CN105188764B - Dna嵌入剂的细胞递送 - Google Patents
Dna嵌入剂的细胞递送 Download PDFInfo
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Abstract
提供了用于将活性剂靶向递送至细胞的组合物和方法。所述组合物包含全部或部分双链合成DNA载体和嵌入在所述DNA载体的双链部分中的活性剂。所述DNA载体还可以被连接至靶向剂。所述组合物可用于将活性剂递送至靶向的细胞类型内,所述细胞类型例如细胞毒性剂。
Description
技术领域
本发明涉及使用DNA载体将活性剂递送至细胞,其中活性剂嵌入至载体的DNA内。DNA载体还可以包括使DNA载体结合至特定细胞类型的靶向剂,从而促进嵌入的活性剂向细胞的递送。
背景
活性剂与DNA之间的非共价相互作用通常通过沟结合或嵌入来产生。具体来说,抗癌疗法的领域已经产生了数种通过嵌入而与DNA相互作用的化疗药物,所述嵌入是涉及在DNA碱基对之间插入平面药物分子的方法。这导致DNA的螺旋形圈数和延长减少。由于良好的疏水键、离子键、氢键以及范德华力,嵌入的缔合常数可以是105M-1至10-11M-1。嵌入通常与稠环结构相关,但非稠环系统也是已知的。嵌入被公认为是成功使用这些化合物作为抗肿瘤剂、抗瘤剂、抗疟疾剂、抗生素剂以及抗真菌剂的重要原因,因为大多数试剂是基因毒性的,特别当存在碱性的、阳离子的或亲电的官能团时。
DNA树枝状聚合物是由相互连接的天然或合成单体寡核苷酸亚单元构造的复杂的、高度分支的分子。DNA树枝状聚合物由DNA单体构建,每个所述DNA单体由共享位于每个链的中心部分(又称“腰”)的序列互补区的两个DNA链形成,但具有四个非互补的单链“臂”用于与其他单体的“臂”杂交。单体从单个单体开始组装成树枝状聚合物,所述单个单体通过互补的臂与另外的单体杂交。另外的层以类似的方式通过单体与先前的层中的单体的臂杂交而添加。通过改变其中臂与另外的单体杂交的层的数目,产生不同大小和形状的DNA树枝状聚合物。为了防止DNA树枝状聚合物随时间推移而分解,可以使用紫外线通过嵌入和活化补骨脂素交联剂将化学“焊点”添加至增长的组件中。在超速离心之后,通常根据树枝状聚合物的大小和分子量在变性蔗糖梯度上对其进行纯化。
DNA树枝状聚合物还可以共价或非共价结合各种不同类型的分子和颗粒,通常通过连接至存在于外层上的未杂交的臂。这些分子和颗粒可以是DNA树枝状聚合物上的信号传导装置和靶向装置,从而允许靶向DNA树枝状聚合物至特异性分子靶标并且通过检测信号传导部分检测树枝状聚合物与靶标的结合。信号产生部分包括大量荧光染料、半抗原、酶和其他分子材料,以及颗粒如金纳米颗粒和量子点。靶向装置包括DNA、RNA和PNA寡核苷酸、抗体、抗体片段、半抗原、适体、肽以及其他靶向装置。因为单体的外层含有许多未杂交的臂,所以DNA树枝状聚合物构建体在各种体外应用中用作信号放大器,通常用于检测特定的核酸和蛋白质,而且也用作电子装置中的检测装置。应用包括对于DNA和蛋白质微阵列、ELISA和ELOSA、Luminex珠粒测定、原位杂交以及其他方面的信号放大。在调查研究中已经广泛发表了标记的和靶向的DNA树枝状聚合物的用途,并且这些材料可作为由GenisphereLLC(Hatfield,PA)出售或生产的商业研究产品而获得。
还已经显示DNA树枝状聚合物具有作为体外和体内应用中的递送装置和转染装置的潜在用途。参见例如,U.S.2005/0089890、WO2008/147526和WO 2010/017544,每个文献以引用的方式整体并入。确切来说,DNA树枝状聚合物与靶向装置(例如,特异于能够引起细胞内吞内化作用事件的细胞表面特征的抗体)结合,所述靶向装置结合靶向接受货物(例如,药物)递送的细胞上的表面特征。被动地与靶向的DNA树枝状聚合物缔合的货物通过与树枝状聚合物的空间缔合而简单地进入细胞,但货物也可以通过许多附接策略(包括连接至外臂)结合树枝状聚合物。
概述
本发明涉及一种用于使用DNA载体将活性剂递送至细胞的改进的方法。DNA载体包含DNA(所述DNA的至少一部分是呈双链形式)和嵌入在双链DNA中的活性剂。在某些实施方案中,DNA载体还包含结合其中待递送活性剂的分子、细胞或组织的靶向剂。在具体实施方案中,DNA载体是具有嵌入在树枝状聚合物的双链部分中的活性剂的DNA树枝状聚合物。当使DNA载体结合至靶向的分子、细胞或组织时,树枝状聚合物通过细胞内化至核内体内,在所述核内体中酸性pH促进活性剂从DNA中释放以产生细胞毒性或其他生物效应。
附图简述
图1A示出用LYSOSENSOR染料标记活细胞。图1B示出含有内化的树枝状聚合物的细胞。图1C是图1A和图1B的合并图,其示出树枝状聚合物被内化至细胞的酸性隔室内。
图2A-2D示出实施例2的结果:未经处理的细胞(图1A),未靶向的阿霉素树枝状聚合物(图2B)、G8mAb缀合的阿霉素树枝状聚合物(图2C和图2D)。
详述
在描述本发明的若干示例性实施方案之前,应了解的是,本发明不限于以下描述中所阐述的构建步骤或处理步骤的细节。本发明能够具有其他实施方案且能够以各种方式实施或进行。
在本说明书全篇中提及“一个实施方案”、“某些实施方案”、“一个或多个实施方案”或“实施方案”意味着结合所述实施方案描述的具体特征、结构、材料或特性包括于本发明的至少一个实施方案中。因此,在本说明书全篇中各个地方出现如“在一个或多个实施方案中”、“在某些实施方案中”、“在一个实施方案中”或“在实施方案中”的短语未必指本发明的同一实施方案。此外,具体特征、结构、材料或特性可以任何适合方式在一个或多个实施方案中加以组合。
在第一方面中,本发明涉及用于将嵌入的活性剂递送至细胞或组织的合成DNA载体,其中DNA是全部或部分双链,并且DNA载体包含嵌入在DNA的双链部分中的活性剂。合成DNA载体的寡核苷酸组分可以是线性或分支的寡核苷酸。DNA载体的每个双链DNA部分的长度为5个碱基对或更长,例如约20-35个碱基对、约35-50个碱基对、约50-100个碱基对或约100-200个碱基对。合成DNA载体在体外被构建,并且优选包含多个双链部分以增加活性剂的负载。包含多个双链部分的合成DNA载体的实例包括DNA树枝状聚合物。如果合成DNA载体包含线性寡核苷酸,则寡核苷酸可以包含单个双链部分或多个双链部分。DNA载体中的所述线性寡核苷酸的总长度通常为至少10个核苷酸,例如长度为10-20个核苷酸、10-50个核苷酸、10-80个核苷酸、10-150个核苷酸或10-200个核苷酸。
在一个实施方案中,合成DNA载体还可以包含结合选择的细胞或组织类型的靶向剂以使DNA载体结合至选择的细胞或组织类型,从而促进活性剂向选择的细胞或组织类型的递送。通过使靶向剂结合至细胞表面蛋白或细胞表面受体,可以使靶向剂结合至选择的细胞或组织类型。适合的靶向剂包括结合细胞表面蛋白或细胞表面受体的抗体、配体或肽。靶向剂通常共价或非共价连接至DNA树枝状聚合物臂(例如,靶向剂连接至互补于树枝状聚合物臂并且与树枝状聚合物臂杂交的寡核苷酸)。
可以由靶向剂靶向的细胞表面受体和蛋白质的实例包括转铁蛋白受体(TfR/CD71)、HIV-1包膜糖蛋白(Env)、疟疾抗原标记以及叶酸受体。优选地,细胞表面受体或蛋白质在感染的、受损的或患病的细胞上特异性表达,或在感染的、受损的或患病的细胞上过表达。靶向剂的具体实例包括抗转铁蛋白抗体或TfR-靶向肽,如结合在各种癌症中过表达的转铁蛋白受体的THRPPMWSPVWP(SEQ ID NO:1)。例如,靶向剂还可以是G8单克隆抗体,所述G8单克隆抗体结合存在于Myo/Nog细胞和它们的衍生物的表面上的大约170kDa的蛋白质。Myo/Nog细胞通过用于骨骼肌特异性转录因子MyoD、骨形态发生蛋白(BMP)抑制剂头蛋白以及细胞表面G8表位的mRNA表达来鉴定。有用的靶向部分的其他实例包括结合PDGF受体、EGFR家族受体、ICAM、CD受体、MMP-9、白细胞介素受体、叶酸受体以及本领域中已知的其他受体的那些靶向部分。
在另一个实施方案中,嵌入在DNA载体的双链部分中的活性剂是化疗剂、抗感染剂、抗疟疾剂以及抗病毒剂或抗真菌剂。所述试剂的具体实例包括黄连素(berberine)(抗真菌的、抗瘤的)、吖啶(例如,普罗黄素(proflavine)或奎纳克林(quinacrine),抗菌的)、癌症化疗剂(例如,道诺霉素(daunomycin)、阿霉素(doxorubicin)、柔红霉素(daunorubicin)、更生霉素(dactinomycin)、顺铂(cisplatin)、卡铂(carboplatin)、伏利拉辛(voreloxin))、白叶藤碱(抗疟疾的细胞毒性的)以及沙利度胺(thalidomide)(治疗多发性骨髓瘤)。
在任何前述实施方案中,DNA载体可以包含DNA树枝状聚合物,包括2层、4层、6层或8层的DNA树枝状聚合物。根据需要选择DNA树枝状聚合物的大小以促进活性剂向细胞和组织的递送(例如,通过循环系统或局部注射),并且使细胞对DNA载体的吸收最大化。通常,DNA载体包含约3-216个寡核苷酸单链。
在第二方面中,本发明涉及包含任何前述实施方案中的DNA载体和至少一种药学上可接受的赋形剂的药物组合物。药学上可接受的赋形剂是任何适合的媒介物,所述媒介物可以用来配制用于向患者、细胞或组织施用以实现治疗或其他生物结果的DNA载体。赋形剂选自与双链DNA相容(即,在递送至选择的细胞或组织之前,它们将不会使DNA载体变性)以及与活性剂和靶向剂(如果存在)相容的那些赋形剂。所述药物赋形剂包括生理缓冲液、糖类和寡糖、稳定剂、增稠剂、润滑剂、蜡、螯合剂、表面活性剂、稀释剂、抗氧化剂、粘合剂、防腐剂、着色剂、乳化剂、脂质胶束或药物制剂的其他常规成分。在某些实施方案中,配制药物组合物用于例如通过注射或输注向患者肠胃外递送。在这种情况下,优选DNA载体包含靶向剂以使得DNA载体由循环引导至希望的细胞或组织类型。在其他实施方案中,药物组合物可以适于口服施用或用于向其中希望递送活性剂的细胞或组织局部施用(例如,外用制剂)。
在一个另外的方面中,本发明涉及制备描述于任何前述实施方案中的DNA载体的方法。在一个实施方案中,DNA载体通过杂交单链DNA以形成全部或部分双链DNA,并且在允许活性剂嵌入至双链部分内的条件下将全部或部分双链DNA与活性剂组合来组装。例如通过色谱法去除多余的活性剂以产生DNA载体。在具体实施方案中,使用自旋柱进行色谱法以去除多余的活性剂。
在替代实施方案中,活性剂可以在单链DNA杂交以组装全部或部分双链DNA过程中并入DNA载体,接着如上所述去除多余的活性剂。
在一个另外的实施方案中,制备DNA载体的方法包括组装DNA树枝状聚合物,在允许活性剂嵌入至DNA树枝状聚合物的双链部分内的条件下将DNA树枝状聚合物与活性剂组合,并且例如通过色谱法(例如,自旋柱)去除多余的活性剂。
在其中DNA载体连接至靶向剂的具体实施方案中,DNA载体由单链DNA组装以形成全部或部分双链DNA,并且将靶向剂连接至组装的DNA。靶向剂与DNA的连接可以通过本领域中已知的共价附接来完成,或所述连接可以是非共价连接如杂交。所述方法包括,例如,通过以下方式的连接:二硫键、N-羟基琥珀酰亚胺(NHS)酯依赖性缩合反应、双官能交联、通过电荷-电荷相互作用使用聚阳离子化合物来将靶向剂桥接至DNA载体、或如描述于WO2010/017544中的与DNA载体直接或间接杂交。例如,将单链捕获寡核苷酸附加至DNA树枝状聚合物的臂,并且将互补于捕获序列的单链寡核苷酸连接至靶向剂。将靶向剂上的互补序列与DNA树枝状聚合物上的捕获序列杂交,以便将靶向剂连接至DNA树枝状聚合物。杂交的捕获序列和互补序列可以任选地被交联。在组装全部或部分双链DNA过程中或在将靶向剂连接至DNA载体之后,可以将活性剂并入DNA载体中。如果靶向剂通过如上所述的捕获序列与DNA载体杂交,则由连接形成的双链DNA也可以嵌入活性剂。
在制备连接至靶向剂的DNA载体的替代实施方案中,在第一步中将单链寡核苷酸附接至靶向剂,并且然后与互补单链寡核苷酸序列杂交以形成DNA载体的全部或部分双链DNA。
在制备DNA载体的任何前述方法中,DNA树枝状聚合物可以通过本领域中已知的任何方法,例如由T.W.Nilsen等,J.Theor.Biol.(1997)187,273-284所述来组装。
在又另外的方面中,本发明涉及将活性剂递送至细胞或组织的方法,所述方法包括使细胞或组织与根据任何前述实施方案的DNA载体接触以使得DNA载体被细胞吸收,并且在细胞内部释放活性剂以对细胞产生细胞毒性或其他生物效应。可以在体内或体外使细胞或组织与DNA载体接触。如果在体外使细胞与DNA载体接触,则细胞将通常在细胞培养物中并且将DNA载体添加至培养基,与细胞一起孵育一段时间以允许结合细胞表面,并且去除多余的DNA载体。在用于内吞作用和释放活性剂的充足时间之后,可以在显微镜下可视化活性剂对细胞的细胞毒性或生物活性。如果在体内使细胞与DNA载体接触,则通过注射、输注、局部施用至希望作用的部位或通过其他适当的方法向患者或动物施用DNA载体,持续一段时间以允许结合细胞表面和内吞作用。可以通过监测生理反应或通过显微镜检查受影响的组织的样品来确定在细胞中释放的活性剂的细胞毒性或生物活性。
在一个另外的方面中,本发明涉及通过使用前述的将活性剂递送至细胞或组织的体内方法向有需要的患者递送DNA载体来治疗疾病或病状的方法。对于治疗特异性,通常将靶向剂选择成特异于其中需要细胞毒性或其他生物活性的细胞类型。将活性剂选择成针对被治疗的疾病或病状是治疗活性的,并且以足以治疗、治愈或改善疾病或病状的症状的量向患者施用DNA载体并且持续足以治疗、治愈或改善疾病或病状的症状的一段时间。通过施用DNA载体可治疗的疾病和病状包括对其可用嵌入至双链DNA内的活性剂的任何疾病或病状。非限制性实例包括癌症、疟疾、真菌性疾病、病毒性疾病、纤维化疾病等。
为了实现治疗,可以例如通过外用涂敷或注射至组织或细胞内来向待治疗的组织或细胞局部施用合成DNA载体。或者,可以例如通过静脉内注射或输注,或通过口服施用来系统地施用合成DNA载体。在任一实施方案中,连接至DNA载体的靶向剂(如果存在)促进DNA载体与待治疗的细胞类型的结合和活性剂向所述细胞类型的递送。
在具体实施方案中,通过施用DNA载体来预防后囊膜混浊(PCO)或降低其发病率,所述DNA载体包含连接至靶向G8表位的单克隆抗体的DNA树枝状聚合物和嵌入DNA的细胞毒素。PCO在白内障手术后的某些成年人和儿童中发生,并且由影响包围晶状体的囊膜的透明度的分化晶状体上皮细胞和成肌纤维细胞引起。当前没有用于预防PCO的有效治疗。已经发现,人晶状体组织中的成肌纤维细胞来源于Myo/Nog细胞,所述Myo/Nog细胞表达骨骼肌特异性转录因子MyoD、骨形态发生蛋白抑制剂头蛋白以及通过G8单克隆抗体(mAb)识别的细胞表面分子。耗尽从经历白内障手术的患者中取得的人晶状体组织的外植体培养物中的Myo/Nog细胞通过使用G8mAb作为靶向剂以及嵌入的阿霉素,用根据本发明的DNA载体(DNA树枝状聚合物)溶解它们来完成。如以下实施例中所示,特异性消除Myo/Nog细胞。从这种数据可以推测,还将防止成肌纤维细胞随时间推移而积累。
实施例
实施例1:G8mAb缀合的树枝状聚合物在横纹肌肉瘤细胞中的内化作用
2层DNA树枝状聚合物制备:如先前所公开制造DNA树枝状聚合物(参见例如,专利5,175,270、5,484,904、5,487,973、6,110,687以及6,274,723,每个所述专利以引用的方式整体并入)。简单来说,DNA树枝状聚合物由DNA单体构建,每个所述DNA单体由共享位于每个链的中心部分的序列互补区的两个DNA链形成。两个链退火以形成单体,所得到的结构可以被描述为具有与四个单链“臂”交界的中心双链“腰”。这种腰加臂结构包含基本的单体。五种单体类型的每一个的末端处的单链臂被设计成以精确和具体的方式彼此相互作用。互补单体的臂之间的碱基配对允许通过依次添加单体层来定向组装树枝状聚合物。树枝状聚合物的每一层的组装包括交联过程,其中DNA的链彼此共价结合,从而形成不受变性条件影响的完全共价分子,所述变性条件以另外的方式将导致树枝状聚合物结构的变形。另外,用作互补捕获寡核苷酸的38个碱基寡核苷酸通过简单的T4DNA连接酶依赖性连接反应连接至可用的树枝状聚合物臂的5'末端,如下所述:
将捕获序列附接至DNA树枝状聚合物:为了将G8单克隆抗体(G8mAb)附接至DNA树枝状聚合物,首先将捕获序列连接至10%-15%的树枝状聚合物臂。使用商业上可获得的化学品(Solulink,www.solulink.com)将针对这种捕获序列的互补寡核苷酸缀合至G8单克隆抗体,并且以一种摩尔比杂交以占据所有可用的捕获序列。如下所概述,每个树枝状聚合物分子附接大约2-5个G8mAb。
通过简单的核酸连接反应使用桥接寡核苷酸将小(15-100个核苷酸)DNA或RNA捕获寡核苷酸(或其他生化类似物)共价附接至树枝状聚合物臂的末端,所述桥接寡核苷酸重叠树枝状聚合物臂与捕获寡核苷酸的相邻部分,由此将捕获寡核苷酸至树枝状聚合物臂的末端。桥接寡核苷酸重叠每个相邻树枝状聚合物臂与捕获寡核苷酸序列的至少5个碱基以促进核酸连接酶(优选T4DNA连接酶)的连接活性,其中优选重叠每个序列至少7个碱基。当树枝状聚合物用于非树枝状聚合物核酸或其他分子的特异性靶向时,桥接寡核苷酸还可以用作其互补序列的核酸阻断剂。
将以下组分添加至微离心管中:
1. 5.4μL(2680ng)1X TE缓冲液中的2层DNA树枝状聚合物(500ng/μL)
2. 2.7μL(134ng)a(-)LIG-BR7桥接寡核苷酸(14聚体)(50ng/μL)
3. 10.2μL 10X连接酶缓冲液
4. 81.7μL不含核酸酶的水
5. 4.0μL(200ng)Cap03捕获寡核苷酸(38聚体)(50ng/μL)
6. 10.0μL(10个单位)T4DNA连接酶(1U/μL)
将前四个反应物添加在一起,加热至65℃并且冷却至室温。然后添加第五个和第六个反应物并且孵育45分钟。通过添加2.8μL的0.5M EDTA溶液来停止连接反应。通过使用100k截留分子量离心过滤器(Millipore Corp.)去除未连接的寡核苷酸,并且在纯化洗涤过程中,将缓冲液改变成pH 7.4的1x无菌PBS。将捕获寡核苷酸连接至树枝状聚合物的第一单链表面臂。
DNA树枝状聚合物的抗体附接和荧光标记:为了靶向细胞表面和开始内化作用,将G8单克隆抗体偶联至DNA树枝状聚合物。将G8单克隆抗体共价附接至互补于捕获序列的寡核苷酸,所述捕获序列先前连接至树枝状聚合物。简单来说,通过3’末端使用商品化学品将捕获序列补体(cplCap03)(5’-TTCTCGTGTTCCGTTTGT ACTCTA AGGTGGATTTTT-3’(SEQ IDNO:2))共价偶联至G8单克隆抗体(So lulink),并且通过HPLC纯化以去除多余的试剂。然后在最后组装试剂(涉及下文3DNA制备部分)的过程中,将这些缀合物杂交至树枝状聚合物捕获序列。另外,制备互补于DNA树枝状聚合物的外表面臂的合成寡核苷酸(IDTTechnologies),所述合成寡核苷酸具有两个荧光标记(Cy3),每一个各自在3'末端和5'末端上。然后将这个寡核苷酸以等于18摩尔寡核苷酸比1摩尔DNA树枝状聚合物的量杂交至以上制备的G8mAb靶向树枝状聚合物的外臂。G8mAb靶向的Cy3标记的DNA树枝状聚合物用于对横纹肌肉瘤细胞进行靶向和内化作用研究。
与Cy3标记的树枝状聚合物缀合的G8单克隆抗体在横纹肌肉瘤细胞中的内化作用:
将人胚胎性和腺泡状横纹肌肉瘤细胞(来自ATCC的细胞系136和细胞系2061)培养在8ml培养基中的100mm组织培养皿(12个盖玻片/板)中的玻璃盖玻片上持续24小时。将136细胞系培养在含有10%胎牛血清的DMEM中。将2061细胞系培养在含有10%FBS的RPMI中。将盖玻片转移至含有1ml以下培养基的35mm组织培养皿(2个盖玻片/皿):
1.含有以1:500稀释(在2uM工作溶液中)的LysoSensor Green DND 189储备溶液(1mM)(Molecular Probes/Invitrogen)的培养基。
2.含有2uM LysoSensor加1:75稀释的G8:4n树枝状聚合物-Cy3的培养基。
将细胞在37℃下在空气中的5%CO2中孵育1小时。用37℃PBS冲洗细胞并且在2%低聚甲醛中固定10分钟。通过落射荧光显微镜可视化细胞。测试各种稀释物和孵育时间。将1:500的Lysosensor加1:75的G8:树枝状聚合物孵育1小时产生最好结果。结果在图lA、图1B和图1C中显示,其示出标记的树枝状聚合物在横纹肌肉瘤细胞中的成功内化作用。图lA示出用在酸性pH下发出绿色荧光的LYSOSENSOR染料标记的细胞(在图lA中集中在细胞内的黑点中)。图1B示出来自细胞内Cy3标记的树枝状聚合物的红色荧光(在图1B中集中在细胞内的黑点中)。图1C是图lA和图1B的合并图像,其示出可视化为黄色的绿色信号和红色信号的共定位,并且在图1C中显示为通过箭头指示的细胞内的黑点。这些结果证实,偶联至DNA树枝状聚合物的G8mAb被内化至细胞的酸性隔室(即,溶酶体)内。
实施例2:靶向耗尽Myo/Nog细胞
2层DNA树枝状聚合物制备:如在实施例1中所述制备具有附接的捕获序列的2层DNA树枝状聚合物。将捕获序列连接至10%-15%的树枝状聚合物臂。
将G8mAb附接至DNA树枝状聚合物:如在实施例1中所述也将G8单克隆抗体(G8mAb)附接至DNA树枝状聚合物。首先,使用商业上可获得的化学品(Solulink,www.solulink.com)将互补于捕获序列(cplCap03)的寡核苷酸的3'末端缀合至G8单克隆抗体。通过HPLC纯化缀合物以去除多余的试剂。然后将捕获序列和捕获序列的补体以一种摩尔比杂交以占据所有可用的捕获序列。如下所概述,每个树枝状聚合物分子附接大约2-5个G8mAb。
阿霉素双链寡核苷酸G8抗体的制备:根据下表,将1.282μg G8mAb寡核苷酸缀合物(如上制备)与1.282μg互补寡核苷酸序列(SEQ ID NO:2)和1500μl的200μM阿霉素溶液组合,并且使用无菌1x PBS将最终体积调节至3000μl。在37℃下孵育混合物持续30分钟。在3mL溶液中的阿霉素最终浓度为100μM。根据操作手册使用Quickspin高容量Sephadex G50柱(Roche)纯化试剂。添加16微升5M NaCl(Life Technologies)以达到86mM NaCl浓度。使用荧光测定法确定最终阿霉素。
阿霉素2层树枝状聚合物(不靶向)的制备:根据下表,将2层Cap03树枝状聚合物(3μg)与1500μl的200μM阿霉素溶液混合,并且使用无菌1x PBS将最终体积调节至3000μl且在37℃下孵育30分钟。在3mL溶液中的阿霉素最终浓度为100μM并且最终树枝状聚合物的浓度为10ng/μl。根据操作手册使用Quickspin高容量Sephadex G50柱(Roche)纯化试剂。添加16微升5M NaCl(Life Technologies)以达到86mM NaCl浓度。使用荧光测定法和紫外/可见光谱法确定最终阿霉素和树枝状聚合物的浓度。
3000.000μl总体积
阿霉素2层G8靶向的树枝状聚合物的制备:根据下表,将2层Cap 03树枝状聚合物(3μg)与1.282μg的G8mAb寡核苷酸缀合物(如上制备)和1500μl的200μM阿霉素溶液混合,并且使用无菌1x PBS将最终体积调节至3000μl。在37℃下孵育混合物持续30分钟。在3mL溶液中的阿霉素最终浓度为100μM并且最终树枝状聚合物的浓度为10ng/μl。根据操作手册使用Quickspin高容量Sephadex G50柱(Roche)纯化试剂。添加16微升5M NaCl(LifeTechnologies)以达到86mM NaCl浓度。使用荧光测定法和紫外/可见光谱法确定最终阿霉素和树枝状聚合物的浓度。
用与嵌入有阿霉素的DNA树枝状聚合物缀合的G8单克隆抗体靶向人晶状体组织中的Myo/Nog细胞:将在白内障手术过程中通过撕囊术去除的人晶状体前组织收集于含有1%盘尼西林/链霉素的DMEM/F12培养基(DF)中。将晶状体组织转移至含有240μl DF的8孔腔室组织培养玻片,所述DF含有以下物质:
1.无添加
2.以1:8稀释于DF中的G8Ab/Cap03寡核苷酸/CPL–阿霉素
3.以1:8稀释于DF中的2层G8Ab/Cap03寡核苷酸/Cpl寡核苷酸
4.以1:8高度稀释于DF中的2层Cap03
晶状体漂浮在腔室中直到它们沉入玻片的底部。将它们在37℃下在空气中的5%CO2中培养24小时。用PBS冲洗组织并且在2%低聚甲醛中固定10分钟,然后用G8抗体和与Alexa 488缀合的抗小鼠IgM以及来自ROCHE TUNEL试剂盒的试剂进行双标记,所述ROCHETUNEL试剂盒在罗丹明通道下发出荧光。
通过落射荧光显微镜确定G8+细胞和TUNEL+细胞的百分比,G8+细胞与TUNEL的百分比,以及TUNEL+细胞与G8的百分比。结果显示于下表中:
注解:这种培养具有大量细胞损失。仅214个细胞留在晶状体上
在未经处理的培养物中少于1%的细胞是TUNEL+(图2A)。另外,通过缺乏红色染色所证明,单独具有阿霉素的树枝状聚合物(DNA:DOX)在Myo/Nog细胞或G8-阴性晶状体细胞中不诱导凋亡(图2B)。然而,具有阿霉素的缀合至G8mAb的树枝状聚合物(G8:DNA:DOX)在Myo/Nog细胞中特异性地诱导细胞凋亡(图2C和图2D,其示出具有不同数目阳性细胞的培养物的两个不同部分)。含G8的细胞被染成绿色,并且经历细胞凋亡的细胞(TUNEL+)被染成红色。图2C中的TUNEL+细胞(红色)通过黑点指示,并且图2D中的TUNEL+细胞(红色)通过深灰点指示。小的黑色斑点是染成绿色的含G8的细胞。
缀合至具有嵌入的阿霉素的双链寡核苷酸的G8mAb还在Myo/Nog细胞中特异性地诱导细胞凋亡(数据未示出)。
实施例3:靶向耗尽胰腺肿瘤细胞
2层DNA树枝状聚合物制备:如实施例1中所述制备2层DNA树枝状聚合物,包括将捕获序列附接至10%-15%的树枝状聚合物臂。
代替G8mAb,将转铁蛋白受体(TfR/CD71)靶向肽THRPPMWSPVWP(TfR肽,SEQ ID NO:1)连接至树枝状聚合物。使用商业上可获得的化学品(Bio-Synthesis,www.biosyn.com)将互补于捕获序列的cplCap03寡核苷酸由3'末端共价偶联至TfR肽,通过HPLC纯化以去除多余的试剂,并且以一种摩尔比杂交以占据所有可用的捕获序列。如下所概括,将大约2-5个肽附接至每个树枝状聚合物。
阿霉素双链寡核苷酸TfR肽的制备:将TfR肽寡核苷酸缀合物(如上制备)杂交至捕获寡核苷酸,并且如在实施例1中所述与阿霉素组合用于制备具有阿霉素的G8mAb双链寡核苷酸缀合物。
阿霉素2层树枝状聚合物(不靶向)的制备:还如先前所描述制备不具有TfR肽靶向的阿霉素2层树枝状聚合物。
阿霉素2层TfR肽靶向的树枝状聚合物的制备:将如上制备的2层TfR肽靶向的树枝状聚合物如在实施例1中所述与阿霉素组合,用于制备具有阿霉素的G8mAb树枝状聚合物缀合物。
体内小鼠研究:为了在小鼠2X105PAN02-Luc细胞(稳定表达萤火虫荧光素酶的鼠胰腺癌细胞)中诱导肿瘤形成,将20ml基质胶(Matrigel)直接注入B6小鼠的胰腺内。四周后,用100μL的1x PBS(阴性对照)、具有阿霉素的TfR肽双链DNA寡核苷酸、具有阿霉素的未靶向的DNA树枝状聚合物或具有阿霉素的TfR肽靶向的DNA树枝状聚合物对小鼠进行眶后注射。每周两次进行注射持续2周,总共4次剂量。在最后一次剂量之后3天使小鼠牺牲,并且将肿瘤、肝脏、脾脏固定于10%福尔马林中持续2小时,并且制备石蜡包埋的切片并且固定在玻片上用于进一步研究。为了确定作为处理结果的细胞死亡水平,使用原位细胞死亡检测试剂盒(Roche)进行TUNEL测定。
在用具有嵌入的阿霉素的TfR靶向的DNA树枝状聚合物和用TfR靶向的双链寡核苷酸处理的小鼠中的肿瘤显示出比PBS缓冲液对照或具有阿霉素的未靶向的DNA树枝状聚合物明显更多的细胞死亡。
虽然在本文中已经参照了具体实施方案来描述本发明,但应了解,这些实施方案仅说明本发明的原理和应用。本领域技术人员将显而易知的是,可在不脱离本发明的精神和范围的情况下对本发明的方法和装置做出各种修改和变更。因此,本发明意图包括在随附权利要求和它们的等效物的范围内的修改和变更。
Claims (12)
1.一种用于将活性剂递送至细胞或组织的组合物,所述组合物包含全部或部分双链合成DNA载体、连接至所述DNA载体的靶向剂和嵌入在所述DNA载体的双链部分中的活性剂,其中所述靶向剂为结合G8抗原的抗体或肽,其中所述活性剂为黄连素、吖啶、道诺霉素、阿霉素、柔红霉素、更生霉素、顺铂、卡铂或沙利度胺。
2.如权利要求1所述的组合物,其中所述双链DNA的长度大于5个碱基对。
3.如权利要求1所述的组合物,其中所述活性剂通过氢键被嵌入在所述双链DNA中。
4.如权利要求1所述的组合物,其中所述合成DNA载体是DNA树枝状聚合物。
5.如权利要求4所述的组合物,其中所述DNA树枝状聚合物包含3-216个杂交的DNA单链。
6.一种药物组合物,其包含如权利要求1-5中任一项所述的组合物和药学上可接受的赋形剂。
7.一种制备用于将活性剂递送至细胞或组织的DNA载体的方法,所述方法包括组装单链DNA寡核苷酸以形成全部或部分双链DNA,将靶向剂连接至所述全部或部分双链DNA,并且使所述全部或部分双链DNA与活性剂接触以使得所述活性剂嵌入至所述DNA的双链部分内,其中所述靶向剂为结合G8抗原的抗体或肽,和其中所述活性剂为黄连素、吖啶、道诺霉素、阿霉素、柔红霉素、更生霉素、顺铂、卡铂或沙利度胺。
8.如权利要求7所述的方法,其中在使所述全部或部分双链DNA与所述活性剂接触的同时组装所述单链DNA寡核苷酸。
9.如权利要求7所述的方法,其中在组装之后使所述全部或部分双链DNA与所述活性剂接触。
10.如权利要求9所述的方法,其还包括在与所述活性剂接触之前将靶向剂连接至所述全部或部分双链DNA。
11.如权利要求6所述的药物组合物在制造用于将活性剂递送至细胞或组织的药物中的用途。
12.如权利要求6所述的药物组合物在制造用于在患者中治疗疾病或病状的药物中的用途。
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