CN113355087B - 对肿瘤细胞外酸性微环境pH响应的超分子荧光探针体系 - Google Patents
对肿瘤细胞外酸性微环境pH响应的超分子荧光探针体系 Download PDFInfo
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Abstract
本发明提供了一种对肿瘤细胞外酸性微环境pH响应的超分子荧光探针体系,该探针体系具有对肿瘤的敏感响应性,即对pH有响应。而细胞实验也表明,这种超分子荧光探针体系对正常细胞具有较低的毒性,而对癌细胞具有较高的毒性,可以作为荧光生物探针用于荧光成像引导癌症手术。这样的结果将激起人们对设计和合成具有产生更高的活性氧效率和性能优异的AIE荧光分子的研究热情,在肿瘤放疗平台的开发上,这也将进一步扩大AIE荧光分子在生物医学研究中的应用。
Description
技术领域
本发明属于癌症治疗技术领域,涉及一种用于光动力疗法的荧光探针体系,具体涉及一种对肿瘤细胞外酸性微环境pH响应的超分子荧光探针体系。
背景技术
荧光生物探针的发展对于诊断和跟踪许多疾病,特别是癌症,具有重要意义,因为这些探针可以对生物环境中的分析物进行敏感、简单和特异的检测。特别是,设计能够对肿瘤微环境刺激作出响应的发光探针,将为智能荧光探针在肿瘤成像方面的发展开辟新的途径。在肿瘤微环境刺激中,pH响应性是一个特别有趣的问题,它已被广泛研究在控制药物递送更好的靶向肿瘤和治疗。这是因为与血液和正常组织(pH约为7.4)相比,肿瘤细胞外环境(pHe)中的pH值呈酸性(6.5-7.2)。迄今为止,大多数pH响应型荧光探针均设计用于靶向pH约5.5的细胞内,只有少数研究集中在对肿瘤细胞外酸性微环境有响应的探针上。
癌症已经成为世界上主要的死亡原因之一。尽管近年来在癌症治疗方面取得了很大进展,包括放射治疗和化疗,但许多患者仍因药物过量或过度放射而遭受癌症耐药性和转移,这意味着非常希望开发更有效的治疗体系。光动力疗法(PDT)是一种有前途的无创性治疗技术,在临床前试验中引起了越来越多的兴趣。光动力疗法需要光敏剂(PS)在光照射下产生活性氧(ROS)引起细胞凋亡。从实验室到临床的转换因其“always-on”模式和不希望的暗细胞毒性而严重受阻。因此,如何在体内调控PS的行为,设计有效的PS是非常重要的。
理想的PS在体循环过程中应该是不发光的,但在癌细胞或肿瘤部位对外部或生物刺激有选择性地发光。然而,传统的PS在生物环境中局部积累时,往往会遭遇聚集性的发光淬灭,难以满足精准癌症治疗的需要。相比之下,聚集诱导发光基元(AIEgens)具有高效的固态发光效率、高的光稳定性和原位活化能力,已被证实是传统PS的良好替代品。由于受到分子内运动(包括旋转和振动)受限,AIEgens在溶液中弱发光或无发光,但在聚集态时发出强烈的荧光。在溶液中,AIEgens的激发态能量是通过分子内运动释放的。这种运动在聚集态被抑制,以激活辐射跃迁,使AIEgens发光。到目前为止,基于AIEgens的PS可以靶向不同的细胞器,如线粒体、脂滴、内质网和细胞膜,显示出很高的癌症治疗潜力。为了避免用药过量,设计了一些自报告体系来实时监测治疗效果。然而,它们也遇到了细胞暗毒性的困扰。
在这样的前提下,AIE-active的PS被设计为具有被抑制的荧光,通过增强非辐射跃迁或切断从单重态到三重态的能量转移以抑制ROS的产生。为此,可采用两种策略来实现这一目标。一种是通过将能量受体或猝灭剂共价连接到周围的PS上的单分子合成。例如,Tang及其同事报道了一系列水溶性AIEgens,其结构中的受体可以加速能量传递,这些能量在水溶液中不存在,一旦停靠在细胞的线粒体上,就会发出强烈的荧光,从而通过PDT引起细胞凋亡。不幸的是,这些PS通常是通过复杂的分子合成制备的,需要繁琐的有机纯化,并且对外部刺激表现出惰性,这极大地限制了它们的高通量测试。另一策略是使用超分子组装体来实现改善的PDT功效。与单一PS体系相比,主-客体相互作用被认为是一种有效的解决体内细胞暗毒性的方法,因为PS的ROS生成和光物理性质可以通过非共价分子相互作用可逆调节,如疏水相互作用、氢键、配位键和静电相互作用。作为主客体结构的一个重要组成部分,规则形状的有机大环,如杯芳烃、环糊精和葫芦脲,由于其固有的空穴可以选择性地完全或部分结合客体分子而被广泛探索。其中,杯芳烃是众所周知的宿主模块分子,并且由于其低毒性和对刺激的多重响应,已成为用于药物递送,生物成像和治疗试剂的理想骨架。
如上所述,大多数pH响应型荧光探针均设计用于靶向pH约5.5的细胞内,只有少数研究集中在对肿瘤细胞外酸性微环境有响应的探针上,而在超分子方面的pH响应型荧光探针更是少有报道。
发明内容
本发明的目的在于开发一种调控活性氧产生效率、细胞暗毒性并且对肿瘤细胞外酸性微环境pH响应的超分子荧光纳米探针体系。
基于此,本发明提供了一种对肿瘤细胞外酸性微环境pH响应的超分子荧光探针体系,其为客体分子和主体分子组合形成的一种超分子络合物,所述超分子络合物对肿瘤细胞外酸性微环境pH产生荧光响应。
作为本发明优选的实施方式,本发明人经过试验研究筛选了一组客体分子的组合,所述客体分子包括如下G1-G7的化合物:
作为本发明优选的实施方式,本发明人经过试验研究筛选了一组主体分子的组合,所述主体分子包括如下H1-H3的化合物:
进一步地,本发明所述的超分子荧光探针体系由客体分子化合物G1-G7中的一种和主体分子化合物H1-H3中的一种组成;具体的,所述客体分子化合物G1-G7和主体分子化合物H1-H3的组合方式选自 体系中的一种。
进一步地,经本发明人研究确认,为了达到更好的应用效果,本发明所述客体分子化合物G1-G7和主体分子化合物H1-H3的组合方式优选的选自 体系中的一种。
需要说明的是,上述及其本发明中所述的表示两种化合物的所形成的超分子络合物。例如,/>表示化合物G1和化合物H1形成的超分子络合物;/>表示化合物G2和化合物H1形成的超分子络合物,其他表述方式与此相同。
进一步地,以物质的量之比计,所述体系中G1:H1=1:1;所述/>体系中G2:H1=1:1;所述/>体系中G3:H1=1:2;所述/>体系中G4:H1=1:2;所述/>体系中G5:H1=1:2;所述/>体系中G6:H1=1:2;所述/>体系中G7:H1=1:1;所述/>体系中G1:H2=1:1;所述/>体系中G2:H2=1:1;所述体系中G3:H2=1:2;所述/>体系中G4:H2=1:2;所述/>体系中G5:H2=1:2;所述/>体系中G6:H2=1:2;所述/>体系中G3:H2=1:1;所述体系中G1:H3=1:1;所述/>体系中G2:H3=1:1;所述/>体系中G3:H3=1:2;所述/>体系中G4:H3=1:2;所述/>体系中G5:H3=1:2;所述体系中G6:H3=1:2;所述/>体系中G3:H2=1:1。
本发明所述的客体分子化合物G3、G5、G6和主体分子化合物H3的制备方法现有文献已有报道,其余化合物经本发明人长期试验研究得出了一种全新的制备路径。其中,所述客体分子G1、G2、G4、G7通过以下方法进行制备:
所述主体分子H1、H2通过以下方法进行制备:
另外,本发明所述的超分子荧光探针体系中除过所必须的由客体分子和主体分子所形成的超分子络合物外,还需要溶剂以承载所述客体分子和主体分子来形成稳定的超分子络合物。经本发明人研究确认,所述溶剂选自四氢呋喃(THF)或二甲基亚砜(DMSO)中的一种或两种组合。
本发明还提供了所述超分子荧光探针体系在肿瘤细胞成像和靶向肿瘤治疗中的应用。特别的是,所述超分子荧光探针体系能够在肿瘤细胞外酸性微环境的pH值为6.5-7.2中产生荧光响应。
需要说明的是,本发明所提供的 前7种超分子荧光探针体系与 后14种超分子荧光探针体系应用相同。经本发明人研究确认,后14种超分子荧光探针体系与前七种相比,应用效果存在差异。但是,本发明欲保护的为上述21种超分子荧光探针体系及其在靶向癌细胞成像和体内肿瘤细胞成像中的应用。因此,在不脱离本发明设计精神的前提下,对本发明所述技术方案作出无创造性改进的方案及其应用,都应落入本发明确定的保护范围内。
与现有技术相比,本发明具有以下有益效果或者优点:
现有技术中大多数的pH响应型荧光探针均设计用于靶向pH约5.5的细胞内,只有少数研究集中在对肿瘤细胞外酸性微环境有响应的探针上。本发明提供了一种超分子荧光探针体系,能够在肿瘤细胞外酸性微环境的pH值为6.5-7.2中产生荧光响应,操作方法更为简单。
从分子分散状态到聚集状态,由于分子内运动受限和系间窜越的增强,杯芳烃衍生物(主体分子)与聚集诱导发光基元(客体分子,AIEgen)之间的主客体络合可以显著提高其发光,具有聚集诱导发光(AIE)的荧光纳米粒子探针的活性氧1O2产生效率也增加。本发明提供了一种对肿瘤细胞外酸性微环境pH响应的超分子荧光探针体系,该探针体系具有对肿瘤的敏感响应性,即对pH有响应。而细胞实验也表明,这种超分子荧光探针体系对正常细胞具有较低的毒性,而对癌细胞具有较高的毒性,可以作为荧光生物探针用于荧光成像引导的癌症手术。这样的结果将激起人们对设计和合成具有产生更高的活性氧效率和性能优异的AIE荧光分子的研究热情,在肿瘤放疗平台的开发上,这也将进一步扩大AIE荧光分子在生物医学研究中的应用。
另外,本发明提供的超分子荧光探针体系能响应肿瘤酸性细胞外微环境,在正常生理条件下携带负电荷,在肿瘤细胞外酸性微环境下,等七种超分子荧光探针体系的表面电荷变为正电荷,使得纳米粒子探针具有被癌细胞内化的能力,并显著开启其荧光。
附图说明
图1中,(A)为白光照射不同时间下ABDA(ABDA的浓度:5×10-6M)在THF中存在或不存在G2分子,以及只有G2分子(G2的浓度:10×10-6M)时在378nm处的吸光度变化;(B)为在THF中白光照射下,在存在G2分子聚集体的情况下,ABDA的紫外可见光谱图(G2的浓度:10×10-6M,ABDA的浓度:5×10-6M),UV测量的时间间隔:3分钟。
图2中,(A)为白光照射不同时间下ABDA(ABDA的浓度:5×10-6M)在THF中存在或不存在络合物,以及只有/>络合物(/>的浓度:10×10-6M)时在378nm处的吸光度变化;(B)为在THF中白光照射下,在存在/>络合物聚集体的情况下,ABDA的紫外可见光谱图(/>的浓度:10×10-6M,ABDA的浓度:5×10-6M),UV测量的时间间隔:2分钟。
图3中,(A)为通过SRB分析法测试细胞存活率:将HeLa细胞与不同浓度的在培养基中孵育24小时的测试结果图;(B)为通过SRB分析法测试细胞存活率:将Humancervical epithelial细胞与不同浓度的/>在培养基中孵育24小时的测试结果图。
图4为G3、和/>在THF中的PL光谱(G3的浓度:10×10-6M,的浓度:10×10-6M,/>的pH值为6.13)。
图5为G2客体分子与H1主体分子在THF中发生自组装之后,在THF的组装体(即超分子络合物)溶液中加入ABDA,于白光照射下产生活性氧1O2。
具体实施方式
下面,结合附图对本发明的技术方案进行进一步的解释说明,但是,本发明并不限于下述的实施方式。
需要说明的是,文中表示两种化合物的所形成的超分子络合物。例如,表示化合物G1和化合物H1形成的超分子络合物;/>表示化合物G2和化合物H1形成的超分子络合物,其他相同的表述方式与此相同。另外,文中如未作特殊说明的符号或名词均为本领域常用的表述方式或释义。
本发明提供了一种对肿瘤细胞外酸性微环境pH响应的超分子荧光探针体系,其为客体分子和主体分子组合形成的一种超分子络合物,所述超分子络合物对肿瘤细胞外酸性微环境pH产生荧光响应。该体系的组分包括客体分子和主体分子。
(1)超分子荧光探针体系化合物的制备
作为本发明优选的实施方式,所述客体分子包括如下G1-G7的化合物:
其中,化合物G3、G5、G6的制备方法在现有报道中已有介绍,化合物G1、G2、G4、G7通过以下方法进行制备:
具体的,其合成方法如下:
化合物3的合成:将化合物1五甘醇(6.00g,25.18mmol)、三乙胺(1.8mL,12.59mmol)溶于30mL二氯甲烷中置于冰浴下搅拌,将甲苯-4-磺酰氯2(1.20g,6.29mmol)分四批加入反应液中,之后室温搅拌3小时后停止反应,饱和碳酸氢钠水溶液洗涤6次,无水硫酸镁干燥,减压蒸出二氯甲烷,柱层析(洗脱剂:二氯甲烷/甲醇=60/1,v/v)分离得2.11g无色油状液体产物3,产率85%。
化合物6的合成:取250mL的三颈烧瓶,装上大磁子,加入锌粉(5.50g,84.13mmol)、4,4’-二羟基二苯甲酮4(3.50g,16.36mmol)和4,4’-二溴二苯甲酮5(5.51g,16.21mmol),搭好回流反应装置,冰浴下,抽真空,充氮气,如此循环三次后,加入70mL新蒸THF,再用玻璃注射器缓慢加入四氯化钛(7mL,63.70mmol),继续冷却10分钟,撤去冰浴,室温搅拌20分钟,升温回流24小时,停止加热,反应完毕,冷却至室温。缓慢加入80mL二氯甲烷,再加入100mL5%稀盐酸搅拌20分钟,之后分液除去水相,有机相再用饱和氯化钠水溶液萃取3次,分出水相,合并有机相,无水MgSO4干燥,过滤,减压蒸发除去溶剂。得到的黄色固体粗产物用乙酸乙酯/石油醚(1/5,v/v)作为洗脱剂,快速柱层析分离得到4.1g白色固体6,收率48%。
化合物7的合成:将化合物6(0.45g,0.86mmol),K2CO3(0.72g,5.17mmol)加入100mL双颈烧瓶中,再放入磁子,搭好回流反应装置,抽真空,充氮气,如此循环三次后,用注射器加入40mL乙腈,室温搅拌0.5小时,随后加入溶于10mL乙腈的化合物3(1.35g,3.44mmol),于100℃下加热回流20小时,停止加热,反应完毕,冷却至室温。过滤除去K2CO3,再减压蒸发除去溶剂乙腈,然后加入30mL二氯甲烷分别用30mL水和30mL饱和氯化钠溶液各萃取一次,合并有机相,无水MgSO4干燥,过滤,减压蒸发除去溶剂,得到黄色粘稠液粗产物,最后用二氯甲烷/甲醇(60/1,v/v)作为洗脱剂,快速柱层析分离得到0.63g黄色粘稠物7,收率75%。
化合物8的合成:取一个50mL的两颈烧瓶,将磁子和化合物7(0.183g,0.190mmol)、吡啶-4-硼酸(0.095g,0.770mmol)、Pd(PPh3)4(0.024g,0.019mmol)、K2CO3(0.264g,1.903mmol)加入其中,搭好回流反应装置,抽真空,充氮气,如此循环三次后,再注入10mL甲苯和5mL乙醇,在100℃下加热回流16小时,反应完毕,将反应液冷却至室温,加硅藻土过滤除去钯催化剂和K2CO3,旋干溶剂,加二氯甲烷再溶解粗产物,饱和食盐水洗涤3次,水相用二氯甲烷再洗涤2次,合并有机相,加入无水MgSO4干燥,减压过滤,减压蒸发除去溶剂得到黄色油状物,最后用二氯甲烷/甲醇(30/1,v/v)作为洗脱剂,快速柱层析分离得到0.138g黄色油状物8,收率75.6%。
化合物G4的合成:取一个50mL的圆底烧瓶,放入磁子,加入溶于10mL乙腈的化合物8(0.138g,0.144mmol),再注入0.855mL碘甲烷,搭好回流反应装置,于100℃下加热回流10小时,反应完毕,冷却至室温。旋干溶剂,做离子交换反应,加入2mL饱和KPF6溶液,再加入10mL丙酮,室温搅拌12小时,交换反应完毕,旋干溶剂得橙红色粗产物,最后用二氯甲烷/甲醇(20/1,v/v)作为洗脱剂,快速柱层析分离得到0.05g橙红色固体产物G4,收率26%。
化合物G1的合成:取一个100mL的两颈烧瓶,将磁子和化合物9(1mL,5.811mmol)、化合物12(1.32g,5.615mmol),再加入25mL乙醇,随后用移液枪加入Et3N(80μL,0.576mmol)、四氢吡咯(0.5mL,6.059mmol)加入其中,搭好回流反应装置,于90℃下加热回流20小时,反应完毕,冷却至室温。抽滤并用乙醇洗涤1~2次,得1.34g暗红色固体粉末G1,收率60.4%。
化合物G2的合成:取一个50mL的两颈烧瓶,将磁子和化合物10(0.233g,0.851mmol)、化合物12(0.207g,0.851mmol),再加入15mL乙醇,随后用移液枪加入Et3N(15μL,0.108mmol)、四氢吡咯(0.5mL,6.059mmol)加入其中,搭好回流反应装置,于90℃下加热回流24小时,反应完毕,冷却至室温。抽滤并用乙醇洗涤1~2次,得暗红色固体粉末粗产物,最后用乙腈-乙醇重结晶得0.21g红色固体粉末G2,收率50%。
化合物G7的合成:取一个100mL的两颈烧瓶,将磁子和化合物11(0.50g,1.843mmol)、化合物12(0.433g,1.843mmol),再加入50mL乙醇,随后用移液枪加入Et3N(40μL,0.288mmol)、四氢吡咯(0.2mL,2.424mmol)加入其中,搭好回流反应装置,于90℃下加热回流18小时,反应完毕,冷却至室温。旋干溶剂,用二氯甲烷/甲醇(40/1,v/v)作为洗脱剂,快速柱层析分离得到0.414g橙黄色固体产物G7,收率46%。
作为本发明优选的实施方式,所述主体分子包括如下H1-H3的化合物:
其中,化合物H3的合成方法在现有文献中已有报道,化合物H1、H2的合成路线如下:
具体的,其合成方法如下:
化合物15的合成:取一个250mL的圆底烧瓶,放入磁子,将化合物13(2.00g,3.082mmol)溶于90mL超干DMF中,加入2.219g(55.48mmol)氢化钠(60%),随后搅拌30分钟后,加入溴代癸烷14(11.5mL,55.48mmol),加热控制温度在85℃反应,反应1小时后会产生白色固体,6小时后停止反应,冷却至室温,加入20mL甲醇(除去多余的NaH),过滤并用甲醇洗涤6次,再用三氯甲烷和甲醇重结晶得2.4g白色固体粉末15,收率64.5%。
化合物16的合成:取一个250mL的圆底烧瓶,放入磁子,将化合物15(2.10g,1.735mmol)溶于120mL超干二氯甲烷中,加入17.5mL冰醋酸,冰浴冷却至0℃,滴加9.3mL(147.65mmol)发烟硝酸(>95%),滴加后溶液立即变成黑色,室温下继续搅拌,待溶液由黑色转化成黄色后加入水淬灭反应,分出有机相,水洗3次,无水硫酸镁干燥,过滤,减压蒸出二氯甲烷,用二氯甲烷和甲醇重结晶,得1.29g淡黄色固体粉末16,收率63.8%。
化合物17的合成:将化合物16(0.5g,0.429mmol)加入100mL圆底烧瓶中,放入磁子,加入45mL无水乙醇,室温下搅拌10分钟,加入0.182gPd/C(5%),搅匀后加入2.1mL(34.32mmol)水合肼(80%),加热回流5小时,停止加热,趁热通过硅藻土过滤,减压蒸出乙醇,固体溶于乙酸乙酯中,水洗两次,无水硫酸镁干燥,过滤,减压蒸出溶剂得0.237g灰白色固体产物17,收率52.8%。
化合物H1的合成:取一个25mL圆底烧瓶,放入磁子,将化合物17(0.1g,0.096mmol)溶于5mL超干二氯甲烷中,室温下滴加溶于5mL超干二氯甲烷中的3,4-二甲基呋喃-2,5-二酮(0.0603g,0.478mmol),滴加完后室温搅拌继续反应约3小时,减压蒸出二氯甲烷,然后三氯甲烷-甲醇重结晶得0.092g黄色晶体H1,收率62.1%。
化合物H2的合成:取一个25mL圆底烧瓶,放入磁子,将化合物17(0.05g,0.0478mmol)、顺丁烯二酸酐(0.0234g,0.239mmol)加入其中,再加入8mL超干二氯甲烷,随后再加入1mL三乙胺、0.1mL吡啶,室温搅拌15小时,减压蒸干溶剂,然后三氯甲烷-甲醇重结晶得0.029g黄色固体H2,收率42.2%。
(2)活性氧的检测方法及所需仪器、试剂:
紫外可见分光光度法根据被测量物质分子对紫外可见波段范围(200~800nm)单色辐射的吸收或反射强度来进行物质的定性、定量或结构分析的一种方法,使用紫外分析仪,利用此法可以在我们合成的分子的溶液中加入指示剂ABDA(9,10-蒽二基-二(亚甲基)二丙二酸)检测合成的分子及超分子络合物产生活性氧的效率。关键在于指示剂ABDA在所合成分子或络合物溶液中发生化学作用后,在白光照射下会发生分解,基于此,在白光照射不同时间间隔内分别测一测其紫外吸收,从紫外吸收的数据结果来证明化合物产生活性氧(1O2)的效率。具体操作如下:
每一个样品,样品浓度配成1.0×10-5M(溶剂THF),ABDA被用作1O2指示剂。将ABDA溶液(溶剂DMSO,1.0×10-2M,20μL)添加到样品溶液(1.0×10-5M,4mL)中,并将白光(4.2mW/cm2)用作照射源。在不同的照射时间记录378nm处的ABDA的吸光度。以G2分子及其络合杯芳烃分子H1测活性氧的数据为例,指示剂ABDA在所合成分子或络合物溶液中发生化学作用后,在白光照射下发生分解产生活性氧1O2,随着其分解产生活性氧1O2,其吸光度也随之降低,到最后分解完曲线吸光度不在变化,吸光度降低越大说明其产生活性氧1O2效率越高。如图1和图2所示(图A中对应的是378nm处不同照射时间的ABDA的吸光度散点图,图B是主客体分子+指示剂ABDA在不同照射时间的波长-吸光度曲线图)。实验结果如图所示:从图1可知,图1(A)中客体分子G2、指示剂ABDA随白光照射时间长短的变化,其吸光度基本不变化或变化较小,而客体分子G2结合指示剂ABDA的混合物随白光照射时间长短的变化,其吸光度变化很大,直到混合物分解饱和后吸光度基本不再变化曲线走势趋于平缓,这说明客体分子G2可以高效产生活性氧1O2;同理,图1(B)说明随白光照射时间(0min到最后42min)的增加,曲线在378nm处的吸光度也随之降低直到混合物分解饱和后吸光度基本不再变化,这也说明客体分子G2可以高效产生活性氧1O2。图2与图1同理,说明主客体络合物可以高效产生活性氧1O2。
(3)细胞暗毒性的实验方法及所需仪器、试剂:
磺酰罗丹明B(SRB)测定法:磺酰罗丹明B是一种蛋白结合染料,可于生物大分子中的碱性氨基酸结合,其在515nm的OD读数与细胞数呈良好的线性关系,故可用作细胞数的定量。利用此法可做细胞毒性的分析。
具体实验过程如下:
在96孔板中以100μL/孔的体积接种细胞,预孵育过夜;根据不同的实验目的处理细胞;倒出培养基并通过添加100μL冷的10%TCA固定细胞。在4℃孵育60分钟或更长时间;丢弃上清液,用去离子水或自来水将板洗涤5次,风干(可以储存直到使用);向每个孔中添加50μL1%乙酸中的0.4%(W/V)SRB(sigmaS9012)溶液,在滴定板振荡器上摇动5分钟;用1%的乙酸将板洗涤5次,风干(可以储存直到使用);加入100μL的10mM非缓冲液三羟甲基氨基甲烷(pH10.5)以溶解结合的染料;在微量滴定板振荡器上混合5分钟,并在自动分光光度板读取器上以490-530nm的单个波长读取光密度(OD值)。
以G2分子络合杯芳烃分子H3做细胞毒性实验的数据为例,图3中图A是培养HeLa细胞(宫颈癌细胞)后,做的细胞毒性实验;图B是培养Humancervicalepithelial细胞(人宫颈上皮细胞)后,做的/>细胞毒性实验。从数据结果看,对癌细胞的杀伤力比较大,即就是其毒性较大;对人正常细胞的杀伤力较小,即就是其毒性较小。
(4)对pH响应的实验方法及所需仪器、试剂:
荧光光谱法具有高灵敏度、操作简易、成本低等优点,可用于检验超分子络合物的pH响应。具体方法过程如下所示:
以G3络合H1(络合当量比1:2)为例,先将G3配制成1.0×10-5M的浓度,再按照其络合当量比加入主体分子H1,这样可配得1.0×10-5M的浓度的主客体络合物溶液,之后使用荧光光谱法测其荧光强度,再其后加入稀盐酸调pH值为6.13,再测荧光强度,观察其荧光强度变化可判断其是否有pH响应。实验结果如图4所示,从图中可知,单一客体分子G3的荧光强度最低,与主体分子H1络合后荧光明显增强,加入稀盐酸调pH值为6.13时测得荧光有所淬灭,说明其对pH有响应。
如上所述,即可较好地实现本发明,上述的实施例仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种改变和改进,均应落入本发明确定的保护范围内。
Claims (8)
1.一种超分子荧光探针体系,其为客体分子和主体分子组合形成的一种超分子络合物,所述超分子络合物对肿瘤细胞外酸性微环境pH产生荧光响应;
所述客体分子为G2的化合物,
,
所述主体分子为H1的化合物,
。
2.根据权利要求1所述的超分子荧光探针体系,其特征在于,客体分子化合物和主体分子化合物的组合方式为G2⊂H1。
3.根据权利要求2所述的超分子荧光探针体系,其特征在于,以物质的量之比计,所述G2⊂H1体系中G2:H1=1:1。
4.根据权利要求1所述的超分子荧光探针体系,其特征在于,客体分子化合物G2通过以下方法进行制备:
。
5.根据权利要求1所述的超分子荧光探针体系,其特征在于,主体分子化合物H1通过以下方法进行制备:
。
6.权利要求1至5中任一项所述超分子荧光探针体系在制备肿瘤细胞成像剂和肿瘤细胞靶向治疗药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述超分子荧光探针体系对肿瘤细胞外酸性微环境pH产生荧光响应。
8.根据权利要求7所述的应用,其特征在于,所述肿瘤细胞外酸性微环境pH为6.5-7.2。
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