CN113521097B - 一种三价铁络合的树状大分子/pDNA复合物及其制备和应用 - Google Patents
一种三价铁络合的树状大分子/pDNA复合物及其制备和应用 Download PDFInfo
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Abstract
本发明涉及一种三价铁络合的树状大分子/pDNA复合物及其制备方法和应用。该复合物为:表面修饰8‑羟基喹啉‑2‑羧酸络合三价铁离子、内部包裹金纳米颗粒的第五代聚酰胺胺PAMAM树状大分子负载p53pDNA。该方法包括:G5.NH2‑HQC制备,{(Au0)25‑G5.NH2‑HQC}制备,Fe‑Au DENP‑HQC制备,三价铁络合的树状大分子/pDNA复合物制备。该方法操作工艺简单,反应条件温和,易于纯化;制备得到的复合物具有良好的生物相容性、单分散性、较低的细胞毒性,同时具有荧光成像功能,可用于肿瘤的铁死亡治疗和基因治疗的联合治疗,在诊疗一体化领域具有潜在的应用价值。
Description
技术领域
本发明属于诊疗一体化功能纳米材料及其制备和应用领域,特别涉及一种三价铁络合的树状大分子/pDNA复合物及其制备方法和应用。
背景技术
癌症是以细胞异常增殖及转移为特点的一大类疾病,已成为危害人类健康的主要疾病之一。近年来关于如何提高早期恶性肿瘤诊断的准确性和治疗效果,特别是如何实现恶性肿瘤“诊疗一体化”方面,成为了纳米医学研究的重点和热点。与此同时,一些新型的肿瘤治疗策略如化学动力学治疗/铁死亡和基因治疗受到了人们的广泛关注。
铁死亡以一种铁依赖性活性氧异常堆积而导致细胞氧化还原稳态失调为特征,是区别于细胞凋亡、细胞坏死、细胞自噬的一种新型细胞程序性死亡方式,其主要特点是铁诱导的脂质活性氧(ROS)积聚导致细胞发生过氧化反应。目前,已有研究证明铁死亡在人类疾病的治疗中的重要性。施等人(Huo M,et al.ACS Nano,2019,13(2):2643-2653)将分散的铁原子构建到氮掺杂的碳纳米材料中,制备PEG化的含单原子铁的纳米催化剂,在酸性肿瘤微环境下,肿瘤部位产生大量有毒的羟基自由基,同时脂质过氧化物快速积累而引起细胞凋亡和铁死亡。另外,陈的研究小组(Zhou Z,et al.Angew.Chem.,Int.Ed.,2017,56(23):6492-6496)开发了一种基于类芬顿反应的纳米系统,可以特异性引起肿瘤部位ROS的产生,进而介导肿瘤铁死亡和细胞凋亡而诱导肿瘤细胞死亡。
基因治疗从本质上来说,就是通过不同的手段将外源基因导入靶细胞,从而达到治疗疾病的目的。为实施有效的基因治疗,安全有效的基因载体是必不可少的。聚酰胺-胺(PAMAM)树状大分子是一类具有纳米级尺寸、结构大小可控、无免疫原性、性质稳定、经修饰后毒性小的超支化合成大分子,其表面具有大量的端基,可以进行不同的功能化修饰,其内部有空腔可以负载药物或无机纳米颗粒。另外,阳离子型PAMAM可通过静电作用有效压缩带负电荷的pDNA等基因药物,用于细胞的基因传递。例如,邱等人使用β-环糊精修饰的第五代PAMAM树状大分子包裹的纳米金颗粒,可有效压缩和递送VEGF和Bcl-2siRNA,用于相关的癌细胞基因沉默,达到癌细胞基因治疗的目的(Qiu J.et al.Nanomaterials,2018,8(3):131)。
检索国内外文献,目前尚未发现关于络合铁离子的树状大分子纳米复合物的制备及其应用于肿瘤荧光成像和铁死亡/基因联合治疗的相关报道。
发明内容
本发明所要解决的技术问题是提供一种三价铁络合的树状大分子/pDNA复合物及其制备和应用,以填补现有技术的空白。
本发明提供一种三价铁络合的树状大分子/pDNA复合物,所述复合物为:表面修饰8-羟基喹啉-2-羧酸络合三价铁离子、内部包裹金纳米颗粒的第五代聚酰胺胺PAMAM树状大分子负载p53 pDNA。
优选地,上述复合物中,所述p53 pDNA为具有人体抑癌基因p53和绿色荧光蛋白EGFP基因的质粒。
本发明还提供一种三价铁络合的树状大分子/pDNA复合物的制备方法,包括:
(1)将8-羟基喹啉-2-羧酸HQC溶解于溶剂中,经EDC和NHS活化,加入到G5.NH2溶液中,搅拌反应,透析,冷冻干燥,得到G5.NH2-HQC;
(2)将步骤(1)中G5.NH2-HQC溶于超纯水中,加入HAuCl4·4H2O水溶液,搅拌,加入含NaBH4冰水溶液反应,透析,冷冻干燥,得到{(Au0)25-G5.NH2-HQC},即Au DENP-HQC;
(3)将步骤(2)中Au DENP-HQC溶于超纯水中,逐滴加入三价铁盐的水溶液搅拌,透析,冷冻干燥,得到三价铁络合的树状大分子Fe-Au DENP-HQC;
(4)将步骤(3)中Fe-Au DENP-HQC与p53 pDNA共同孵育,得到Fe-Au DENP-HQC/p53pDNA复合物,即三价铁络合的树状大分子/pDNA复合物。
优选地,上述方法中,所述步骤(1)中溶剂为DMSO;G5.NH2溶液的溶剂为超纯水。
优选地,上述方法中,所述步骤(1)中HQC、EDC和NHS的摩尔比为1:8~10:8~10;G5.NH2与HQC的摩尔比为1:25~35。
优选地,上述方法中,所述步骤(1)中EDC和NHS的溶剂均为超纯水。
优选地,上述方法中,所述步骤(1)中活化时间为2-4h。
优选地,上述方法中,所述步骤(1)中搅拌反应温度为室温,搅拌反应时间为12~48h。
优选地,上述方法中,所述步骤(1)、(2)和(3)中透析的条件为:用截留分子量为8000~14000的透析袋透析2-3天。
优选地,上述方法中,所述步骤(2)中G5.NH2-HQC与HAuCl4·4H2O摩尔比为1:20~30;HAuCl4·4H2O与NaBH4的摩尔比为1:4~1:6。
优选地,上述方法中,所述步骤(2)中搅拌为:冰水浴条件下搅拌15~30min。
优选地,上述方法中,所述步骤(2)中反应是在冰水浴条件下反应2~3h。
优选地,上述方法中,所述步骤(3)中三价铁盐为FeCl3;Au DENP-HQC与三价铁盐的摩尔比为1:28~1:35。
优选地,上述方法中,所述步骤(3)中搅拌温度为室温,搅拌时间为2~4h。
优选地,上述方法中,所述步骤(4)中Fe-Au DENP-HQC与p53 pDNA的N/P为2:1~20:1,其中N/P比为树状大分子的伯胺基与质粒p53骨架上磷酸基团的摩尔比。
优选地,上述方法中,所述步骤(4)中p53 pDNA为带有增强的绿色荧光蛋白EGFP基因的质粒。
优选地,上述方法中,所述步骤(4)中共同孵育时间为15-30分钟。
本发明还提供一种三价铁络合的树状大分子/pDNA复合物在制备肿瘤的荧光成像和铁死亡/基因联合治疗的诊疗剂中的应用。
本发明基于第五代聚酰胺胺树状大分子(G5.NH2)作为载体,在其表面修饰三价铁离子的配体络合三价铁离子,在其内部包裹纳米金颗粒,同时通过静电吸附负载p53质粒,形成复合物,得到可用于肿瘤的铁死亡和基因治疗的联合治疗以及荧光成像诊断的纳米材料。实验结果表明,Fe-Au DENP-HQC/p53 pDNA不仅可以用于荧光成像,而且兼具肿瘤的铁死亡治疗和基因治疗两种治疗模式,可实现肿瘤诊疗一体化。
本发明使用核磁共振氢谱(1H NMR)、紫外可见吸收光谱(UV-vis)、电感耦合等离子体-原子发射光谱法(ICP-OES)、动态光散射(DLS)和透射电镜测试(TEM)等方法表征制备得到的三价铁络合的树状大分子,然后通过CCK-8法来评价该纳米材料的细胞毒性,采用凝胶阻滞实验和绿色荧光蛋白表达实验来确定载体的基因压缩和转染效果,再利用流式细胞仪、共聚焦显微镜来评价该纳米材料对肿瘤细胞活性氧水平、脂质过氧化物、谷胱甘肽水平的影响,最后采用Western Blot评价对负载了p53质粒的功能化树状大分子复合物介导的蛋白表达效果,最后在小鼠体内评价了其荧光成像功能及联合治疗的抗肿瘤效果,具体测试结果如下:
(1)1H NMR表征
核磁氢谱分析结果如图2所示,2.2-3.4ppm是G5的亚甲基特征峰,6.5-8.5ppm是HQC的特征峰,并通过积分计算得到每个树状大分子上连接了14.6个HQC。
(2)UV-vis测试结果
UV-vis测试结果如图3所示,G5.NH2-HQC在251nm左右有吸收峰,说明HQC成功连接在G5上;附图3中B中,Au DENP-HQC在520nm左右有紫外吸收,相比之下,G5.NH2-HQC则无此特征吸收峰,说明成功合成了有等离子体共振吸收峰的纳米金颗粒;Fe-Au DENP-HQC、HQC-Fe均在260nm左右有吸收峰,表明三价铁离子通过与HQC螯合连接在Au DENP-HQC上,证明功能化树状大分子的成功合成。
(3)ICP-OES测试结果
称取0.5mg的Fe-Au DENP-HQC粉末加入1mL王水(浓硝酸:浓盐酸体积比=1:3配置)消化3小时后,加入3mL超纯水稀释,进行原子发射光谱ICP-OES测试,结果如表1所示,每摩尔G5树状大分子约包裹了25.0mol的Au元素并螯合了20.0mol的Fe元素。
(4)透射电镜(TEM)测试
TEM测试结果如图4所示,分析结果可知,Fe-Au DENP-HQC的尺寸均一、分散性良好,内部包裹的金纳米颗粒平均粒径为1.9nm。
(5)铁离子释放测试
取本发明制备的Fe-Au DENP-HQC,将其配置成浓度为2mg/mL的溶液,然后取0.5mL置于截留分子量为8000-14000的透析袋中,将透析袋放在含有9mL PBS溶液(pH=5.5、6.5、7)的管子中,并将管子置于37℃的摇床中,分别在第1、2、4、8、12、24小时测外部缓冲液中铁离子的含量。实验结果如图5所示,在pH=7.4时,Fe-Au DENP-HQC较稳定,几乎没有铁离子释放,随pH减小,铁离子释放量逐渐增加,当pH=5.5时,24小时内铁离子释放量为21.86%,是pH=7.4的PBS溶液中铁离子释放量的16倍。实验结果说明,Fe-Au DENP-HQC具有pH响应性的铁离子释放能力,有助于在弱酸性环境中缓慢释放铁离子用于铁死亡治疗。
(6)材料稳定性测试
取实施例1中的Fe-Au DENP-HQC分别溶于超纯水、PBS和培养基中配置成0.3mg/mL的溶液,通过纳米激光粒度仪测得不同溶液的水合粒径(附图6中A),显示材料在不同溶剂中具有良好的分散性和溶解性。常温下放置7天,随着时间的推移,Fe-Au DENP-HQC在不同溶液中没有沉淀产生,且7天内不同时间点的水动力学直径没有发生明显的变化(附图6中B),证明了合成的材料具有良好的胶体稳定性。
(7)亚甲基蓝(MB)褪色测试
以氧化环境下MB降解为基础,采用经典比色法检测羟基自由基(·OH)的生成。将Fe-Au DENP-HQC溶解在pH=6.5的磷酸盐缓冲液中,再加入到含有H2O2(10mM)和MB(10μg/mL)的水溶液中。在25℃下记录2小时内不同时间间隔点该溶液在664nm处的吸光度,记录MB的降解情况。实验结果如图7所示,在反应120分钟后,MB降解了36.1%,表明Fe-Au DENP-HQC具有较好的·OH产生能力。
(8)凝胶阻滞实验
取本发明制备的Fe-Au DENP-HQC,将其配置成浓度为2mg/mL的溶液,采用定氮试剂盒测定Fe-Au DENP-HQC的氨基的个数,每个G5树状大分子表面的伯胺基个数约为34.2。然后将Fe-Au DENP-HQC与p53 pDNA按照不同的氮磷比(N/P=0.125、0.25、0.5、1、2、4)孵育15-30分钟,孵育完之后进行琼脂糖凝胶电泳(如图8中A所示),实验结果表明,Fe-Au DENP-HQC在N/P为0.5及以上的条件下就可以完全包裹pDNA,说明该材料具有很好的pDNA包裹能力。
将Fe-Au DENP-HQC与p53 pDNA(N/P=0.125、0.25、0.5、1、2、4)复合,室温孵育30分钟,然后加入至1mL的PBS进行Zeta电势和水动力学直径测试。如图8中B和C,测试结果表明Fe-Au DENP-HQC在N/P大于等于0.5的条件下就可以完全压缩p53 pDNA。
(9)Zeta电势及水动力学直径测试结果
将实施例1中Fe-Au DENP-HQC与p53 pDNA按不同N/P比(N/P=2、5、10、15、20)复合,最终体积为100μL,室温孵育30min,然后加入1mL的PBS,测试复合物的电势和粒径。测试结果如图9所示,Fe-Au DENP-HQC/p53 pDNA复合物的粒径大小和电势都在合适的细胞转染范围内(131.9-162.8nm和29.2-39.1mV)。
(10)CCK-8细胞活力测试结果
附图10中A中,与对照组(PBS缓冲液组)相比,在实验浓度范围内([Fe]=0-1000μM),经Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53处理的L929细胞活力没有受到明显影响,细胞活力均保持70%以上。在达到最高浓度1000μM时,Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA处理后的L929的细胞活力分别为70.6%和71.7%,这充分说明实施例1中合成的Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA均具有良好的细胞相容性。
纳米材料对PANC-1癌细胞的毒性测试实验结果如图10中B所示,Fe-Au DENP-HQC/p53pDNA组对PANC-1细胞的致死效果最显著,当铁的浓度高达1000μM时,细胞活力仅为6.3%,在加入铁死亡抑制剂(DFO)后,Fe-Au DENP-HQC组PANC-1细胞的存活率大幅提升,细胞活力为27.6%,这说明其通过铁死亡的方式可引起肿瘤细胞死亡。Fe-Au DENP-HQC组的肿瘤细胞杀伤效果较弱,细胞活力为12.6%,说明p53 pDNA具有增强铁死亡治疗的效果,表明联合治疗对肿瘤细胞的生长具有良好的抑制效果。
(11)EGFP蛋白表达实验
将PANC-1细胞种于12孔板上,于37℃、5%CO2培养箱中培养24小时,更换新鲜培养基,加入实施例1中所得的Fe-Au DENP-HQC与p53 pDNA在不同N/P下(N/P=0、2、5、10、15、20)形成的复合物,转染PANC-1细胞4小时,然后更换新鲜培养基继续培养,24小时后通过荧光显微镜观测绿色荧光蛋白表达情况(如附图所示11中A)。同样的,通过流式细胞仪定量检测EGFP蛋白的表达(如附图11中B所示)。在N/P=15时,EGFP表达量最高,转染效率最好。
(12)活性氧(ROS)检测
收集对数生长期的PANC-1细胞,以1.5×105细胞每孔的密度将其种植于激光共聚焦显微镜皿中,在37℃、5%CO2培养箱中过夜,待细胞贴壁后,然后更换新鲜培养基,以PBS为空白对照组,Fe-Au DENP-HQC、Fe-Au DENP-HQC/p53 pDNA、Fe-Au DENP-HQC+铁死亡抑制剂(DFO)作为实验组,在培养箱中培育24小时,培养结束后用PBS清洗三次。在避光条件下,每孔加2μL ROS探针和2000μL DMEM培养基,在培养箱中孵育20分钟,孵育结束后用PBS清洗三次,接着用2.5%的戊二醛固定15分钟,固定后用DAPI染色10分钟,然后在油镜下观察细胞(如附图所示12中A)。同样通过流式细胞仪定量检测细胞内活性氧水平(如附图所示12中B)。检测结果显示,Fe-Au DENP-HQC/p53 pDNA复合物组细胞的绿色荧光信号高于Fe-AuDENP-HQC,Fe-Au DENP-HQC+DFO和PBS组荧光值相差不大,说明基因治疗对铁死亡治疗的协同促进作用,同时也说明Fe-Au DENP-HQC/p53 pDNA复合物将铁死亡治疗和基因治疗整合在一个纳米平台上,能够实现增强的联合治疗。
(13)脂质过氧化物(LPO)检测
以1.5×105细胞每孔的密度种植PANC-1细胞于激光共聚焦显微镜皿中,在37℃、5%CO2培养箱中过夜,待细胞贴壁后,然后更换新鲜培养基,以PBS为空白对照组,Fe-AuDENP-HQC、Fe-Au DENP-HQC/p53 pDNA、Fe-Au DENP-HQC+DFO作为实验组,在培养箱中培育24小时,培养结束后用PBS清洗三次。在避光条件下,每孔加1μL LPO探针和500μL DMEM培养基,孵育20分钟,孵育结束后用PBS清洗三次,然后用2.5%的戊二醛固定15分钟,固定后用DAPI染色10分钟,然后在油镜下观察细胞(如图13所示)。实验结果表明,相比于PBS组,Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA复合物处理的细胞均显示了较强的绿色荧光信号。此外,Fe-Au DENP-HQC/p53 pDNA组细胞的绿色荧光信号要明显高于Fe-Au DENP-HQC组,说明本发明制备的Fe-Au DENP-HQC能很好地转染p53 pDNA。实验结果表明Fe-Au DENP-HQC能够使得癌细胞内产生一定量的ROS,发挥铁死亡治疗效果。另一方面,p53 pDNA的存在能够通过基因治疗增强胞内ROS的产生,从而进一步提高细胞内的LPO水平。
(14)谷胱甘肽(GSH)检测
收集对数生长期的PANC-1细胞,以1.5×105细胞每孔的密度将其种植于6孔板中,在37℃、5%CO2培养箱中过夜,待细胞贴壁后,更换新鲜培养基,以PBS为空白对照组,不同铁浓度的Fe-Au DENP-HQC溶液作为实验组,在培养箱中培育24小时,培养结束后用PBS清洗三次,用胰蛋白酶消化细胞,1000rpm、5min离心后,用500μL PBS溶液重悬,再次离心收集细胞沉淀,使用GSH和GSSG检测试剂盒(购自碧云天生物技术公司)检测细胞内GSH水平。实验结果如图14所示,相比于PBS组,Fe-Au DENP-HQC处理的细胞GSH水平均发生了不同程度的降低,并且随着Fe浓度的增加,细胞内GSH水平越低。这是由于Fe浓度的大小影响细胞内ROS的产生水平,从而影响细胞内GSH的消耗,进一步影响肿瘤细胞的铁死亡效果。
(15)体外Western Blot实验结果
如附图15所示。以PBS为空白对照组,β-actin作为内参蛋白。实验结果表明,在实验组和对照组中内参蛋白量表达都很正常,相比于PBS组,Fe-Au DENP-HQC、Fe-Au DENP-HQC/EGFP pDNA、Fe-Au DENP-HQC/p53 pDNA处理的实验组p53、PTEN蛋白有明显的上调,SLC7A11、GPX-4蛋白表达量明显降低。相比于Fe-Au DENP-HQC和Fe-Au DENP-HQC/EGFPpDNA组,Fe-Au DENP-HQC/p53 pDNA组中p53和PTEN蛋白上调、SLC7A11和GPX-4蛋白降低的更为明显。Fe-Au DENP-HQC和Fe-Au DENP-HQC/EGFP pDNA的蛋白表达量相差不大。这一结果也证明了本发明合成的载体可以有效地携带pDNA进入细胞并且能够显著促进p53质粒的转染及表达,从而达到基因治疗的目的。
(16)体内抗肿瘤效果评价
将取3-4周龄的雌性裸鼠,每只皮下种植5×106PANC-1细胞,构建肿瘤模型,肿瘤体积达到50mm3左右。取实施例1中制备的Fe-Au DENP-HQC用无菌PBS缓冲液配置,同时将实验荷瘤裸鼠随机分为四组(对照组、材料组、基因组、基因对照组),随后通过瘤内注射的方式向每只荷瘤裸鼠瘤内注射100μL溶液,其中pDNA的用量为10μg/只/次:第一组瘤内注射PBS(对照组),第二组瘤内注射Fe-Au DENP-HQC/EGFP pDNA(基因对照组),第三组瘤内注射Fe-Au DENP-HQC(材料组),第四组瘤内注射Fe-Au DENP-HQC/p53 pDNA(基因组)。之后,间隔3天给药一次,总共给药三次,记录21天内小鼠体重(A)和小鼠肿瘤体积(B)及解剖后的肿瘤图片(C)。实验结果如图16所示,与对照组相比,其余三组裸鼠的肿瘤体积均得到了有效抑制,基因组的肿瘤抑制效果最好,材料组和基因对照组的肿瘤抑制效果相差不大且肿瘤抑制率均小于基因组,实验结果证明本发明中合成的Fe-Au DENP-HQC/p53 pDNA复合物能应用于动物体内肿瘤的铁死亡/基因联合治疗。
(17)荧光成像效果评价
构建裸鼠PANC-1肿瘤模型,向荷瘤裸鼠瘤内注射裸p53 pDNA和Fe-Au DENP-HQC/p53pDNA的PBS溶液(100μL,其中pDNA的用量为20μg/只),72h后评价肿瘤部位的荧光成像效果。如图17所示,在注射前(附图17中A和B),小鼠体内未产生荧光信号,在注射72h后(附图17中C和D),小鼠的主要器官出现了不同强度的荧光信号。此外,注射Fe-Au DENP-HQC/p53pDNA的实验组肿瘤荧光信号明显强于注射裸pDNA的实验组,说明本发明制备的Fe-AuDENP-HQC能很好地转染p53 pDNA,并用于小鼠肿瘤的荧光成像。
有益效果
(1)本发明操作工艺简单,反应条件温和,易于纯化,所用的合成原料均为环境友好型材料,具有产业化的实施前景;
(2)制备得到的三价铁络合的树状大分子/pDNA复合物中的铁离子可以和肿瘤细胞内的H2O2发生芬顿反应,引起ROS的含量升高,诱发铁死亡;包裹的金纳米粒子能够提高基因转染效率。另一方面,该纳米材料能够较好的转染p53 pDNA进行荧光成像,从而实现肿瘤诊疗一体化应用研究;
(3)制备得到的三价铁络合的树状大分子/pDNA复合物具有良好的水溶性、生物相容性。实验结果表明显示该纳米材料不仅具有良好的荧光成像效果,同时可以把铁死亡治疗和基因治疗两种治疗模式集中在一个纳米平台上,增强了对癌细胞和肿瘤的治疗效果,为联合治疗提供一种新方法,该复合物在肿瘤荧光成像和肿瘤治疗领域具有潜在的应用价值。
附图说明
图1为本发明三价铁络合的树状大分子/pDNA复合物的合成流程及应用示意图。
图2为本发明制备的G5.NH2-HQC的核磁共振氢谱(1H NMR)图。
图3为本发明制备的G5.NH2-HQC、Au DENP-HQC、Fe-Au DENP-HQC、HQC-Fe的UV-vis图(A)以及Au DENP-HQC和G5.NH2-HQC放大的图谱(B)。
图4为本发明制备的Fe-Au DENP-HQC高分辨透射电镜图片(A)以及对应的粒径分布图(B)。
图5为本发明制备的Fe-Au DENP-HQC在PBS溶液中的铁离子释放曲线图。
图6为本发明制备的Fe-Au DENP-HQC溶解在不同介质中通过纳米激光粒度仪测得的水合粒径图(A)、Fe-Au DENP-HQC溶解在水中7天内的水合粒径变化曲线(B);其中插图为颗粒在不同介质和不同时间点时的照片。
图7为本发明制备的Fe-Au DENP-HQC产生·OH对MB降解随时间变化的曲线。
图8中(A)为本发明制备的Fe-Au DENP-HQC复合p53 pDNA在不同N/P下的凝胶阻滞电泳图谱,(B)和(C)分别为通过纳米激光粒度仪测得的复合物在不同N/P条件下的水合粒径图与表面电势图。
图9为本发明制备的Fe-Au DENP-HQC与p53 pDNA在不同N/P下(N/P=2、5、10、15、20)的复合物通过纳米激光粒度仪测得的水合粒径图(A)与表面电势图(B)。
图10为本发明Fe-Au DENP-HQC、Fe-Au DENP-HQC/p53 pDNA、Fe-Au DENP-HQC+DFO([Fe]=0-1000μM)与L929细胞(A)、PANC-1细胞(B)孵育24h后通过CCK-8法测得的细胞活力。
图11为本发明制备的Fe-Au DENP-HQC/p53 pDNA复合物在不同N/P比例(N/P=2、5、10、15、20)下处理PANC-1细胞后EGFP绿色荧光表达的荧光显微镜照片(A)及定量结果(B)。
图12为ROS探针染色PANC-1细胞的激光共聚焦成像图(A)及定量结果(B)。
图13为LPO探针染色PANC-1细胞后的激光共聚焦显微镜图片。
图14为本发明制备的Fe-Au DENP-HQC处理PANC-1细胞后细胞内GSH水平检测图。
图15为PANC-1细胞分别经过PBS、Fe-Au DENP-HQC、Fe-Au DENP-HQC/EGFP pDNA和Fe-Au DENP-HQC/p53 pDNA复合物转染癌细胞后,p53、GPX-4、PTEN、SLC7A11蛋白表达的Western Blot结果分析图:A为电泳条带图,B为定量分析结果图,其中A中:1.PBS,2.Fe-AuDENP-HQC/EGFP pDNA,3.Fe-Au DENP-HQC,4.Fe-Au DENP-HQC/p53 pDNA。
图16为实施例1中PBS、Fe-Au DENP-HQC、Fe-Au DENP-HQC/p53 pDNA、Fe-Au DENP-HQC/EGFP pDNA材料通过瘤内注射至小鼠肿瘤部位,记录21天内小鼠体重(A)和小鼠肿瘤体积(B)及解剖后的肿瘤图片(C),其中C中:(1)PBS,(2)Fe-Au DENP-HQC/EGFP pDNA,(3)Fe-Au DENP-HQC,(4)Fe-Au DENP-HQC/p53 pDNA。
图17为裸p53 pDNA和Fe-Au DENP-HQC/p53 pDNA的PBS溶液注射荷瘤鼠前(A,B)及注射72h后(C,D)的荧光成像图,其中A,C对应裸p53 pDNA,B、D对应Fe-Au DENP-HQC/p53pDNA。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
除非特殊说明,否则所有化学试剂都是可商购的,无需进一步纯化即可直接使用。树状大分子G5购自美国Dendritech公司。氯化铁(III)购自Adamas试剂有限公司(中国上海)。8-羟基喹啉-2-羧酸购自上海易势化工有限公司(中国上海)。HAuCl4·4H2O购自国药控股化学试剂有限公司(中国上海)。NaBH4,EDC和NHS购自百灵威科技有限公司(中国上海)。DFO购自Sigma-Aldrich。p53 pDNA购自和元生物技术(上海)股份有限公司(中国上海)。DMEM培养基、胎牛血清(FBS,GIBCO)、青霉素-链霉素(HyClone,Thermo Scientific,Logan,UT)和胰蛋白酶0.25%溶液(HyClone)购自杭州吉诺生物医学技术有限公司(中国杭州)。C11 BODIPY 581/591购自上海宏叶生物科技有限公司(中国上海)。Cell Counting Kit-8(CCK-8)和Annexin V-FITC/PI细胞凋亡检测试剂盒购自7Sea Biotech Co.,Ltd.(中国上海)。GSH与GSSH检测试剂盒,ROS检测试剂盒和GPX-4抗体购自上海碧云天生物公司(中国上海)。p53,PTEN和SLC7A11抗体购自上海拓然生物科技有限公司(中国上海)。PANC-1细胞(人类胰腺癌细胞系)由复旦大学附属肿瘤医院赠送,购买自American Type CultureCollection(ATCC)。4-5周龄的裸鼠购自上海斯莱克实验动物中心(中国上海)。所有实验中使用的电阻率高于18.2MΩ.cm的水均通过实验室水净化系统(Cascada I,PALL,北京,中国)进行净化。截留分子量(MWCO)为8000-14000的再生纤维素透析膜购自Fisher(宾夕法尼亚州匹兹堡)。
实施例1
(1)称取20mg的第五代PAMAM树状大分子(G5.NH2)(Mw=26010)和4.35mg的HQC(Mw=189.17),22.05mg的EDC和13.35mg的NHS。首先将HQC和EDC分别溶于5mL DMSO和2mL超纯水中,使其充分溶解,将EDC溶液加入HQC溶液中搅拌半个小时,然后将称好的NHS溶于5mL超纯水中,待其充分溶解后,加入上述反应中,搅拌三个小时后,加入溶解于5mL超纯水中的第五代PAMAM树状大分子(G5.NH2),室温搅拌一天,用截留分子量为8000~14000的透析袋透析三天,冷冻干燥,得到G5.NH2-HQC粉末。
(2)分别称取10mg的G5.NH2-HQC(Mw=28656)、6.6mg的NaBH4,先将G5.NH2-HQC溶于5mL超纯水中,加入119.75μL的HAuCl4·4H2O水溶液(30mg/mL),冰水浴中搅拌半个小时,然后迅速加入NaBH4溶液(2mg/mL),反应3h后,用截留分子量为8000~14000的透析袋透析三天,冷冻干燥,得到干燥的Au DENP-HQC。
(3)称取10mg Au DENP-HQC(Mw=33581)溶于5mL超纯水中,待其充分溶解后加入溶于5mL超纯水中的FeCl3(1.35mg)室温搅拌反应三小时,用截留分子量为8000~14000的透析袋透析三天,冷冻干燥,得到Fe-Au DENP-HQC。
(4)称取Fe-Au DENP-HQC,配置成2mg/mL的超纯水溶液,在不同的N/P比条件下(N/P=0.125、0.25、0.5、1、2、4),分别与1μg p53 pDNA(具有EGFP基因,能够表达绿色荧光蛋白)通过静电作用形成Fe-Au DENP-HQC/p53 pDNA复合物。
称取3mg HQC(Mw=189.17)溶于5mL DMSO中,待其充分溶解后加入溶于5mL超纯水中的FeCl3(2.57mg)室温搅拌反应三小时,用截留分子量为8000~14000的透析袋透析三天,冷冻干燥,得到HQC-Fe。
实施例2
对实施例1步骤(3)中制备的Fe-Au DENP-HQC及制备过程中的相关中间产物进行表征。
(1)称取5mg实施例1步骤(1)中的G5.NH2-HQC,将其溶于500μL D2O中,进行核磁氢谱分析(如图2所示)。如图2所示,其中2.2~3.4ppm为第五代树状大分子的亚甲基特征峰,6.5-8.5ppm是HQC的特征峰,通过积分计算得到每个树状大分子上连接了14.6个HQC。
(2)取实施例1制备得到的G5.NH2-HQC、Au DENP-HQC、Fe-Au DENP-HQC、HQC-Fe配置成0.3mg/mL的水溶液,测量紫外吸收,结果如图3所示。紫外结果表明,G5.NH2-HQC纳米颗粒在251nm左右有吸收峰,此结果说明HQC成功连接在G5上;附图3中B中,Au DENP-HQC在520nm左右处有紫外吸收,相比之下,G5.NH2-HQC则无此特征吸收峰,说明成功合成了有独特等离子体共振吸收峰的纳米金颗粒;Fe-Au DENP-HQC、HQC-Fe均在260nm左右有吸收峰,说明Fe通过与HQC螯合连接在Au DENP-HQC上。
(3)取0.2mg/mL的Fe-Au DENP-HQC水溶液进行水动力学直径和Zeta电势测试,如表1所示,Fe-Au DENP-HQC水动力学直径在158.2nm,Zeta电势为31.8mV,单分散性系数较小为0.308。
(4)称取0.5mg Fe-Au DENP-HQC加入1mL王水(浓硝酸:浓盐酸体积比=1:3配置)消化3小时后,加入3mL超纯水稀释,进行原子发射光谱ICP-OES测试,如表1所示,每摩尔Fe-Au DENP-HQC约含25.0摩尔Au元素和20.0摩尔Fe元素。
(5)为了对制备得到的纳米颗粒的形貌和尺寸进行表征,取实施例1制备的Fe-AuDENP-HQC 1mg溶解在1mL超纯水中,并取5μL滴在铜网表面,进行TEM测试(如图4所示)。TEM结果显示Fe-Au DENP-HQC尺寸均一、分散性良好,金核平均粒径约为1.9nm。
表1
实施例3
铁离子在肿瘤微环境中的释放影响纳米材料的治疗效果。取本发明制备的Fe-AuDENP-HQC,将其配置成浓度为2mg/mL的溶液,然后取0.5mL置于截留分子量为8000-14000的透析袋中,将透析袋放在含有9mL水的离心管中,并将离心管置于37℃的摇床中,分别在第1、2、4、8、12、24小时测外部缓冲液中铁离子的含量。实验结果如图5所示,在pH=7.4时,Fe-Au DENP-HQC较稳定,几乎没有铁离子释放,随pH减小,铁离子释放量逐渐增加,当pH=5.5时,铁离子24小时释放量为21.86%,是生理pH溶液中铁离子释放量的16倍。实验结果说明,Fe-Au DENP-HQC具有pH响应性的铁离子释放能力,能够在弱酸性的肿瘤环境中缓慢释放铁离子用于癌细胞铁死亡治疗。
实施例4
将实施例1步骤(3)制备的Fe-Au DENP-HQC分别溶解于超纯水、PBS和DMEM培养基中配置成0.3mg/mL的溶液,通过纳米激光粒度仪测得不同溶液的水合粒径(图6中A),拍照如图6中A内嵌图片,显示了材料在不同溶剂中具有良好的分散性。常温下放置7天,随着时间的推移,Fe-Au DENP-HQC在不同溶液中没有沉淀产生,且7天内不同时间点的水动力学直径没有发生明显的变化(附图6中B),证明了合成的材料具有良好的胶体稳定性。
实施例5
以氧化环境下MB降解为基础,采用经典比色法检测·OH的生成。将Fe-Au DENP-HQC溶解在pH=6.5的磷酸盐缓冲溶液中,再加入到含有H2O2(10mM)和MB(10μg/mL)的水溶液中。在25℃下记录2小时内不同时间间隔点该溶液在664nm处的吸光度,记录MB的降解情况。实验结果如图7所示,在反应120分钟后,MB降解36.1%,表明Fe-Au DENP-HQC具有较好的·OH产生能力。
实施例6
凝胶阻滞实验被用来表征载体对pDNA包裹的能力,取实施例1制备的Fe-Au DENP-HQC,将其配置成浓度为2mg/mL的溶液,采用定氮试剂盒测定Fe-Au DENP-HQC的氨基的个数,每个G5树状大分子表面的伯胺基个数为34.2个。然后将Fe-Au DENP-HQC与p53 pDNA按照不同的氮磷比(N/P=0.125、0.25、0.5、1、2、4)孵育15-30分钟,孵育完之后进行琼脂糖凝胶电泳(如图8中A所示,其中条带1:DNA marker;条带2:裸p53 pDNA;条带3:N/P=0.125:1;条带4:N/P=0.25:1;条带5:N/P=0.5:1;条带6:N/P=1:1;条带7:N/P=2:1;条带8:N/P=4:1),实验结果表明Fe-Au DENP-HQC在N/P为0.5及以上时就可以完全包裹pDNA,说明该材料具有很好的pDNA包裹能力。
将Fe-Au DENP-HQC与p53 pDNA(N/P=0.125、0.25、0.5、1、2、4)复合,室温孵育30分钟,然后加入至1mL的PBS溶液进行Zeta电势和水动力学直径测试。如图8中B和C,测试结果同样表明Fe-Au DENP-HQC在N/P大于等于0.5的条件下就可以完全压缩p53pDNA。
实施例7
通过表面电势和粒径来表征Fe-Au DENP-HQC/p53 pDNA复合物。将实施例1中Fe-Au DENP-HQC与p53 pDNA按不同N/P比(N/P=2、5、10、15、20)复合,最终体积为100μL,室温孵育30min,然后加入1mL的PBS。采用纳米粒度仪对其表面电势和粒径进行了表征,测试结果如图9所示,Fe-Au DENP-HQC/p53 pDNA复合物的粒径大小和电势都在合适的细胞转染范围内(131.9-162.8nm和29.2-39.1mV)。
实施例8
通过CCK-8比色法来评价Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA对细胞增殖的影响,以L929细胞和PANC-1细胞为细胞模型评价实施例1制备的Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA材料对细胞增殖的影响。用无菌PBS配置不同浓度的Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA的PBS溶液。PANC-1细胞种植于96孔板分别与Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA(Fe浓度为0、50、100、200、400、600、800、1000μM)在37℃下共培养24h,然后换为含10μL CCK-8溶液的培养液100μL,继续在37℃下培养3小时,然后在450nm处测其吸光值,并根据此值计算细胞的活力(如图10)。附图10中A,与对照组(PBS缓冲液组)相比,在实验浓度范围内,经Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53处理的L929细胞活力没有受到明显影响,细胞活力均保持70%以上,在达到最高浓度1000μM时,Fe-AuDENP-HQC和Fe-Au DENP-HQC/p53 pDNA处理后的L929的细胞活力分别为70.6%和71.7%,这充分说明实施例1中合成的Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA具有良好的细胞相容性。
纳米材料对PANC-1细胞的毒性测试实验(图10中B),Fe-Au DENP-HQC/p53 pDNA组对PANC-1细胞的致死效果最显著,当铁的浓度高达1000μM时,细胞活力仅为6.3%,在加入DFO后,Fe-Au DENP-HQC组PANC-1细胞的存活率大幅提升,细胞活力为27.6%,说明其通过铁死亡的方式引起肿瘤细胞死亡。Fe-Au DENP-HQC组的肿瘤细胞杀伤效果较弱,细胞活力为12.6%,说明p53 pDNA具有增强铁死亡治疗的效果,表明联合治疗对肿瘤细胞的生长具有良好的抑制效果。
实施例9
将PANC-1细胞种于12孔板上,于37℃、5%CO2培养箱中培养24小时,更换新鲜培养基,加入实施例1中所得的Fe-Au DENP-HQC与p53 pDNA在不同N/P下(N/P=0、2、5、10、15、20)复合物,混合均匀,转染PANC-1细胞,在培养基箱中培育4小时,然后更换新鲜培养基继续培养,24小时后通过荧光显微镜观测绿色荧光蛋白(EGFP)表达情况(如附图所示11中A)。同样的,通过流式细胞仪定量检测EGFP蛋白的表达(如附图所示11中B)。如图11所示,在N/P=15时,EGFP表达量最高,转染效率最好。
实施例10
为了进一步验证本发明合成的Fe-Au DENP-HQC/p53 pDNA纳米复合物具有铁死亡治疗和基因治疗的联合治疗效果,将Fe-Au DENP-HQC、Fe-Au DENP-HQC/p53 pDNA、Fe-AuDENP-HQC+DFO三组材料与细胞共孵育后,采用共聚焦显微镜来进一步观察细胞内ROS水平。收集对数生长期的PANC-1细胞,以1.5×105细胞每孔的密度将其种植于激光共聚焦显微镜皿中,在37℃、5%CO2培养箱中过夜,待细胞贴壁后,然后更换新鲜培养基,以PBS为空白对照组,Fe-Au DENP-HQC、Fe-Au DENP-HQC/p53 pDNA、Fe-Au DENP-HQC+DFO作为实验组,在培养箱中培育24小时,培养结束后用PBS清洗三次。在避光条件下,每孔加2μL ROS探针和2000μL DMEM培养基,在培养箱中孵育20分钟,孵育结束后用PBS清洗三次,然后用2.5%的戊二醛固定15分钟,固定后用DAPI染色10分钟,然后在油镜下观察细胞的绿色荧光信号(如附图所示12)。检测结果显示,在测定实验条件下,Fe-Au DENP-HQC/p53 pDNA复合物的绿色荧光信号高于Fe-Au DENP-HQC,Fe-Au DENP-HQC+DFO和PBS组几乎没有荧光信号,这说明基因治疗对铁死亡治疗的促进作用,同时也说明Fe-Au DENP-HQC/p53 pDNA复合物能够将铁死亡治疗和基因治疗整合在一个纳米平台上,实现联合治疗。
实施例11
采用激光共聚焦显微镜进一步观察细胞内LPO表达水平。以1.5×105细胞每孔的密度PANC-1细胞种植于激光共聚焦显微镜皿中,在37℃、5%CO2培养箱中过夜,待细胞贴壁后,然后更换新鲜培养基,以PBS为空白对照组,Fe-Au DENP-HQC、Fe-Au DENP-HQC/p53pDNA、Fe-Au DENP-HQC+DFO作为实验组,在培养箱中培育24小时,培养结束后用PBS清洗三次。在避光条件下,每孔加1μL LPO探针和500μL DMEM培养基,在培养箱中孵育20分钟,孵育结束后用PBS清洗三次,然后用2.5%的戊二醛固定15分钟,固定后用DAPI染色10分钟,然后在油镜下观察细胞的荧光信号(如图13所示)。实验结果表明,相比于PBS组,Fe-Au DENP-HQC和Fe-Au DENP-HQC/p53 pDNA复合物处理的细胞均显示了较高的绿色荧光信号,此外,Fe-Au DENP-HQC/p53 pDNA的荧光强度要高于Fe-Au DENP-HQC,说明本发明制备的Fe-AuDENP-HQC能很好的转染p53 pDNA。实验结果表明Fe-Au DENP-HQC能够使得癌细胞内产生一定量的ROS,发挥铁死亡治疗效果。另一方面,p53pDNA的存在能够通过基因治疗增强胞内ROS的产生,从而进一步提高细胞内的LPO水平。本实验也证明了基因治疗与铁死亡治疗的协同促进作用。
实施例12
收集对数生长期的PANC-1细胞,以1.5×105细胞每孔的密度将其种植于6孔板中,在37℃、5%CO2培养箱中过夜,待细胞贴壁后,然后更换新鲜培养基,以PBS为空白对照组,不同Fe浓度的Fe-Au DENP-HQC溶液作为实验组,在培养基箱中培育24小时,培养结束后用PBS清洗三次,用胰蛋白酶消化细胞,1000rpm、5min离心后,用500μL PBS溶液重悬,再次离心收集细胞沉淀,使用GSH和GSSG检测试剂盒(购自碧云天生物技术公司)检测细胞内GSH水平。实验结果如图14所示,在测定实验条件下,相比于PBS组,Fe-Au DENP-HQC处理的细胞GSH水平均发生了不同程度的降低,并且随着Fe浓度的增加,细胞内GSH水平越低。由于Fe浓度的大小影响细胞内ROS的产生水平,从而影响细胞内GSH的消耗,进一步影响肿瘤细胞的铁死亡效果。
实施例13
采用Western Blot来评价PANC-1细胞内铁死亡相关蛋白表达情况。相关研究表明,p53基因是铁死亡调节机制中的一个关键基因,其表达升高会引起p53、PTEN蛋白表达量增加,SLC7A11、GPX-4蛋白表达量降低,从而促进肿瘤细胞发生铁死亡。如附图15所示。以PBS为空白对照组,β-actin作为内参蛋白,在实验组和对照组中内参蛋白量表达都很正常,相比于PBS组,Fe-Au DENP-HQC、Fe-Au DENP-HQC/EGFP pDNA、Fe-Au DENP-HQC/p53 pDNA处理的实验组p53、PTEN蛋白有明显的上调,SLC7A11、GPX-4蛋白表达量明显降低。相比于Fe-Au DENP-HQC和Fe-Au DENP-HQC/EGFP pDNA组,Fe-Au DENP-HQC/p53 pDNA组中p53和PTEN蛋白上调、SLC7A11和GPX-4蛋白降低的更为明显,Fe-Au DENP-HQC和Fe-Au DENP-HQC/EGFPpDNA的蛋白表达量相差不大。这一结果也证明了本发明合成的纳米材料可以有效地携带pDNA进入细胞并且能够显著促进p53质粒的转染及表达,从而达到基因治疗的目的。这也充分证明了基因治疗对铁死亡治疗的促进作用。
实施例14
将取3-4周龄的雌性裸鼠,每只皮下种植5×106PANC-1细胞,构建肿瘤模型,肿瘤体积达到50mm3左右。取实施例1中制备的Fe-Au DENP-HQC用无菌PBS缓冲液配置,同时将实验荷瘤裸鼠随机分为四组(对照组、材料组、基因组、基因对照组),随后通过瘤内注射的方式向每只荷瘤裸鼠瘤内注射100μL溶液,其中pDNA的用量为10μg/只/次:第一组瘤内注射PBS(对照组),第二组瘤内注射Fe-Au DENP-HQC/EGFP pDNA(基因对照组),第三组瘤内注射Fe-Au DENP-HQC(材料组),第四组瘤内注射Fe-Au DENP-HQC/p53 pDNA(基因组)。之后,间隔3天给药一次,总共给药三次,记录21天内小鼠体重(A)和小鼠肿瘤体积(B)及解剖后的肿瘤图片(C)。实验结果如图16所示,与对照组相比,其余三组裸鼠的肿瘤体积均得到了有效抑制,基因组的肿瘤抑制效果最好,材料组和基因对照组的肿瘤抑制效果相差不大且肿瘤抑制率均小于基因组,实验结果证明本发明中合成的Fe-Au DENP-HQC/p53 pDNA复合物能应用于动物体内肿瘤的铁死亡/基因联合治疗。
实施例15
构建裸鼠PANC-1肿瘤模型,向荷瘤裸鼠瘤内注射裸p53 pDNA或Fe-Au DENP-HQC/p53pDNA的PBS溶液(100μL,其中pDNA的用量为20μg/只),72h后评价肿瘤部位的荧光成像效果。如图17所示,在注射前(附图17中A和B),小鼠体内未产生荧光信号,在注射72h后(附图17中C和D),小鼠的主要器官出现了不同强度的荧光信号。此外,注射Fe-Au DENP-HQC/p53pDNA的实验组肿瘤荧光信号明显强于注射裸pDNA的实验组,说明本发明制备的Fe-AuDENP-HQC能很好的转染p53 pDNA,Fe-Au DENP-HQC/p53 pDNA复合物可用于体内肿瘤的荧光成像诊断。
Claims (10)
1.一种三价铁络合的树状大分子/pDNA复合物,其特征在于,所述复合物为:表面修饰8-羟基喹啉-2-羧酸络合三价铁离子、内部包裹金纳米颗粒的第五代聚酰胺胺PAMAM树状大分子负载p53 pDNA,所述p53 pDNA为具有人体抑癌基因p53和绿色荧光蛋白EGFP基因的质粒。
2.一种三价铁络合的树状大分子/pDNA复合物的制备方法,包括:
(1)将8-羟基喹啉-2-羧酸HQC溶解于溶剂中,经EDC和NHS活化,加入到G5.NH2溶液中,搅拌反应,透析,冷冻干燥,得到G5.NH2-HQC;
(2)将步骤(1)中G5.NH2-HQC溶于超纯水中,加入HAuCl4·4H2O水溶液,搅拌,加入含NaBH4冰水溶液反应,透析,冷冻干燥,得到{(Au0)25-G5.NH2-HQC},即Au DENP-HQC;
(3)将步骤(2)中Au DENP-HQC溶于超纯水中,逐滴加入三价铁盐的水溶液搅拌,透析,冷冻干燥,得到Fe-Au DENP-HQC;
(4)将步骤(3)中Fe-Au DENP-HQC与p53 pDNA共同孵育,得到Fe-Au DENP-HQC/p53pDNA复合物,即三价铁络合的树状大分子/pDNA复合物,所述p53 pDNA为具有人体抑癌基因p53和绿色荧光蛋白EGFP基因的质粒。
3. 根据权利要求2所述的制备方法,其特征在于,所述步骤(1)中溶剂为DMSO;G5.NH2溶液的溶剂为超纯水;HQC、EDC和NHS的摩尔比为1 : 8~10 : 8~10;G5.NH2与HQC的摩尔比为1 :25~35。
4. 根据权利要求2所述的制备方法,其特征在于,所述步骤(1)中活化时间为2-4 h;搅拌反应温度为室温,搅拌反应时间为12~48h。
5. 根据权利要求2所述的制备方法,其特征在于,所述步骤(2)中G5.NH2-HQC与HAuCl4·4H2O的摩尔比为1 : 20~30HAuCl4·4H2O与NaBH4的摩尔比为1 : 4~1 : 6。
6.根据权利要求2所述的制备方法,其特征在于,所述步骤(2)中搅拌为:冰水浴条件下搅拌15~30min;反应是在冰水浴条件下反应2~3h。
7. 根据权利要求2所述的制备方法,其特征在于,所述步骤(3)中三价铁盐为FeCl3;AuDENP-HQC与三价铁盐的摩尔比为1 : 28~1 : 35。
8.根据权利要求2所述的制备方法,其特征在于,所述步骤(3)中搅拌温度为室温,搅拌时间为2~4h。
9. 根据权利要求2所述的制备方法,其特征在于,所述步骤(4)中Fe-Au DENP-HQC与p53 pDNA的N/P为2 : 1~20 : 1,其中N/P比为树状大分子的伯胺基与质粒p53骨架上磷酸基团的摩尔比;共同孵育时间为15-30分钟。
10.一种如权利要求1所述的复合物在制备肿瘤的荧光成像和铁死亡/基因联合治疗的诊疗剂中的应用。
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