CN116059356A - 一种具有自产氧能力的dna纳米酶及其制备方法与应用 - Google Patents
一种具有自产氧能力的dna纳米酶及其制备方法与应用 Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明提供了一种具有自产氧能力的DNA纳米酶及其制备方法与应用,包括DNA纳米花、血红素辅酶和光敏剂Ce6,DNA纳米花由引物链和底物链通过滚环扩增形成,DNA纳米花在高钾的Tris缓冲液中形成AS1411G四链体空间结构,将血红素辅酶嵌入至AS1411G四链体结构中,光敏剂Ce6通过π‑π作用堆叠在AS1411G四链体结构中,形成具有自产氧能力的DNA纳米酶;该DNA纳米酶高效稳定负载血红素辅酶和光敏剂Ce6,通过EPR效应富集于肿瘤组织,其中AS1411G四链体识别高表达核仁素的肿瘤细胞实现靶向定位;在肿瘤中高过氧化氢的环境下,血红素辅酶高效催化产氧,显著缓解肿瘤微环境乏氧的情况;同时光敏剂可以将产生的氧转化为具有肿瘤细胞毒性的活性氧ROS,进一步对肿瘤细胞进行杀伤。
Description
技术领域
本发明涉及纳米材料和纳米生物医药领域,具体涉及一种具有自产氧能力的DNA纳米酶及其制备方法与应用。
背景技术
癌症是影响人类生命健康和社会发展的重大问题。化学疗法作为临床上治疗癌症的主要手段,但肿瘤微环境的异质性和单一化疗造成低疗效、严重不良反应并形成肿瘤耐药性。因光动力疗法(photodynamic therapy,PDT)具有可控性好、毒性低和微创性等优点,从而避免高剂量诱导的不良反应和肿耐药性。
然而,PDT在实际应用中存在一些障碍。由于肿瘤细胞增殖异常、血管结构缺失及肿瘤组织血流不足,肿瘤微环境常表现为低氧、弱酸性及过表达的过氧化氢、细胞因子等特点。肿瘤乏氧上调肿瘤缺氧诱导因子-1α(HIF-1α)的表达水平,导致肿瘤细胞对化疗、PDT等疗法产生耐药性。光敏剂转化氧气为具有细胞毒性的活性氧(ROS)进行光动力治疗,从而加剧肿瘤乏氧程度且严重影响ROS生成效率,最终导致PDT疗效低和预后不良。此外, ROS通过结合DNA、脂质等生物大分子,引起细胞器功能障碍及损伤,最终导致肿瘤细胞死亡。因ROS的半衰期较短(<200ns)以及扩散范围较小(20nm),定位于细胞器的ROS比在细胞膜或细胞质内的更具肿瘤杀伤能力,因此保证光敏剂有效的传递是实现光动力疗效的重要条件。
为解决肿瘤缺氧,已发展多种供氧策略以提高肿瘤组织的氧气水平。通过递送全氟化碳、血红蛋白或者红细胞膜等携氧材料,可直接改善肿瘤乏氧情况。然而,这些递氧材料通常存在携氧能力差、稳定性差,如血红蛋白半衰期短等缺陷,导致制剂递氧效率低。而响应肿瘤微环境的自产氧策略恰能解决此类问题,靶向递送过氧化氢酶、二氧化锰纳米粒、片及普鲁士蓝纳米粒等具有催化活性的酶和纳米材料,分解肿瘤细胞内过表达的H2O2,上调胞内氧气水平,进而提高光动力疗效,且在肿瘤缺氧治疗中疗效显著。但以上制剂均存在应用限制: (1)过氧化氢酶在体内存在不同程度的失活;(2)纳米材料组成成分不可控性和生物安全性。因此,构建安全高效的递送载体,在实现光敏剂靶向递送的同时解决肿瘤乏氧问题,是实现光动力治疗研究领域亟待解决的问题。
发明内容
为解决上述技术问题,本发明提出了一种具有自产氧能力的DNA纳米酶及其制备方法与应用,其目的是为了构建一种安全高效的递送载体,将过血红素辅酶和光敏剂高效组合成为纳米材料,能够实现体内稳定的靶向肿瘤组织,并且能在肿瘤细胞处有效释放、协同作用,改善肿瘤微环境乏氧情况,提高肿瘤光动力治疗的效果。
为了实现上述目的,本发明提供了一种具有自产氧能力的DNA纳米酶,包括DNA纳米花、血红素辅酶和光敏剂Ce6,所述DNA纳米花由引物链和底物链通过滚环扩增形成,所述引物链如SEQ ID NO.1所示:
GTGGTGGTGTTGGTGGTGGT;
所述底物链如SEQ ID NO.2所示:
CCACCAACACCACCACCACCTTTGACACACTAGCGATACGCGTATCGCTATGGCATATCGTACGATATGCCAGTGTGTCTTTCCACCA;
所述DNA纳米花具有AS1411 G四链体结构,所述血红素辅酶嵌入至AS1411 G四链体结构中,所述光敏剂Ce6通过π-π作用堆叠在AS1411 G四链体结构中。
作为优选,所述DNA纳米酶的形状为球形,粒径为100~200nm。
基于一个总的发明构思,本发明还提供了一种具有自产氧能力的DNA纳米酶的制备方法,包括以下步骤:
S1、制备DNA纳米花:将底物链和引物链混合,经退火进行互补配对形成带缺口的环状DNA,然后加入DNA连接酶反应形成一个完整的环状DNA,最后引入DNA聚合酶进行 PCR扩增,形成若干个拷贝链的DNA长链,DNA长链通过碱基配对和自组装方式合成DNA 纳米花;
S2、AS1411 G四链体结构形成:取DNA纳米花溶于含高钾离子的Tris缓冲液中,室温孵育,形成带AS1411 G四链体空间结构的DNA纳米花;
S3、组装形成DNA纳米酶:将步骤S2制得的带AS1411 G四链体空间结构的DNA纳米花与血红素辅酶混合、室温孵育4~6h,然后加入光敏剂Ce6涡旋混匀,继续室温孵育3~4h,离心收集沉淀即得具有自产氧能力的DNA纳米酶。
作为优选,所述步骤S1中退火温度为95℃水浴10~30min,然后缓慢降温至25℃,所述降温时间大于2.5h。
作为优选,所述步骤S1中DNA连接酶为T4-DNA连接酶,所述DNA聚合酶为phi29 DNA聚合酶。
作为优选,所述步骤S2中Tris缓冲液为Tris-HCl、NaCl和KCl混合液,pH为7~8。
作为优选,所述步骤S3中DNA纳米花与血红素辅酶的混合摩尔比为1:40~50;所述DNA 纳米花与光敏剂Ce6的混合摩尔比为1:500~600。
一种如权利要求1~2任一项所述的具有自产氧能力的DNA纳米酶或者如权利要求3~7 任一项所述制备方法制得的具有自产氧能力的DNA纳米酶在制备抗肿瘤光动力治疗药物中的应用。
与现有技术相比,本发明具有以下有益效果:
1、本发明提供的DNA纳米酶高效稳定负载血红素辅酶和光敏剂Ce6,通过EPR效应富集于肿瘤组织,其中AS1411 G四链体识别高表达核仁素的肿瘤细胞实现靶向定位;在肿瘤中高过氧化氢的环境下,血红素辅酶高效催化产氧,显著缓解肿瘤微环境乏氧的情况;同时光敏剂可以将产生的氧转化为具有肿瘤细胞毒性的活性氧ROS,进一步对肿瘤细胞进行杀伤,血红素辅酶与光敏剂协同作用,显著增强肿瘤光动力治疗的效果;
2、该DNA纳米酶中的DNA纳米花的关键结构为AS1411 G-四链体,G-四链体由富含鸟嘌呤(G)的寡核酸链通过四个相邻鸟嘌呤碱基间氢键连接形成,其中,血红素辅酶可嵌入至芳香族平面中,形成具有过氧化氢酶活性的DNA酶,催化肿瘤组织中富含的过氧化氢,产生氧气;与此同时,光敏剂Ce6可通过π-π作用堆叠在其芳香族平面,从而实现将血红素辅酶和光敏剂Ce6稳定负载于DNA纳米花结构中,形成稳定性良好的DNA纳米酶,并利用AS1411G-四链体对肿瘤的高特异性识别,实现肿瘤靶向递送;
3、该DNA纳米酶具有良好的抗酶降解性能,能够实现体内的稳定递送并靶向肿瘤细胞,对心、肝、脾、肺、肾等无明显副作用,具有良好的生物安全性;
4、该DNA纳米酶制备方法简单可控,形成粒径100~200nm的球形纳米粒子,具有较好的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明的DNA纳米酶构建原理图;
图2为本发明实施例1中DNA纳米花合成中的滚环扩增示意图;
图3为本发明实施例1中滚环反应不同时间所形成的纳米花扫描电镜图;
图4为本发明实施例1中DNA纳米花中***血红素辅酶,并包载光敏剂的表征图,其中图4A为动态光闪射图;图4B为光敏剂包载后荧光图;图4C为透射电镜图;图4D为电镜元素分析图;
图5为本发明实施例2中DNA纳米酶催化产氧并增强光敏剂的光动力效果的表征图,其中图5A为DNA纳米酶催化下H2O2的消耗情况图;图5B为DNA纳米酶催化下O2的产生图;图5C为DNA纳米酶催化下,促光动力作用的自由基生成图(通过SOSG试剂盒检测);图5D为DNA纳米酶催化产氧增强光动力效果的机制图;图5E为验证纳米酶的氧气的生成性能;图5F为纳米酶对DNA酶活性的保护性能;
图6为本发明实施例3中DNA纳米酶的跨细胞转运、细胞核靶向、以及肿瘤细胞选择性表征图,其中图6A为时间依赖性的细胞摄取,从共定位结果来看,纳米酶主要聚集于细胞核,图6B为DNA纳米酶可选择性地被肿瘤细胞摄取结果图,图6C为细胞摄取的定量结果图;
图7为本发明实施例3中DNA酶对肿瘤细胞的杀伤表征图,其中图7A为MTT实验表征细胞毒性结果图;图7B为活死细胞染色表征细胞毒性结果图;图7C为荧光染色表征胞内自由基的生成结果图;
图8为本发明实施例4中DNA酶在肿瘤组织中的靶向富集表征图,以游离光敏剂为对照,表征纳米粒在肿瘤组织中的富集,其中图8A为离体成像结果,图8B为荧光定量结果;
图9为本发明实施例4中DNA酶的体内抗肿瘤活性表征图,其中图9A为治疗过程中动态监测肿瘤的体积,图9B为治疗后的肿瘤体积大小图,图9C为肿瘤体重的定量结果;
图10为本发明实施例4中DNA酶的生物安全性评估表征图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段;若未特别指明,实施例中所用试剂均为市售。
以下实施例中,所采用的仪器及生产厂家的详细信息参见表1:
表1:主要仪器名称及生产厂家
仪器名称 | 生产厂家 |
CP225型电子天平 | 德国赛多利斯(Sartorius)公司 |
BP224S型电子天平 | 德国赛多利斯(Sartorius)公司 |
涡旋混合仪 | 上海青浦沪西分析仪器厂 |
掌上离心机 | 美国Scilogex公司 |
TGL 20M台式高速冷冻离心机 | 长沙英泰仪器有限公司 |
Nano ZS90粒径/电位分析仪 | 英国Malvern仪器有限公司 |
UV-2600紫外分光光度计 | 日本岛津公司 |
PHS 3C酸度计 | 上海仪电科学仪器股份有限公司 |
Infinite M200PRO多功能酶标仪 | 奥地利TECAN公司 |
Tecnai G2 F20型透射电子显微镜 | 美国FEI公司 |
MIN4-UVF纯水机 | 湖南科尔顿水务有限公司 |
SHA B水浴恒温振荡器 | 常州澳华仪器有限公司 |
<![CDATA[Forma Series II型CO<sub>2</sub>培养箱]]> | 美国Thermo Fisher公司 |
IX70型倒置荧光显微镜 | 日本Olympus公司 |
SW-CJ-2FD型垂直净化工作台 | 苏州净化设备有限公司 |
IVIS Lumina III小动物活体成像*** | 美国PerkinElmer公司 |
4℃冰箱 | 中国海尔医用冷藏冰箱 |
-20℃冰箱 | 中国海尔医用低温保存冰箱 |
-80℃冰箱 | 中国海尔医用低温保存冰箱 |
以下实施例中,所采用的主要试剂名称及生产厂家参见表2:
表2:主要试剂名称及生产厂家
试剂名称 | 生产厂家 |
DNA(HPLC纯化级) | 生工生物工程(上海)股份有限公司 |
氧化铜纳米酶 | 北京德科岛金科技有限公司 |
核苷 | 美国sigma公司 |
3,3’,5,5’-四甲基联苯胺(TMB) | 美国sigma公司 |
无水乙酸钠 | 国药集团化学试剂有限公司 |
冰乙酸 | 国药集团化学试剂有限公司 |
2-氨基-2-(羟甲基)-1,3-丙二醇(Tris) | 国药集团化学试剂有限公司 |
二水合磷酸二氢钠 | 国药集团化学试剂有限公司 |
氯化钠 | 上海麦克林生化科技有限公司 |
30%过氧化氢 | 西陇化工股份有限公司 |
实施例1
DNA纳米花(DF)的制备
其基本流程图如图2所示:经一步退火法,底物链和引物链的两端碱基进行互补配对,形成一个带缺口的环状DNA。加入T4-DNA连接酶,修复底物链上的缺口,形成一个完整的环形DNA。最后引入phi29 DNA酶,识别环状DNA的引物链,进行无限复制,扩增出具有若干个拷贝链的DNA长链,DNA长链以DNA碱基配对和液晶自组装方式合成DNA纳米花。其中引物链的序列为:GTGGTGGTGTTGGTGGTGGT,SEQ ID NO.1;
底物链的序列为:
CCACCAACACCACCACCACCTTTGACACACTAGCGATACGCGTATCGCTATGGCATATCGTACGATATGCCAGTGTGTCTTTCCACCA,SEQ ID NO.2。
具体操作:在100μL T4-DNA连接酶缓冲液(5mM Tris-HCl,1mM MgCl2,0.1mM ATP,1mM二硫苏糖醇)中加入引物链(终浓度为1.2μM)和底物链(终浓度为0.6μM),吹打混匀后置于95℃水浴锅加热10min,转移至装有95℃热水的250mL烧杯中,缓慢降温至 25℃,且降温时间为3h。精密吸取2.5μL T4连接酶(10U/μL)与上述DNA退火产物涡旋混匀,25℃孵育4h以闭合其环状DNA的缺口。
在100μL RCA缓冲液(50mM Tris-HCl,10mM(NH4)2SO4,10mM MgCl2,4mM二硫苏糖醇)中加入DNA环合产物(终浓度为0.3μM),phi29 DNA聚合酶(终浓度为1U/μL), dNTP(终浓度为2mM)和BSA,混合均匀并置于30℃水浴锅中孵育3h。扩增反应结束后,置于75℃孵育10min,高温使phi29聚合酶失活以终止反应。通过扫描电镜监测纳米花的合成,结果如图3所示,由图3可知:随着反应时间的延长,纳米花的粒径越来越大,但粒子数越来越少,据此确定最优反应时间为3h。以16000rpm离心15min收集DNA纳米花,超纯水洗涤两次以去除多余盐分,超声细胞仪分散(40W,12s),置于-20℃长期储存。
实施例2
载光敏剂的DNA纳米酶(CH/DF)的制备
取实施例1制得的适量DNA纳米花(DF)与等体积2×缓冲液(20mM Tris-HCl,40mMNaCl,40mMKCl,pH 7.6)混合,室温孵育1h使纳米花结构中AS411区域序列在高钾离子条件下形成G-四链体空间结构,并通过π-π作用结合血红素辅酶(hemin)、光敏剂Ce6芳香族配体。DNA纳米花与hemin(以下简称H)以摩尔比1:40的比例均匀混合,室温孵育4 h,16000rpm离心10min,沉淀用等体积缓冲液超声复溶,即得H/DF;将H/DF与Ce6(简称为C)以摩尔比1:600的比例涡旋混匀,室温孵育4h后离心收集沉淀,即得载光敏剂的 DNA纳米酶(CH/DF)。
实验例1
载光敏剂的DNA纳米酶(CH/DF)的表征
取10μL DNA纳米酶CH/DF样品,用超纯水稀释100倍,分别采用动态光激光散射法(DLS)及激光多普勒测速法(LDV),测定其水合粒径、粒径分布,结果如图4A所示,所制得CH/DF粒径均匀,平均水化粒径为300nm左右
通过荧光光谱仪监测光敏剂Ce6的包载,如图4B所示,Ce6载入纳米花之后,其荧光发射光谱显著降低。
扫描电子显微镜(SEM)表征:取出新制备的王水中浸泡过夜的硅片,用水和丙酮清洗干净并烘干,于硅片表面滴加10μl DNA纳米花溶液,样品置于70℃烘箱干燥2h。SEM成像前,用金涂覆样品以增强其导电性。结果如图4C所示,纳米粒呈现均匀球形结构,干燥后粒径约为100nm:
透射电子显微镜(TEM)及能谱分析(EDS)表征:取适量DNA纳米花和CH/DF超声分散,滴加于铜网上,置于70℃烘箱烘干30min,用于TEM成像并通过EDS分析制剂中各元素组成。结果如图4D所示,纳米粒为均匀球形,结构中的N/P元素主要归属于DNA,而 Fe元素来自于血红素的包载。
综合以上结果可知,DNA纳米花成功包载了血红素辅酶和光敏剂Ce6,形成了粒径均一的球形纳米粒。
实验例2
DNA纳米酶(CH/DF)的产氧及稳定性检测
1、过氧化氢酶活性检测
基于Góth原理,H2O2结合钼酸铵形成稳定且不可逆的黄色复合物,根据紫外谱图中405 nm处的吸光度计算H2O2浓度。采用钼酸铵法检测DNA纳米花的过氧化酶活性,具体操作为:吸取0.5mLH2O2(1mM)与0.1mL CH/DF(0.5μM)于1.5mL EP管混合均匀,室温反应1min,再加入0.5mL钼酸铵溶液(32.4mM)以终止反应,静置10min后使用紫外分光光度计检测A405nm。结果如图5A所示,证实了CH/DF可消耗试管中的H2O2。
2、自产氧能力考察
为进一步研究CH/DF的自产氧性能,采用便携式溶解氧仪实时监测体系的溶氧水平。主要操作如下:吸取4mL CH/DF(0.5μM)于10mL烧杯,加入H2O2储备液稀释至终浓度为1mM。检测过程中保持低速搅拌,研究CH/DF在光动力治疗中氧气的动态变化。结果如图 5B所示,表明了CH/DF可快速催化H2O2产生O2。
3、ROS产生能力考察
SOSG荧光探针不受其他活性氧物质如羟基自由基(·OH)或超氧阴离子(·O2–)影响,可特异性结合单线态氧(1O2),从而降低背景干扰。吸取CH/DF(0.5μM)、SOSG(2.5 μM)和H2O2(1mM)于96孔板,均匀混合,补加缓冲液至终体积为100μL。使用功率为 0.75W/cm2的660nm激光照射孔板1min,每10s记录SOSG的荧光强度(Ex=504nm,Em=524 nm)。结果如图5C所示,在光照条件下,SOSG的荧光快速增强,证明了CH/DF的光动力活性。
4、酶稳定性考察
本实验旨在于研究DNA纳米花与游离G-四链体(CH/G4)的抗酶降解能力,具体操作如下:G-四链体按照实施例2的方法荷载hemin、Ce6,制备CH/G4,然后将CH/G4和CH/DF 两种制剂分别与10%FBS共孵育12h,模拟酶降解过程。按照上述方法考察经酶处理后两种制剂的自产氧性能及ROS产生能力。结果如图5E和图5F所示,CH/G4经FBS处理后,其活性降低较明显,而CH/DF依然维持系原始活性,证实了DNA纳米花的抗酶降解能力。
综合本实验例的上述结果可知,DNA纳米酶(CH/DF)能够高效催化H2O2产生O2,同时CH/DF还可催化生成单线态氧1O2,协助光动力治疗(PDT),其基本原理如图4D所示;同时,CH/DF能够有效抵抗酶降解,结构稳定,能够便于体内实现有效递送。
实验例3
1、细胞摄取实验:将A549或HEK-293细胞以每孔2.5×104个细胞密度接种于24孔板 (0.5mL细胞悬液/孔),置于细胞培养箱培养24h。吸去废旧培基,加入CH/DF,分别孵育1、2或4h。PBS清洗三次,4%多聚甲醛与细胞共孵育20min。固定后吸弃,加入DAPI(1 μg/mL)染色细胞核。孵育10min后用PBS清洗两遍,置于细胞成像仪并观察荧光分布。如图6A所示,随着孵育时间的延长,纳米花在胞内的荧光逐渐增强,证明了其时间依耐性的细胞摄取;图6B和图6C显示,纳米粒在肿瘤细胞中的荧光强度明显高于正常细胞,证实了制剂CH/DF的肿瘤靶向选择性。
2、细胞毒性实验:将A549细胞以密度5×103个/孔接种于96孔板中,于细胞培养箱中孵育24h。吸弃旧培基,PBS清洗两遍,然后分组使用DF、C/DF、CH/DF、C/DF+L和CH/DF+L 进行处理,其中C/DF+L和CH/DF+L为使用光照的对照组;另外选取不同浓度的上述样品溶液分组处理细胞进行对比。孵育24h后激光组给予0.75W/cm2激光照射1min,继续培养24h。每孔加入20μL MTT(5mg·mL-1)溶液,孵育4h,吸弃上清,再加入100μL DMSO,置于恒温水浴振荡箱中90rpm/min振荡10min。使用酶标仪检测每孔570nm波长处吸光度(A) 值,依据存活率(%)=(A样品组-A调零组)/(A空白组-A调零组),计算各组细胞存活率。结果如图7A所示:DF没有细胞毒性,表明材料的生物安全性;包载光敏剂Ce6后,C/DF 体现出一定的光毒性;进一步包载血红素,毒性增强;在光照条件下,CH/DF体现出最强的细胞毒性,证实了其自产氧能力对光动力治疗的协同增效。
3、活/死细胞染色实验:采用商售Calcein-AM/PI试剂盒进行活/死细胞染色实验,钙荧光素乙酰氧基甲酯(Calcein-AM)作为一种标记活细胞的荧光染料,易穿过活细胞膜。入胞后Calcein-AM被胞内的酯酶水解成非膜渗透型荧光分子Calcein,在细胞内滞留并发出绿色荧光。碘化丙啶(PI)不能进入活细胞,因死细胞的细胞膜通透性好,PI入胞后与细胞核处DNA结合,发出强烈的红色荧光。如上述方法制备获得密度为5×104cells/mL的A549细胞悬液,按照每孔5千个细胞的密度,将细胞接种于96孔板中,置于细胞培养箱中孵育24h。分组加入DF、C/DF、CH/DF、C/DF+L和CH/DF+L干预24h后,激光组外加光源照射1min。继续孵育24h,除去旧培养基,PBS轻轻清洗2~3次,防止细胞被吹走。精密吸取Calcein-AM 工作液10μl于10mLPBS中,涡旋混匀,再加入PI工作液10μl,充分混匀。每孔加入100μl 染色液,孵育30min后吸弃上清,用荧光显微镜观察绿色及红色荧光分布情况。结果如图7B,通过染色,活细胞呈现绿色,凋亡的细胞呈现红色,通过染色观察,各个制剂的细胞毒性结果与MTT实验高度一致。
4、胞内ROS水平考察:本实验使用DCFH-DA试剂盒监测细胞内ROS表达水平。其工作原理为,DCFH-DA可自由跨膜进入胞内,被内源性酯酶水解生成无荧光的DCFH,因 DCFH无法穿过细胞膜,胞内ROS将胞内分布的DCFH迅速氧化为具有强烈荧光的DCF,其绿色荧光强度与ROS水平呈正比。将A549细胞以每孔5×104个/mL的密度接种于24孔板中,设置对照组、DF组、C/DF组、H/DF组、CH/DF组、CD/DF+L组和CHD/DF+L组。 10nM样品与细胞共孵育4h后,预冷PBS清洗3次。用无胎牛血清的培基稀释DCFH-DA (10μM),加入孔中,孵育30min,然后外加660nm激光照射1min(0.75W/cm2)。最后使用细胞成像仪观察细胞内荧光分布情况。结果如图7C,经光照后,C/DF及CH/DF组的细胞内荧光显著增强,证实了光敏剂的光动力效应。与此同时,CH/DF具有更强的胞内荧光信号,归因于该纳米酶的自产氧活性对光动力学增效能力。
实施例4
(1)荷瘤裸鼠造模:裸鼠由中南大学实验动物学部统一在符合动物实验要求的条件下购买并饲养。所有的动物护理和处理均由当地伦理委员会批准,并按照中国相关动物保护法的指导方针实施。在裸鼠右前侧进行皮下接种事先准备好的含有A549细胞、PBS和基质胶的混悬液,每只裸鼠的接种量约为5x 106个细胞,然后正常饲养接种后的裸鼠,待肿瘤体积生长到合适大小后再用于后续的动物实验。
(2)活体成像:将Cy5标记的DNA纳米酶通过尾静脉注射进荷瘤裸鼠体内。同时设置对照组,对照组给与当量的Cy5尾静脉注射。在注射后1h和24h时间点,对各组裸鼠进行小动物活体成像。图8A为离体成像结果,图8B为荧光定量结果。相比于游离Ce6,CH/DF 具有更强的肿瘤荧光信号,证实了纳米制剂的肿瘤靶向富集能力。
(3)体内抗肿瘤实验:按照上述方法建立皮下荷A549异体移植的肿瘤裸鼠模型,待肿瘤长到约60mm3时,给予不同治疗,接下来对每组荷瘤裸鼠进行标记、体重称量和瘤径测定,然后根据设置的组别,对每只荷瘤裸鼠进行尾静脉给药。同时在治疗期间内隔天进行体重称量和瘤径测定。治疗第16天后,把荷瘤裸鼠安乐处死,取出肿瘤称重。图9A为治疗过程中动态监测肿瘤的体积,图9B为治疗后的肿瘤体积大小图,图9C为肿瘤体重的定量结果。结果表明,各个分组制剂的抗肿瘤疗效排序为CH/DF+L>C/DF+L>CH/DF>C/DF≈DF,其中在光照条件下CH/DF具有最强的抗肿瘤能力,进一步证实了该纳米酶的肿瘤治疗潜力。治疗后,取各组老鼠的主要器官,并进行病理观察,结果如图10所示:治疗后,小鼠的各个器官均无明显改变,证实了DNA纳米酶制剂的生物安全性。
以上所述实施例,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明的技术范围内,根据本发明的技术方案及其构思加以等同替换或改变,都应涵盖在本发明的保护范围内。
Claims (8)
1.一种具有自产氧能力的DNA纳米酶,其特征在于,包括DNA纳米花、血红素辅酶和光敏剂Ce6,所述DNA纳米花由引物链和底物链通过滚环扩增形成,所述引物链如SEQ ID NO.1所示:
GTGGTGGTGTTGGTGGTGGT;
所述底物链如SEQ ID NO.2所示:
CCACCAACACCACCACCACCTTTGACACACTAGCGATACGCGTATCGCTATGGCATATCGTACGATATGCCAGTGTGTCTTTCCACCA;
所述DNA纳米花具有AS1411 G四链体结构,所述血红素辅酶嵌入至AS1411 G四链体结构中,所述光敏剂Ce6通过π-π作用堆叠在AS1411 G四链体结构中。
2.根据权利要求1所述的具有自产氧能力的DNA纳米酶,其特征在于,所述DNA纳米酶的形状为球形,粒径为100~200nm。
3.一种如权利要求1~2任一项所述具有自产氧能力的DNA纳米酶的制备方法,其特征在于,包括以下步骤:
S1、制备DNA纳米花:将底物链和引物链混合,经退火进行互补配对形成带缺口的环状DNA,然后加入DNA连接酶反应形成一个完整的环状DNA,最后引入DNA聚合酶进行PCR扩增,形成若干个拷贝链的DNA长链,DNA长链通过碱基配对和自组装方式合成DNA纳米花;
S2、AS1411 G四链体结构形成:取DNA纳米花溶于含高钾离子的Tris缓冲液中,室温孵育,形成带AS1411 G四链体空间结构的DNA纳米花;
S3、组装形成DNA纳米酶:将步骤S2制得的带AS1411 G四链体空间结构的DNA纳米花与血红素辅酶混合、室温孵育4~6h,然后加入光敏剂Ce6涡旋混匀,继续室温孵育3~4h,离心收集沉淀即得具有自产氧能力的DNA纳米酶。
4.根据权利要求3所述的制备方法,其特征在于,所述步骤S1中退火温度为95℃水浴10~30min,然后缓慢降温至25℃,所述降温时间大于2.5h。
5.根据权利要求3所述的制备方法,其特征在于,所述步骤S1中DNA连接酶为T4-DNA连接酶,所述DNA聚合酶为phi29 DNA聚合酶。
6.根据权利要求3所述的制备方法,其特征在于,所述步骤S2中Tris缓冲液为Tris-HCl、NaCl和KCl混合液,pH为7~8。
7.根据权利要求3所述的制备方法,其特征在于,所述步骤S3中DNA纳米花与血红素辅酶的混合摩尔比为1:40~50;所述DNA纳米花与光敏剂Ce6的混合摩尔比为1:500~600。
8.一种如权利要求1~2任一项所述的具有自产氧能力的DNA纳米酶或者如权利要求3~7任一项所述制备方法制得的具有自产氧能力的DNA纳米酶在制备抗肿瘤光动力治疗药物中的应用。
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