CN110279713A - 一种用于靶向治疗肿瘤的药物联合制剂及其制备方法 - Google Patents
一种用于靶向治疗肿瘤的药物联合制剂及其制备方法 Download PDFInfo
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Abstract
本发明提供一种用于靶向治疗肿瘤的药物联合制剂及其制备方法。由Apt‑CS/rGO/Ag+‑DNA复合纳米材料和Vc构成,其中,Apt‑CS/rGO/Ag+‑DNA复合纳米材料中,Apt‑CS表示核酸适配体‑壳聚糖耦合物;Ag+‑DNA表示Ag+‑DNA耦合物。本发明利用肿瘤细胞内产生ROS较多的特性,使用特异性核酸适配体负载Ag+/DNA/rGO复合纳米材料,靶向运输到肿瘤部位;在肿瘤细胞中,在Vc刺激下,rGO协同H2O2共同催化Ag+还原成Ag纳米颗粒,利用Ag纳米颗粒的生物学毒性,从而实现对肿瘤的治疗作用。
Description
技术领域
本发明属于医药领域,具体涉及一种用于靶向治疗肿瘤的药物联合制剂及其制备方法。
背景技术
如今,肿瘤已经成为了威胁中国居民生命健康最大的因素,无论男性还是女性,恶性肿瘤死亡率均逐渐升高。目前,肿瘤治疗效果差、复发转移率高且肿瘤治疗副作用大、精准性差等原因,导致肿瘤治疗难度大。因此,聚焦我国高发、特色和具有优势研究基础的癌种,并针对其进行相应的靶向治疗是提高患者生存率的一个关键突破点。
银纳米颗粒可以作为抗菌剂和抗癌剂,并被广泛应用于生物医学领域。它可以选择性地破坏胞内线粒体呼吸链,引起活性氧簇ROS的积累和中断ATP的生成,来造成核酸链损伤。
AgNP对肿瘤细胞DNA造成多种影响,从而抑制肿瘤细胞的增殖。AgNPs通过破坏细胞的胞质分裂和细胞核分裂(细胞和细胞核的分裂)有效地抑制细胞生长,导致单细胞中未分裂的巨核和多个细胞核,抑制细胞增殖。
AgNP能够产生活性氧簇(ROS),造成肿瘤细胞的损伤。AgNP具有抗白血病活性,能够抑制急性髓细胞性白血病(AML)细胞生长。AgNP能够有效杀死AML细胞,并且AgNP作用细胞后能够使ROS水平显著提高,诱导氧化应激,线粒体跨膜电位耗散,DNA损伤,最终细胞进入凋亡过程。
AgNP同样对肝细胞造成极大的影响,如AgNP能使小鼠肝细胞还原型谷胱甘肽耗竭,线粒体膜电位下降,ROS水平上升。胶体银处理增加了超氧化物歧化酶活性。这可能导致氧化还原失衡,显着增加SOD活性以响应高水平ROI分子的产生,并且过氧化氢酶和谷胱甘肽过氧化物酶的活性缺乏可能使过氧化氢(H2O2)的毒性作用导致细胞死亡。
肿瘤的生长依赖于新生血管的生成,抑制血管的生成有利于抑制肿瘤的生长。AgNP的抗肿瘤机制也能与抗血管生相关。例如,AgNP有效降低牛视网膜血管内皮细胞(BREC)的细胞活力,并且能够阻滞血管内皮生长因子(VascμLar Endothelial GrowthFactor,VEGF)诱导的BREC细胞增殖,并且在体内外试验中,均能显著的抑制新生血管的形成,阻止P13K/Akt信号通路的激活,引发细胞凋亡。这表明AgNP是一种有效的抗血管生成分子,其抗血管生成特性在癌症治疗中具有潜在的应用价值。
然而如何准确高效地将AgNP靶向引导到肿瘤部位,是目前迫切需要解决的问题。
发明内容
本发明的目的是提供一种用于靶向治疗肿瘤的药物联合制剂及其制备方法。
本发明所提供的用于靶向治疗肿瘤的药物联合制剂,由Apt-CS/rGO/Ag+-DNA复合纳米材料和维生素C(Vc)构成,
其中,所述Apt-CS/rGO/Ag+-DNA复合纳米材料与维生素C单独包装;
其中,Apt-CS/rGO/Ag+-DNA复合纳米材料中,Apt-CS表示对所述肿瘤有特异性的核酸适配体-壳聚糖耦合物;Ag+-DNA表示Ag+-DNA耦合物;rGO表示还原型氧化石墨烯;
所述核酸适配体可针对不同的肿瘤选择对应的核酸适配体;
所述肿瘤为癌,所述癌为人乳腺癌时,所述核酸适配体为核仁素适配体AS1411,具体可为Cy5荧光标记的核仁素适配体AS1411。
所述癌为人肺癌时,所述核酸适配体为Endoglin适配体或核仁素适配体AS1411,具体可为Cy5荧光标记的Endoglin适配体或核仁素适配体AS1411。
所述癌为肝癌时,所述核酸适配体为TLS11a,具体可为Cy5荧光标记的TLS11a适配体。
所述DNA用于负载银离子,并与石墨烯通过π-π共轭形成复合纳米材料;
具体地,所述DNA的序列可为:GCCGCGTGCGGCCGGTGCCGAGAGAGAGAGAGGAGAGAGA。
所述Apt-CS/rGO/Ag+-DNA复合纳米材料通过包括如下步骤的方法制备得到:
1)制备Ag+-DNA耦合物溶液;
2)制备还原型氧化石墨烯溶液;
3)将还原型氧化石墨烯溶液加入到Ag+-DNA偶合物溶液中,涡旋,孵育,离心,收集沉淀得到rGO/Ag+-DNA,将rGO/Ag+-DNA分散到水中得到rGO/Ag+-DNA溶液;
4)将壳聚糖分散到冰醋酸溶液中,加入rGO/Ag+-DNA溶液,搅拌反应,离心,收集沉淀,得到CS/rGO/Ag+-DNA纳米材料,将核酸适配体与CS/rGO/Ag+-DNA纳米材料中的CS偶联,得到Apt-CS/rGO/Ag+-DNA复合纳米材料。
上述方法步骤1)中,制备Ag+-DNA耦合物的操作如下:将硝酸银溶解于水中,将DNA加入到硝酸银溶液中,涡旋,孵育,得到Ag+-DNA溶液;
其中,硝酸银中银离子与DNA的配比可为:0.2mmol:1OD(体积10μL);
所述涡旋的时间可为5-10min;
所述孵育的温度可为22-27℃,时间可为0.5-4h,具体可为1-2h;
上述方法步骤2)中,制备还原型氧化石墨烯溶液的操作包括:将氧化石墨超声剥离得到氧化石墨烯片,采用水合肼还原,得到还原型氧化石墨烯(rGO)溶液。
上述方法步骤3)中,所述还原型氧化石墨烯的溶液中还原型氧化石墨烯与Ag+-DNA偶合物溶液的配比可为:0.05mg:1mL;其中,1mL Ag+-DNA偶合物溶液为通过将0.2mmol的硝酸银溶解于1mL水中,将1OD DNA加入到所述硝酸银溶液孵育后制得的溶液;
所述涡旋的时间可为5-10min;
所述孵育的温度可为22-27℃,时间可为30-40min;
所述离心的条件为:10000res/min离心10min;
上述方法步骤4)中,所述冰醋酸溶液可为(质量浓度)1%的冰醋酸溶液;
所述壳聚糖与rGO/Ag+-DNA溶液的配比可为:1mg:1mL;其中,1mL rGO/Ag+-DNA溶液通过将50μL 1mg/mL还原型氧化石墨烯溶液加入到1mL Ag+-DNA偶合物溶液中孵育,去除上清液,再将得到的GO/Ag+-DNA沉淀分散到1mL水中制得;
所述搅拌反应的时间可为6h;
所述离心的条件可为:12000res/min 15min;
步骤4)中,所述偶联以戊二醛为偶联剂实现;
所述戊二醛以溶液的形式加入,所述戊二醛溶液的质量浓度可为2.5%;
所述核酸适配体的浓度可为1OD;
所述CS/rGO/Ag+-DNA纳米材料中的CS与核酸适配体的配比可为:1mg:100μL。
上述药物联合制剂还可进一步包括一个记录数据的载体。
所述记录数据的载体记载有所述药物联合制剂的给药方法,即,先将Apt-CS/rGO/Ag+-DNA纳米材料给药;2-7min(具体可为5min)后再将Vc给药,即可。
所述Apt-CS/rGO/Ag+-DNA纳米材料可通过局部注射给药;
所述Vc可通过注射或口服给药。
上述药物联合制剂在制备靶向治疗肿瘤的药物中的应用和/或在制备肿瘤核磁成像造影剂中的应用也属于本发明的保护范围。
所述应用中,所述药物联合制剂中Apt-CS/rGO/Ag+-DNA纳米材料中的核酸适配体根据待治疗肿瘤的类型确定;
所述核酸适配体可针对不同的肿瘤选择对应的核酸适配体;
所述肿瘤为癌,所述癌为人乳腺癌时,所述核酸适配体为核仁素适配体AS1411,具体可为Cy5荧光标记的核仁素适配体AS1411。
所述癌为人肺癌时,所述核酸适配体为Endoglin适配体或核仁素适配体AS1411,具体可为Cy5荧光标记的Endoglin适配体或核仁素适配体AS1411。
所述癌为肝癌时,所述核酸适配体为TLS11a,具体可为Cy5荧光标记的TLS11a适配体。
Vc在刺激肿瘤细胞释放过氧化氢,过氧化氢催化Apt-CS/rGO/Ag+-DNA纳米材料中银离子在肿瘤部位的还原沉积中的应用也属于本发明的保护范围。
本发明利用肿瘤细胞内产生ROS较多的特性,使用特异性核酸适配体负载Ag+/DNA/rGO复合纳米材料,靶向运输到肿瘤部位;在肿瘤细胞中,在Vc刺激下,rGO协同H2O2共同催化Ag+还原成Ag纳米颗粒,利用Ag纳米颗粒的生物学毒性,从而实现对肿瘤的治疗作用。
附图说明
图1为利用电化学工作站检测荷瘤鼠肿瘤部位H2O2的实验过程示意图。
图2为利用电化学工作站检测荷瘤鼠肿瘤局部注射Vc刺激产生的过氧化氢,a正常裸鼠局部注射100μL生理盐水;b正常裸鼠局部注射100μL 5mM的Vc;c荷瘤鼠肿瘤局部注射100μL 5mM的Vc。
图3为肿瘤细胞在Vc刺激下释放过氧化氢催化生成银单质,10μL5mMVc刺激HepG2细胞释放H2O2还原单质Ag随Ag+浓度的变化;a:10mMAg+,b:20mMAg+,c:50mMAg+,d:100mMAg+,e:200mMAg+,f:400mMAg+。
图4为本发明实施例制备的Apt-CS/rGO/Ag+-DNA复合纳米材料的红外表征图,其中a表示rGO/Ag+-DNA,b表示CS/rGO/Ag+-DNA,c表示Apt-CS/rGO/Ag+-DNA。
图5为100μl 1mg/mL Apt-CS/rGO/Ag+-DNA复合纳米材料与MCF-7、A549和L02细胞孵育后,PBS洗涤,在10μl 5mM Vc刺激下释放过氧化氢催化生成银单质的共聚焦显微镜图。
图6为100μlPBS、100μl 1mg/mL RS-CS/rGO/Ag+-DNA复合纳米材料、100μl 1mg/mLApt-CS/rGO/Ag+-DNA复合纳米材料不同时间的体内肿瘤靶向荧光活体成像图。
具体实施方式
下面通过具体实施例对本发明进行说明,但本发明并不局限于此。
下述实施例中所使用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试剂、生物材料等,如无特殊说明,均可从商业途径得到。
下述实施例中采用的DNA为上海生工公司的产品,其序列为:GCCGCGTGCGGCCGGTGCCGAGAGAGAGAGAGGAGAGAGA
实施例1
利用电化学工作站检测荷瘤鼠肿瘤部位的H2O2
(1)电极的预处理
金电极表面依次使用直径为0.3μm、0.05μm的Al2O3匀浆在麂皮上将其表面进行打磨,除去表面及周围的污物,再依次用超纯水、乙醇、超纯水、乙醇进行超声处理5min,吹干电极,将电极置0.5M H2SO4中进行循环伏安扫描,然用纯水冲洗干净,最后将电极置于Fe(CN)6 3-/4-溶液中分别进行循环伏安扫描和交流阻抗扫描,用纯水冲洗晾干备用。
(2)电极修饰
在处理好的电极表面滴加20μL浓度分别为1mg/mL的壳聚糖/石墨烯/血红素复合材料,37℃放置1h,PBS洗涤三次,在-0.2V下电沉积铂纳米颗粒90s,PBS洗涤,如此循环3次。
(3)检测荷瘤鼠肿瘤部位H2O2
荷瘤小鼠肿瘤部位去皮(大小约为1mm2),将工作电极、参比电极、对电极分别固定于肿瘤去皮部位,尾静脉和肿瘤局部分别注射100μL(0.5mM)Vc,利用电化学工作站i-t法检测H2O2。
结果与讨论:图1为利用电化学工作站检测荷瘤鼠肿瘤部位H2O2的实验过程示意图;
正常裸鼠尾静脉注射100μL生理盐水后电化学工作站几乎未观察到信号变化,正常裸鼠尾静脉注射100μL 5mM的Vc后,修饰电极检测响应电流稍微波动,荷瘤鼠尾静脉注射100μL 5mM的Vc后,产生约800pA的电流信号,对比正常裸鼠尾静脉注射生理盐水及Vc可知,荷瘤鼠肿瘤细胞在Vc刺激下,肿瘤细胞会释放过氧化氢,释放的过氧化氢在修饰电极的催化作用下分解产生电流信号。
正常裸鼠布局注射100μL生理盐水后电极信号基本没有变化(图2a),正常裸鼠局部注射100μL 5mM的Vc后,修饰电极检测响应电流约50pA(图2b),荷瘤鼠尾静脉注射100μL5mM的Vc后,产生约1000pA的电流信号(图2c),对比正常裸鼠局部注射生理盐水及Vc可知,荷瘤鼠肿瘤细胞在Vc刺激下,肿瘤细胞会释放过氧化氢,释放的过氧化氢在修饰电极的催化作用下分解产生电流信号,同时可以看到,荷瘤鼠局部注射Vc比尾静脉注射Vc刺激释放的过氧化氢更多,其原因可能是由于尾静脉注射Vc在血液循环后,浓度稀释,对肿瘤细胞的刺激没有局部强烈,故局部产生的电流信号比尾静脉强。
利用电化学工作站检测H2O2还原DNA修饰的银离子
金电极表面依次使用直径为0.3μm、0.05μm的Al2O3匀浆在麂皮上将其表面进行打磨,除去表面及周围的污物,再依次用超纯水、乙醇、超纯水、乙醇进行超声处理5min,吹干电极,将电极置0.5M H2SO4中进行循环伏安扫描,然用纯水冲洗干净,最后将电极置于Fe(CN)6 3-/4-溶液中分别进行循环伏安扫描和交流阻抗扫描,用纯水冲洗晾干备用。
在处理好的电极表面滴加20μL浓度分别为1mg/mL的还原性石墨烯(rGO),37℃放置1h,PBS洗涤一次,将电极浸泡在体积为300μL不同浓度的DNA修饰的Ag+(100mM),H2O2(100mM)溶液中,避光孵育3min,用pH8.6的甘氨酸-NaOH缓冲液进行磁力搅拌洗涤5min;在5mL 1mol/L KCl溶液中,以洗涤后的电极为工作电极、铂电极为对电极、饱和甘汞电极为参比电极,在电化学工作站上进行线性扫描LSV,扫描范围为记录Ag的溶出峰电流。
结果与讨论:修饰rGO的电极(20μL 1mg/mL)浸没在体积为300μL的50μM DNA、100mM Ag+、10mM H2O2溶液中,避光3min后洗涤,电化学工作站的修饰电极溶出峰响应信号可达40μA;修饰rGO电极(20μL 1mg/mL)浸没在体积为300μL的5μM DNA、100mM Ag+、10mMH2O2溶液中,避光3min后洗涤,电化学工作站的修饰电极溶出峰响应信号可达4.5μA;修饰rGO的电极(20μL 1mg/mL)浸没在体积为300μL的100mM Ag+、10mM H2O2溶液中,避光3min后洗涤,电化学工作站的修饰电极溶出峰响应信号可达3.5μA;裸电极浸泡在5μM DNA、1000mMAg+、10mM H2O2体系溶液中,避光3min后洗涤,电极溶出峰响应信号可达2.1μA;裸电极浸泡在100mM Ag+、10mM H2O2溶液体系中,避光3min后洗涤,电极溶出峰几乎无响应信号;裸电极浸泡在Ag+溶液中避光3min后洗涤,电极溶出峰几乎无响应信号。电极分别置于pH=8.6的甘氨酸溶液作为反应介质检测阳极溶出峰。由于DNA与Ag+形成耦合物,Ag+浓度增强,H2O2催化还原Ag+产生的电流信号大;因此,利用H2O2可高效催化修饰DNA的银离子。
Vc刺激细胞产生银单质
将HepG2(肝癌细胞)用PBS轻轻洗涤,计数,培养皿细胞数为1×106,加入1mL培养基轻轻晃动,在培养皿中加入100μL 100mM Ag+,5min后加入10μL Vc(5mM)刺激下细胞,观察细胞形态及生成的银单质;在培养皿中加入10μL Vc(5mM)刺激下细胞,然后加入100μL100mM Ag+,观察细胞形态及生成的银单质;在培养皿中加入100μL 100mM Ag+溶液,5min后加入10μL Vc(5mM),随时间观察细胞形态及产生的银单质。
图3为肿瘤细胞在Vc刺激下释放过氧化氢催化生成银单质,10μL 5mM Vc刺激HepG2细胞释放H2O2还原单质Ag随Ag+浓度的变化;a:10mM Ag+,b:20mM Ag+,c:50mM Ag+,d:100mM Ag+,e:200mM Ag+,f:400mM Ag+。
结果与讨论:相比于未处理的细胞,肿瘤细胞加入Ag+溶液,利用Vc刺激后,细胞凋亡,同时产生大量的黑色银单质;肿瘤细胞Vc刺激,5min后再加入Ag+溶液产生的黑色银单质,细胞凋亡;从图可以看到,先加入Ag+溶液的细胞产生的银单质更多。肿瘤细胞加入100μL Ag+溶液,利用Vc刺激后,随着时间的推移,细胞产生的银单质越来越多,12min后银单质数量不再增多,细胞慢慢凋亡;肿瘤细胞加入不同浓度的Ag+溶液,利用Vc刺激后,随着浓度的增加,细胞产生的银单质越来越多,细胞慢慢凋亡(如图3a-f)。
实施例2、Apt-CS/rGO/Ag+-DNA复合纳米材料的制备
1)称取34mg硝酸银溶解于1mL的超纯水,将溶解的10μL 1OD DNA加入硝酸银溶液(DNA终浓度为2.5μM),涡旋5min,室温孵育1h;
2)取1mg氧化石墨烯(GO)溶于1mL超纯水,利用细胞破碎超声仪超声6h,3000res/min离心,去除沉淀,加入10μL水合肼(NH2-NH2),涡旋10min,60℃水浴4h,离心10000rpm,去除上清液,洗涤两次,分散成1mg/mL还原型氧化石墨烯溶液备用;
3)取50μL还原性石墨烯溶液加入1mL步骤1)制备的Ag+-DNA溶液中,涡旋5min,室温孵育30min,10000res/min离心10min,去除上清液,洗涤两次,合成GO/Ag+-DNA,分散于1mL超纯水中备用;
4)取10mg壳聚糖(CS)分散于10mL 1%(质量浓度)的冰醋酸溶液中,取1mL加入10mL超纯水,搅拌溶解,直到溶液无气泡产生,然后加入1mL上述rGO/Ag+-DNA溶液,搅拌反应6h,离心12000res/min 15min去除上清液,得到CS/rGO/Ag+-DNA纳米材料,加入50μL2.5%(质量浓度)戊二醛溶液和100μL 1OD Cy5荧光标记的核仁素适配体AS1411,搅拌反应1h,加入1%的BSA搅拌30min,12000res/min离心去除上清液,洗涤2次,得到Apt-CS/rGO/Ag+-DNA复合纳米材料。
图4为红外表征图,其中a表示rGO/Ag+-DNA,b表示CS/rGO/Ag+-DNA,c表示Apt-CS/rGO/Ag+-DNA。
利用红外光谱仪压片技术分别检测Ag+-DNA、rGO/Ag+-DNA、CS/rGO/Ag+-DNA、Apt-CS/rGO/Ag+-DNA复合纳米材料的红外吸收。从图4红外吸收光谱图中可以看出,Apt-CS/rGO/Ag+-DNA复合纳米材料合成成功。
利用拉曼技术检测rGO、Apt-CS/rGO/Ag+-DNA复合纳米材料的拉曼吸收。Apt-CS/rGO/Ag+-DNA复合纳米材料与rGO在1348cm-1和1592cm-1存在D、G带,表明该复合纳米材料合成成功。
利用XPS表征分析检测rGO、Apt-CS/rGO/Ag+-DNA复合纳米材料的元素组成。Apt-CS/rGO/Ag+-DNA复合纳米材料组成元素中含有P、Ag、C、O、N等,而rGO中有的C、O、N元素,表明该复合纳米材料合成成功。
利用AFM显微镜观察Apt-CS/rGO/Ag+-DNA复合纳米材料。Apt-CS/rGO/Ag+-DNA复合纳米材料厚度大约为30-40nm。
3、复合纳米材料的活性研究
复合纳米材料与细胞靶向结合:
将细胞离心重悬均匀的铺于孔板中,培养6h,计数,用吸管将培养基去除,PBS洗涤两次,加入100μL Apt-CS/rGO/Ag+-DNA复合纳米材料,轻微晃动孔板,使纳米材料均匀分布于细胞,孵育10min,PBS洗涤两次,加入10μL 5mM Vc,轻微晃动孔板,使Vc溶液均匀分布于细胞,显微镜观察细胞靶向性及银沉积现象。
细胞爬片实验:
胰酶消化及收集生长期的MCF-7、A549、L02细胞,用PBS制成细胞悬浮液,浓度为每毫升1×106个细胞,细胞铺入六孔板,中央滴入5μL PBS,将盖玻片置于六孔板内,轻压盖玻片使之紧贴孔板,将各组细胞悬浮液按200μL/孔均匀涂于六孔板内的盖玻片上,与细胞培养箱内培养2h,待细胞贴壁后,各孔中加入1至2mL的培养基,与培养箱培养12h,吸出培养基,PBS洗涤2次,加入2μL Apt-CS/rGO/Ag+-DNA复合纳米材料,避光孵育10min,洗涤后加入2μL的5mM的Vc,孵育10min,WB缓冲液洗涤各孔3次,每次5min,PBS洗涤3次,加入适量DAPI细胞核染色5min,PBS洗涤3次,将盖玻片盖到滴有抗荧光猝灭剂的载玻片上,荧光显微镜观察。
图5为100μL 1mg/mL Apt-CS/rGO/Ag+-DNA复合纳米材料与MCF-7、A549和L02细胞孵育后,PBS洗涤,在10μL 5mM Vc刺激下释放过氧化氢催化生成银单质的共聚焦显微镜图。
如图5所示,由于适配体AS1411对MCF-7和A549细胞具有靶向性,细胞与材料结合、洗涤,加入Vc后,Vc刺激细胞产生大量的过氧化氢,过氧化氢将Ag+还原为Ag单质,细胞内看到大量的黑色银单质;由于L02细胞没有靶向性,L02细胞与材料孵育后,并未与细胞结合,PBS洗涤,再次加入Vc后,由于与细胞结合的材料几乎没有,因此没有黑色的单质银产生。说明Apt-CS/rGO/Ag+-DNA复合纳米材料可以靶向MCF-7(人乳腺癌细胞)和A549(人肺癌细胞)细胞,并在Vc刺激下产生单质银。
PI(碘化丙啶)染料观测细胞死亡:
将MCF-7、A549、293T细胞离心重悬均匀的铺于孔板中,培养6h,计数,用吸管将培养基去除,PBS洗涤两次,加入100μL Apt-CS/rGO/Ag+-DNA复合纳米材料,轻微晃动孔板,使纳米材料均匀分布于细胞,孵育10min,PBS洗涤两次,加入100μL 1mg/mL PI染料,轻微晃动孔板,加入10μL 5mMVc,轻微晃动孔板,使Vc溶液均匀分布于细胞,共聚焦显微镜观察细胞形态。
加入AS1411-Ag+-rGO,MCF-7细胞周围出现绿色荧光,说明FITC标记的AS1411-Ag+-rGO靶向MCF-7细胞,加入维生素C后,在5分钟时细胞出现红色荧光及大量的黑色银。在10-20分钟时,细胞红色荧光达到最亮,说明细胞膜的通透性发生了改变,细胞出现凋亡现象。
Apt-CS/rGO/Ag+-DNA复合纳米材料体外毒性检测:
取对数生长期的MCF-7、A549、293T细胞,用DMEM培养基掉这么好密度后接种于96孔板,每孔100μL培养基,细胞数在每孔1×104个细胞,设置空白孔和调零孔,实验分为Apt-CS/rGO/Ag+-DNA、Apt-CS/rGO/Ag+-DNA+Vc和空白组和调零组,每组3个复孔,毒性作用的浓度分别为0.01,0.1,0.5,1,5,10mg/mL。将细胞置于细胞培养箱培养0.5,1h;每孔加入20μLMTT,培养箱培养2h,去除上清液,每孔加入200μL DMSO,避光震荡10min,用酶标仪测OD值。
单独加入AS1411-Ag+-rGO后,随着浓度的增加,细胞活力减弱,AS1411-Ag+-rGO与维C联合加入后,明显可以看到细胞的活力比单独AS1411-Ag+-rGO弱,其由于维C刺激细胞产生过氧化氢,过氧化氢将AS1411-Ag+-rGO中的银离子还原为银单质,从而使细胞的活力减低,图中1mg/mL AS1411-Ag+-rGO和2mM维C对细胞的毒性最强。
体内抗肿瘤研究:
收集A549细胞,将约1×107的细胞注射到裸鼠背部皮下;采取随机数字表的方法,将荷瘤鼠随机分为4组,每组8只;待肿瘤体积长至约为100mm3时(种瘤10天后),分别尾静脉注射纳米材料及Vc,计量如下:PBS(0.01M,pH=7.4)100μL、Apt-CS/rGO/Ag+-DNA(1mg/mL)100μL、RS-CS/rGO/Ag+-DNA(RS是指随机DNA链,其序列为ATACCAGCTTATTCAATTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGATAGTAAGTGCAATCT:主要是作对照实验用的,也是由上海生工公司提供)(1mg/mL)100μL,2小时后尾静脉注射Vc 10mm 100μl,观察复合纳米材料在小鼠体内的荧光动态分布。
图6为100μL PBS、100μL 1mg/mL RS-CS/rGO/Ag+-DNA复合纳米材料、100μL 1mg/mL Apt-CS/rGO/Ag+-DNA复合纳米材料不同时间的体内肿瘤靶向荧光活体成像图。
结论:利用红外光谱仪分析可知,Apt-CS/GO/Ag+-DNA复合纳米材料合成成功,合成的Apt-CS/GO/Ag+-DNA复合纳米材料可靶向A549细胞,在Vc的刺激下可产生银单质,将该Apt-CS/GO/Ag+-DNA复合纳米材料尾静脉注射到荷瘤小鼠体内,在Vc刺激下,荷瘤小鼠的肿瘤部位会产生黑色的银单质,其具有成为肿瘤治疗制剂的潜在价值。
Claims (9)
1.一种药物联合制剂,由Apt-CS/rGO/Ag+-DNA复合纳米材料和维生素C构成,其中,所述Apt-CS/rGO/Ag+-DNA复合纳米材料与维生素C单独包装;
Apt-CS/rGO/Ag+-DNA复合纳米材料中,Apt-CS表示核酸适配体-壳聚糖耦合物;Ag+-DNA表示Ag+-DNA耦合物;rGO表示还原型氧化石墨烯。
2.根据权利要求1所述的药物联合制剂,其特征在于:所述核酸适配体针对不同的肿瘤选择对应的核酸适配体;
所述肿瘤为癌,所述癌为人乳腺癌时,所述核酸适配体为核仁素适配体AS1411;所述癌为人肺癌时,所述核酸适配体为Endoglin适配体或核仁素适配体AS1411;所述癌为肝癌时,所述核酸适配体为TLS11a。
3.根据权利要求1所述的药物联合制剂,其特征在于:所述Apt-CS/rGO/Ag+-DNA复合纳米材料通过包括如下步骤的方法制备得到:
1)制备Ag+-DNA耦合物溶液;
2)制备还原型氧化石墨烯溶液;
3)将还原型氧化石墨烯溶液加入到Ag+-DNA偶合物溶液中,涡旋,孵育,离心,收集沉淀得到rGO/Ag+-DNA,将rGO/Ag+-DNA分散到水中得到rGO/Ag+-DNA溶液;
4)将壳聚糖分散到冰醋酸溶液中,加入rGO/Ag+-DNA溶液,搅拌反应,离心,收集沉淀,得到CS/rGO/Ag+-DNA纳米材料,将适配体与CS/rGO/Ag+-DNA纳米材料中的CS偶联,得到Apt-CS/rGO/Ag+-DNA复合纳米材料。
4.根据权利要求1-3中任一项所述的药物联合制剂,其特征在于:所述药物联合制剂进一步包括一个记录数据的载体;
所述记录数据的载体记载有所述药物联合制剂的给药方法,即,先将Apt-CS/rGO/Ag+-DNA纳米材料给药;2-7min后再将维生素C给药,即可。
5.根据权利要求4所述的药物联合制剂,其特征在于:所述Apt-CS/rGO/Ag+-DNA纳米材料通过局部注射给药;
所述维生素C通过注射或口服给药。
6.权利要求1-5中任一项所述的药物联合制剂在制备靶向治疗肿瘤的药物中的应用,其中,所述药物联合制剂中Apt-CS/rGO/Ag+-DNA纳米材料中的核酸适配体根据待治疗肿瘤的类型确定。
7.权利要求1-5中任一项所述的药物联合制剂在制备肿瘤核磁成像造影剂中的应用;其中,所述药物联合制剂中Apt-CS/rGO/Ag+-DNA纳米材料中的核酸适配体根据肿瘤的类型确定。
8.根据权利要求6或7所述的应用,其特征在于:所述肿瘤为癌,所述癌为人乳腺癌时,所述核酸适配体为核仁素适配体AS1411;所述癌为人肺癌时,所述核酸适配体为Endoglin适配体或核仁素适配体AS1411;所述癌为肝癌时,所述核酸适配体为TLS11a。
9.Vc在刺激肿瘤细胞释放过氧化氢,过氧化氢催化Apt-CS/rGO/Ag+-DNA纳米材料中银离子在肿瘤部位的还原沉积中的应用。
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