。
实验结果如图6所示，显示LPR的细胞摄取大约是顺铂的2倍，四价铂的6倍，大大提高了细胞摄取量。
实施例14：本发明制备的具有运输siRNA功能的铂肽共聚物的细胞DNA铂化。
按照1×106个细胞/孔的密度将HeLa细胞接种于6孔板中，在细胞培养箱中培养12h，分别将顺铂、四价羧基铂和LPR以40μΜPt的最终浓度添加到孔中，在37℃孵育6h后，用冷PBS洗涤细胞三次，并使用DNA提取试剂盒提取DNA，使用Nanodrop 2000分光光度计检测DNA产量，再通过ICP-MS测定Pt含量。DNA-Pt加合量表示为每μg DNA pg Pt。
实验结果如图7所示，显示LPR的DNA铂化量大约是顺铂的1.5倍，四价铂的3倍，有效提高细胞的DNA铂化量，从而证明可以增强对癌细胞的杀伤力。
实施例15：本发明制备的具有运输siRNA功能的铂肽共聚物的细胞毒性
将HeLa细胞以3×103个细胞/孔的密度接种在96孔板中，于细胞培养箱中孵育过夜。设置实验组、对照组和空白组，实验组接种细胞且加入不同种类及不同浓度的药物，对照组接种细胞但不加入药物，空白组不接种细胞且不加药物。将细胞在细胞培养箱中继续培养48h。然后每孔加入20μLMTT溶液(5mg/mL)，继续培养4h。随后，每孔加入150μL二甲基亚砜(DMSO)，于37℃震荡箱中震荡15min，使结晶物充分溶解。使用Bio-Rad 680酶标仪在490nm处测量各孔的吸光度A值。细胞存活率(％)＝[实验组A值-空白组A值]/[对照组A值-空白组A值]。
实施例16：本发明制备的具有运输siRNA功能的铂肽共聚物的细胞毒性
将A549细胞以3×103个细胞/孔的密度接种在96孔板中，于细胞培养箱中孵育过夜。设置实验组、对照组和空白组，实验组接种细胞且加入不同种类及不同浓度的药物，对照组接种细胞但不加入药物，空白组不接种细胞且不加药物。将细胞在细胞培养箱中继续培养48h。然后每孔加入20μL MTT溶液(5mg/mL)，继续培养4h。随后，每孔加入150μL二甲基亚砜(DMSO)，于37℃震荡箱中震荡15min，使结晶物充分溶解。使用Bio-Rad 680酶标仪在490nm处测量各孔的吸光度A值。细胞存活率(％)＝[实验组A值-空白组A值]/[对照组A值-空白组A值]。
通过实施例15和实施例16表明本发明制备的具有运输siRNA功能的铂肽共聚物具有良好的癌细胞抑制作用，铂类药物和siRNA可以协同杀死癌细胞，增强对肿瘤的治疗效果。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种具有运输siRNA功能的铂肽共聚物的制备方法，其特征在于：
以具有抗肿瘤作用的羧基铂为交联分子，与多肽进行交联形成铂肽共聚物，再利用静电相互作用与siRNA混合组装，最后用负电荷脂质体包裹，形成稳定的纳米复合物；
所述多肽包含2-4个赖氨酸残基以及2-4个精氨酸残基；
所述siRNA具有抑制细胞增殖的功能；
所述负电荷脂质体包括生育酚聚乙二醇琥珀酸酯脂质体或双十六烷基磷酸酯脂质体。
2.根据权利要求1所述的制备方法，其特征在于包括如下步骤：
步骤1：铂肽交联
将多肽溶于吡啶水溶液或pH＝7.4的PBS溶液中；再将Pt(IV)-COOH、N-羟基丁二酰亚胺和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐溶于水中反应10～60min；然后将两种溶液混合均匀，室温避光搅拌12～24h，冻干除去溶剂后，分别用氯仿和丙酮提纯除去有机小分子杂质，再通过凝胶色谱柱除去杂质，得到铂肽交联产物Pt-AA；
步骤2：Pt-AA-RNA
将铂肽交联产物Pt-AA和RNA在常温下混合均匀后在超声中组装；
步骤3：Liposome-Pt-AA-RNA
3a、阴离子脂质体：称取大豆卵磷脂、生育酚聚乙二醇琥珀酸酯和胆固醇，并将其溶解在氯仿中，通过旋转蒸发除去溶剂，形成均匀的脂质膜，然后加水，并通过超声处理和过滤膜得到阴离子脂质体；
3b、将阴离子脂质体水化，与Pt-AA-RNA混合后进行超声得到Liposome-Pt-AA-RNA。
3.根据权利要求2所述的制备方法，其特征在于：
步骤1中，Pt(IV)-COOH添加的摩尔量是多肽摩尔量的0.5～4倍，N-羟基丁二酰亚胺添加的摩尔量是多肽摩尔量的4～8倍，1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐添加的摩尔量是多肽摩尔量的10～20倍。
4.根据权利要求2所述的制备方法，其特征在于：
步骤2中，铂肽交联产物Pt-AA和RNA的质量比为50:1～200:1。
5.根据权利要求2所述的制备方法，其特征在于：
步骤3中，脂质体和Pt-AA-RNA的质量比为2：1～16：1。
6.根据权利要求1-5中所述的任一项制备方法制得的具有运输siRNA功能的铂肽共聚物的应用，其特征在于：是在癌症靶向治疗过程中作为化疗和基因协同治疗的纳米传递系统使用。
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4931553A (en) * | 1988-05-11 | 1990-06-05 | Gill Devinder S | Platinum-polymer complexes and their use as antitumor agents |
| WO2010118200A2 (en) * | 2009-04-08 | 2010-10-14 | Brian Salvatore | Liposomal formulations of tocopheryl amides |
| CN101870726A (zh) * | 2010-06-04 | 2010-10-27 | 臧林泉 | 一种多肽-顺铂偶合物及其制备方法与应用 |
| US20140127284A1 (en) * | 2012-10-18 | 2014-05-08 | The Regents Of The University Of California | Micro-rnas and micro-rna inhibitors to modulate blood vessel growth, patterning, tumor growth and malignant disease and method for making and using them |
| US20180250332A1 (en) * | 2015-09-14 | 2018-09-06 | Nippon Kayaku Kabushiki Kaisha | Polymer Conjugate Of Hexa-Coordinated Platinum Complex |
| CN111135187A (zh) * | 2018-10-16 | 2020-05-12 | 国家纳米科学中心 | 一种多肽-顺铂前药复合物、其自组装纳米递送体系及其制备方法和应用 |
| CN111494411A (zh) * | 2020-05-21 | 2020-08-07 | 中国医学科学院放射医学研究所 | 一种原位自组装四价铂药物及其制备方法与应用 |
| WO2021216849A1 (en) * | 2020-04-22 | 2021-10-28 | Oncovolution, Llc | Peptide platinum complexes and methods of use thereof |
-
2021
- 2021-12-27 CN CN202111610856.7A patent/CN114209848B/zh active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4931553A (en) * | 1988-05-11 | 1990-06-05 | Gill Devinder S | Platinum-polymer complexes and their use as antitumor agents |
| WO2010118200A2 (en) * | 2009-04-08 | 2010-10-14 | Brian Salvatore | Liposomal formulations of tocopheryl amides |
| CN101870726A (zh) * | 2010-06-04 | 2010-10-27 | 臧林泉 | 一种多肽-顺铂偶合物及其制备方法与应用 |
| US20140127284A1 (en) * | 2012-10-18 | 2014-05-08 | The Regents Of The University Of California | Micro-rnas and micro-rna inhibitors to modulate blood vessel growth, patterning, tumor growth and malignant disease and method for making and using them |
| US20180250332A1 (en) * | 2015-09-14 | 2018-09-06 | Nippon Kayaku Kabushiki Kaisha | Polymer Conjugate Of Hexa-Coordinated Platinum Complex |
| CN111135187A (zh) * | 2018-10-16 | 2020-05-12 | 国家纳米科学中心 | 一种多肽-顺铂前药复合物、其自组装纳米递送体系及其制备方法和应用 |
| WO2021216849A1 (en) * | 2020-04-22 | 2021-10-28 | Oncovolution, Llc | Peptide platinum complexes and methods of use thereof |
| CN111494411A (zh) * | 2020-05-21 | 2020-08-07 | 中国医学科学院放射医学研究所 | 一种原位自组装四价铂药物及其制备方法与应用 |
Non-Patent Citations (5)
| Title |
|---|
| HAIQIN SONG ET AL: "A cross-linked polymeric micellar delivery system for cisplatin(IV) complex", 《EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS》, vol. 83, pages 63 - 75 * |
| JIHOON KIM ET AL: "Polymeric biomaterials for the delivery of platinum-based anticancer drugs", 《BIOMATERIALS SCIENCE》, vol. 7, pages 1002 - 1017 * |
| 余敬谋等: "核交联聚合物胶束递药系统的研究进展", 《药学学报》, vol. 49, pages 183 - 189 * |
| 李麦云: "铂肽共聚物运输siRNA和RNF11蛋白与单功能铂配合物的相互作用研究", 《中国优秀硕士论文电子期刊网》, pages 3 * |
| 焦自学;陈杰;田华雨;陈学思;: "聚乙烯亚胺介导siRNA和顺铂协同抗肿瘤治疗", 高分子学报, no. 01 * |
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