CN113788861A - 一种金属铱(ⅲ)配合物及其制备方法和应用 - Google Patents
一种金属铱(ⅲ)配合物及其制备方法和应用 Download PDFInfo
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- CN113788861A CN113788861A CN202111199897.1A CN202111199897A CN113788861A CN 113788861 A CN113788861 A CN 113788861A CN 202111199897 A CN202111199897 A CN 202111199897A CN 113788861 A CN113788861 A CN 113788861A
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Abstract
本发明提供了一种金属铱(Ⅲ)配合物及其制备方法和应用,本发明提供的具有式(1)或式(2)结构的抗肿瘤铱(Ⅲ)配合物能够特异性靶向线粒体或溶酶体,有较低的暗毒性和较高的光毒性,在肿瘤细胞,特别是非小细胞肺癌细胞中具有非常高的光毒性指数。同时,该抗肿瘤金属铱(Ⅲ)配合物可作为光敏剂参与光动力疗法。相比传统光敏剂,该抗肿瘤金属铱(Ⅲ)配合物具有水溶性好,光稳定性高,低暗毒性等优点,实现诊断和治疗的一体化。
Description
技术领域
本申请涉及生物医药技术领域,特别涉及一种金属铱(Ⅲ)配合物及其制备方法和应用。
背景技术
癌症是人类最大的杀手之一,其有效的治疗方法仍非常有限。顺铂及其衍生物药物的问世给癌症患者带来福音,但这类药物对正常组织的毒副作用以及癌细胞易形成耐药性等缺点限制了它们的使用。
光动力疗法(Photodynamic Therapy,PDT)作为一种临床批准的微创疗法。光敏剂、光源和氧气是光动力治疗的三大要素。光敏剂是光动力疗法的关键因素之一。光敏剂吸收光子跃迁至激发态,再直接或间接将能量传递给氧,产生活性氧物种(ROS),使细胞结构受损。PDT在目前癌症治疗中受到广泛认可。PDT可以通过诱导肿瘤细胞的凋亡或者坏死,直接破坏肿瘤;可以损害肿瘤脉管系统间接杀死肿瘤;或通过启动机体免疫功能来杀伤肿瘤。PDT借助光纤等介入技术进行治疗,可避免手术造成的创伤;进入组织的药物达到一定浓度并受到光照,才能产生光毒反应;主要攻击光照区的病变组织,且病变细胞对光敏药物不产生耐药性,不会因为多次治疗的人增加毒性反应。总之光动力疗法具有创伤小、副作用小、选择性好、可重复治疗、治愈率高的优点。但目前临床光敏剂也存在它不可忽视的缺陷,如水溶性、光稳定性低、癌细胞选择性差、氧依赖性强、激发和发射波长较短、双光子吸收截面低等,于是新的光动力诊疗剂的研究越来越引起人们的重视。
铱元素,原子序数为77,原子量为192.22,属于元素周期表Ⅷ族过渡元素。铱的合金具有极高的熔点和抗腐蚀性,应用于航天、汽车和医药行业。金属铱(Ⅲ)离子可以与O^O,C^N和N^N的双齿配体形成稳定配合物,形成具有磷光发射特性的配合物,广泛应用于有机致电发光,生物荧光探针,化学传感器和催化合成等领域。在肿瘤治疗中,相比于经典金属配合物类的抗肿瘤药顺铂,金属铱(Ⅲ)配合物具有稳定性高、水溶性好,优异的磷光性能、配位点多、易于改造等特点。此外,根据其磷光寿命长、对氧气敏感的特点,使它可以作为光敏剂用于光动力治疗中。(参考文献:[1]高盼.金属铱(Ⅲ)配合物的功能设计及抗肿瘤作用机制的研究[D].暨南大学.)
现有技术中多以金属铱(Ⅲ)配合物作为抗肿瘤药物成分,如申请公布号CN107400146A的发明专利申请提供了一种抗肿瘤金属铱(Ⅲ)配合物及其制备方法和应用,该金属铱(Ⅲ)配合物可作用于A549细胞抑制其生长。然而,现有技术提供的金属铱(Ⅲ)配合物在用于光动力疗法时,其抗肿瘤活性仍有待提高。
发明内容
本发明所要解决的技术问题在于,提供一种金属铱(Ⅲ)配合物及其制备方法和应用。该金属铱(Ⅲ)配合物具有优异的光物理性质,低暗毒性和高光毒性,可作为光动力诊疗剂靶向非小细胞肺癌细胞,实现诊断和治疗的一体化。
为了解决上述问题,一方面,一种金属铱(Ⅲ)配合物,所述金属铱(Ⅲ)配合物包括[Ir(ppy)2(L)]+或[Ir(thpy)2(L)]+结构,其中,所述[Ir(ppy)2(L)]+的结构式如式(1)所示,所述[Ir(thpy)2(L)]+的结构式如式(2)所示:
进一步的,所述金属铱(Ⅲ)配合物的化学式为[Ir(ppy)2(L)]PF6或[Ir(thpy)2(L)]PF6。
另一方面,所述的金属铱(Ⅲ)配合物的制备方法,所述方法包括如下步骤:
步骤一、将4-二苯氨基苯甲醛和N-溴代琥珀酰亚胺按摩尔比为1:(2-3)的比例混合,室温搅拌反应20-30h,分离获得产物A;
步骤二、将所述产物A和4-吡啶硼酸以摩尔比为1:(3-3.5)的比例混合,加入钯催化剂、碳酸钾和1,4-二氧六环,于100-115℃下反应15-25h,提纯获得产物B;
步骤三、将所述产物B和1,10-邻菲罗啉-5,6-二酮、醋酸铵以摩尔比为1:(1-2):(25-35)的比例溶于冰醋酸中,加热至110-130℃回流3-5h,调pH至5.5-6.5,提纯获得主配体L;
步骤四、将所述主配体L与环金属铱氯桥前体以摩尔比为1:(0.4-0.6)的比例混合溶解,避光通氮气,60-70℃回流搅拌7-9h,冷却后加入六氟磷酸铵搅拌1-2h,抽滤,提纯获得所述金属铱(Ⅲ)配合物。
进一步的,所述的制备方法,所述环金属铱氯桥前体为[Ir(ppy)2Cl]2或[Ir(thpy)2Cl]2。
进一步的,所述的制备方法,所述步骤二中,所述钯催化剂、碳酸钾与产物A的摩尔比为(17-19):(2-4):1;和/或,
所述1,4-二氧六环的用量为(8-12)mL 1,4-二氧六环:1mmol产物A。
进一步的,所述的制备方法,所述步骤四中,所述六氟磷酸铵与主配体L的摩尔比为1:0.08。
在一种实施方式中,所述步骤一中,4-二苯氨基苯甲醛溶于四氢呋喃溶液中;所述步骤二中,钯催化剂为四(三苯基膦)钯;所述步骤四中,主配体L与环金属铱氯桥前体溶于DCM/MeOH=2:1中。
另一方面,一种光敏剂,包括所述的金属铱(Ⅲ)配合物。
另一方面,所述的金属铱(Ⅲ)配合物,和/或,所述的光敏剂在制备抗肿瘤药物中的应用;优选的,所述抗肿瘤药物包括适用于光动力疗法的药物。
进一步的,所述的应用,所述抗肿瘤药物适用的肿瘤细胞包括人宫颈癌细胞株HeLa、人非小细胞肺癌细胞株A549、人非小细胞肺癌细胞株A549R和人乳腺癌细胞株MCF-7。
进一步的,所述的应用,所述金属铱(Ⅲ)配合物定位于肿瘤细胞的线粒体和/或溶酶体;优选的,式(1)所示的结构式定位于线粒体,式(2)所示的结构式定位于溶酶体。
所述金属铱(Ⅲ)配合物作为光敏剂药物具有pH相应的磷光发光性质。该光敏剂药物具有较低的体外细胞暗毒性和较高的光毒性。
被细胞摄取后,该光敏剂药物能够定位于线粒体和溶酶体中,经光照后能有效得诱导胞内活性氧物种(ROS)水平上调和线粒体膜电位下降,上调的ROS水平可以对生物大分子通过氧化反应进行破坏损伤,线粒体膜电位下降可以导致线粒体功能损伤,进而导致肿瘤细胞凋亡。特别在肺癌细胞中具有高光毒性指数。
与现有技术相比,本发明的有益效果为:
1、本发明提供的新型磷光金属铱(Ⅲ)配合物可作为治疗癌症的光敏剂药物,该光敏剂药物具有pH响应的磷光发光性质,较低的体外细胞暗毒性和较高的光毒性,使得本申请金属铱(Ⅲ)配合物可实现光敏剂在光动力治疗过程中的诊断和治疗一体化。
2、本发明提供的新型磷光金属铱(Ⅲ)配合物相比传统光敏剂具有水溶性好,光稳定性高的优点。
3、本发明提供的新型磷光金属铱(Ⅲ)配合物被细胞主动摄取后,光敏剂药物能够定位于线粒体和溶酶体中,经光照后能有效诱导胞内ROS水平上调和线粒体膜电位下降,上调的ROS水平可以对生物大分子通过氧化反应进行破坏损伤,线粒体膜电位下降可以导致线粒体功能损伤,进而导致肿瘤细胞凋亡。
4、本发明提供的新型磷光金属铱(Ⅲ)配合物表现出优异的光动力疗效,特别是在人非小细胞肺癌细胞中具有非常高的光毒性指数,实验数据显示该光敏剂药物是一种肺癌靶向的高效光动力诊疗试剂。
附图说明
此处所说明的附图用来提供对本申请的进一步理解,构成本申请的一部分,本申请的示意性实施例及其说明用于解释本申请,并不构成对本申请的不当限定。在附图中:
图1是[Ir(ppy)2(L)]+合成路线图;
图2是[Ir(thpy)2(L)]+合成路线图;
图3是配体L的ESI-MS图谱;
图4是配体L的1H NMR图谱;
图5是配合物Ir-ppy的ESI-MS图谱;
图6是配合物Ir-ppy的1H NMR图谱;
图7是配合物Ir-thpy的ESI-MS图谱;
图8是配合物Ir-thpy的1H NMR图谱;
图9是配合物Ir-ppy的吸收和发射光谱图;
图10是配合物Ir-thpy的吸收和发射光谱图;
图11是配合物Ir-ppy的磷光pH响应图;
图12是配合物Ir-thpy的磷光pH响应图;
图13是HeLa细胞对配合物Ir-ppy摄取随时间的变化情况图;
图14是HeLa细胞对配合物Ir-thpy摄取随时间的变化情况图;
图15是配合物Ir-ppy和Ir-thpy与MTR(A)或LTR(B)在HeLa细胞中的共定位成像图;
图16是不同条件下HeLa细胞对配合物Ir-ppy的摄取情况图;
图17是不同条件下HeLa细胞对配合物Ir-thpy的摄取情况图;
图18是425nm光照条件下,配合物Ir-ppy、Ir-thpy在空气饱和磷酸氢二钠-柠檬酸缓冲液中对ABDA的光氧化。ABDA在波长为377nm(pH 3.0)、378nm(pH 5.0)和380nm(pH 6.4和7.4)处的吸光度随时间变化曲线;
图19是配合物Ir-ppy诱导细胞凋亡的流式细胞术结果图;
图20是配合物Ir-thpy诱导细胞凋亡的流式细胞术结果图;
图21是胞内ROS水平检测结果图;
图22是配合物Ir-ppy(A)和Ir-thpy(B)诱导线粒体膜电位变化结果图。
具体实施方式
为了更清楚的阐释本申请的整体构思,下面以实施例的方式进行详细说明。在下文的描述中,给出了大量具体的细节以便提供对本申请更为彻底的理解。然而,对于本领域技术人员来说显而易见的是,本申请可以无需一个或多个这些细节而得以实施。在其他的例子中,为了避免与本申请发生混淆,对于本领域公知的一些技术特征未进行描述。
实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。
如未特殊说明,在以下实施方式中,所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例中部分试剂如表1所示:
表1实施例所用部分试剂
其中:甲醇、二氯甲烷、石油醚、四氢呋喃、乙醇、乙酸、乙酸铵、磷酸氢二钠、磷酸二氢钾、氯化钾、氯化钠、碳酸钾和氘代试剂等均为市售;实验用水均为超纯水;电喷雾质谱(ESI-MS)采用美国Thermo LCQ-DECA-XP液相色谱-质谱联用仪获得;核磁共振氢谱(1HNMR)采用Bruker Avance 400spectrometer获得,采用TMS作为内标;核磁共振碳谱(13CNMR)采用Varian INOVA-500NB获得,采用TMS作为内标;元素分析(EA)采用德国Elementar公司Vario EL元素分析仪获得;紫外可见光谱(UV-Vis)采用美国Varian Cary 100UV/Vis分光光度计获得;荧光光谱采用爱丁堡FLS920稳态荧光光谱仪获得;多功能酶标仪采用德国Infinite M200 Pro;激光共聚焦采用德国Carl Zeiss LSM 710;流式细胞术采用美国BDFACS Calibur流式细胞仪测得。
实施例所用细胞系及培养条件:人宫颈癌细胞株(HeLa)、人肺癌细胞株(A549)、耐顺铂人肺癌细胞细胞株(A549R)、人乳腺癌细胞株(MCF-7)和人正常肝脏细胞株(LO2)均来自中山大学实验与动物中心。细胞使用含10%胎牛血清,且添加100U/mL青霉素和100μg/mL链霉素的DMEM(高糖型)或RPMI 1640培养基进行培养,放置于含5%CO2和95%空气的37℃湿润恒温细胞培养箱中。A549R使用添加顺铂的RPMI 1640培养基培养,以保持其对顺铂的耐药性。
实施例1药物的合成
(1)前体的合成:
环金属铱氯桥前体[Ir(ppy)2Cl]2和[Ir(thpy)2Cl]2采用文献方法[Journal ofthe American Chemical Society,2011,133(29):11231-9.]合成。
(2)配体的合成:
取10.93g(0.04mol)4-二苯氨基苯甲醛溶于100mL四氢呋喃,再加入18.5g(0.104mol)N-溴代琥珀酰亚胺,室温搅拌反应24h后终止反应,减压旋干溶剂后柱色谱分离(DCM/PE=1:1,v/v),得到亮黄色溴代产物9.1g,产率53%。
取溴代产物1.8g(0.004mol)和4-吡啶硼酸1.54g(0.0125mol),加入85mg(0.074mmol)四(三苯基膦)钯催化,再加入12mL溶有1.72g(0.012mol)碳酸钾的去离子水和1,4-二氧六环40mL,在氮气氛围中搅拌,110℃反应20h。过滤出去黑色固体,滤液减压旋蒸至少量后,用二氯甲烷萃取,有机相用水洗两遍,减压旋蒸至油状后加少量甲醇,静止过夜,大量黄色固体析出,抽滤后得1.6g产物A,产率90%。
取产物A860 mg(2.01mmol)与1,10-邻菲罗啉-5,6-二酮420mg(2.00mmol)以及4.6g(60mmol)醋酸铵,溶于20mL冰醋酸,加热至120℃回流4h。冷却至室温后加30mL水稀释,用浓氨水中和至pH为6左右,抽滤、水洗、乙醇洗。柱色谱提纯(DCM/MeOH=20:1),得橙黄色主配体(L)810mg,产率65%。
配体L的ESI-MS图谱如图3所示,配体L的1H NMR图谱如图4所示。
1H NMR(400MHz,DMSO)δ13.84(s,1H),9.04(d,J=7.4,3.7Hz,2H),9.01(d,J=8.0Hz,1H),8.93(d,J=7.6Hz,1H),8.63(d,J=4.6,1.6Hz,4H),8.33(t,J=16.7Hz,2H),7.86(d,J=8.7Hz,6H),7.72(d,J=4.6,1.6Hz,4H),7.35(d,J=8.7Hz,2H),7.27(d,J=8.7Hz,4H).ESI-MS(MeOH):m/z calcd for[M+H]+,618.24;found:618.45.Elementalanalysis calcd(%)for C41H27N7·CH3OH·CH3COOH:C,68.28;H,5.50;N,11.38;found:C,68.30;H,5.73;N,10.99.
(3)配合物[Ir(thpy)2(L)]PF6的合成
取主配体226mg(0.366mmol)与204mg(0.186mmol)[Ir(thpy)2Cl]2,溶于6mL DCM/MeOH=2:1,避光通氮气,65℃回流搅拌8h。冷却后加入600mg(3.68mmol)NH4PF6搅拌1h,抽滤,滤液减压旋蒸后柱色谱纯化(DCM/MeOH=10:1),得到黄色固体[Ir(thpy)2(L)]PF6(以下简称Ir-thpy)265mg,产率57%。
[Ir(thpy)2(L)]+合成路线图如图2所示。
配合物Ir-thpy的ESI-MS图谱如图7所示,配合物Ir-thpy的1H NMR图谱如图8所示。
1H NMR(400MHz,DMSO)δ14.75(s,1H),8.63(dd,J=4.6,1.6Hz,4H),8.39(d,J=8.7Hz,2H),8.14–8.07(m,4H),7.86(d,J=8.7Hz,4H),7.76(d,J=3.9Hz,4H),7.72(dd,J=7.7,3.1Hz,6H),7.44(d,J=5.9Hz,2H),7.34(d,J=8.8Hz,2H),7.28(d,J=8.7Hz,4H),6.81(dd,J=10.1,4.7Hz,2H),6.28(d,J=4.7Hz,2H),5.76(s,2H).ESI-MS(MeOH):m/zcalcd for[M-PF6]+,1130.24;found:1130.52.Elemental analysis calcd(%)forC59H39N9S2PF6Ir·CH3OH·H2O:C,54.37;H,3.42;N,9.51;found:C,54.82;H,3.67;N,9.58.
(4)配合物[Ir(ppy)2(L)]PF6的合成
用制备[Ir(thpy)2(L)]PF6同样方法得到[Ir(ppy)2(L)]PF6(以下简称Ir-ppy),淡黄色固体280mg,产率52%。
[Ir(ppy)2(L)]+合成路线图如图1所示。
配合物Ir-ppy的ESI-MS图谱如图5所示,配合物Ir-ppy的1HNMR图谱如图6所示。
1HNMR(400MHz,DMSO)δ15.00(s,1H),9.59(s,1H),9.16(s,1H),8.61(d,J=6.0Hz,4H),8.43(d,J=8.5Hz,2H),8.27(d,J=8.3Hz,2H),8.13(s,2H),8.07–8.01(m,2H),7.96(d,J=7.2Hz,2H),7.89(d,J=7.4Hz,2H),7.84(d,J=8.6Hz,4H),7.71(d,J=6.0Hz,4H),7.53(d,J=5.6Hz,2H),7.30(d,J=7.4Hz,2H),7.26(d,J=8.3Hz,4H),7.06(t,J=7.6Hz,2H),7.00(t,J=6.7Hz,2H),6.95(t,J=6.9Hz,2H),6.30(d,J=7.0Hz,2H).ESI-MS(MeOH):m/z calcd for[M-PF6]+,1118.32;found:1118.72.Elemental analysis calcd(%)forC63H43N9Ir·(CH3CH2)O·CH3OH·H2O:C,58.87;H,4.29;N,9.09;found:C,58.78;H,4.44;N,9.10.
实施例2配合物的光物理性质
脂水分配系数测定实验:
正辛醇和水的混合溶液在摇床上混合24小时,静置分层后取体积相等的两相溶液加入配合物,在摇床上混合48小时。静置分层,得到配合物的两相溶液,出去沉淀。用甲醇等浓度稀释两相分别测定紫外可见吸收值,通过式3计算lgPo/w值:
lgPo/w=lg(Ao/Aw) 式3
Ao及Aw分别为配合物特定波长下正辛醇及水相中的吸光度。
磷光寿命测定实验:
磷光寿命用英国爱丁堡公司生产的组合式荧光寿命与稳态荧光光谱仪检测。在室温条件下,分别将样品溶于3mL PBS缓冲液、乙腈和二氯甲烷中,测试浓度为10μM。用405nm激发,收集其在450-800nm范围内光谱数据。磷光数据进行拟合分析,用标准回归分析法,得到样品的最佳指数曲线。
磷光量子产率测定实验:
用三联吡啶钌作为标准物,Ru(bpy)3Cl2在PBS、CH2Cl2、CH3CN中的磷光量子产率分别为0.028、0.062、0.059,按照文献方法[Pure Appl.Chem.,Vol.83,No.12,pp.2213–2228,2011.]测得配合物的磷光量子产率,通过式4计算ΦPL值:
ΦPL(Ru)为三联吡啶钌的量子产率,I是配合物染料的积分面积,A是配合物和染料的吸光度值,η是溶剂的折射率。
通过前述方法合成了配体L和配合物Ir-ppy和Ir-thpy,经柱层析纯化后都通过了质谱、核磁和元素分析表征。
测试了室温条件下配合物Ir-ppy和Ir-thpy在PBS、乙腈和二氯甲烷中的紫外可见吸收光谱与磷光发射光谱,如图9和图10所示。
Ir-ppy和Ir-thpy的紫外可见吸收光谱中有两个主要的吸收峰,在250-330nm范围的吸收峰是自旋允许的配体内部电子跃迁(Intraligand Transition,1IL),在320-440nm范围的吸收峰是金属到配体的电荷跃迁(metal-to-ligand charge transfer,1MLCT)。两个配合物在二氯甲烷到乙腈再到PBS中的吸收强度是逐渐变小的,在这些溶剂中的稀溶液都呈浅黄色。
根据配合物的发射光谱来看,从二氯甲烷到乙腈再到PBS,配合物的最大发射波长有所红移,Ir-ppy在PBS中的发射强度较高于乙腈中的,而Ir-thpy在PBS中发射强度比在乙腈中要小。配合物的光物理数据总结于表2。
表2室温条件下配合物Ir-ppy和Ir-thpy的光物理数据
通过测试在不同pH的磷酸氢二钠-柠檬酸缓冲液中Ir-ppy和Ir-thpy的磷光强度,结果如图11和图12所示,Ir-ppy和Ir-thpy在酸性条件下有较强的磷光,而在偏碱性条件下磷光强度是随碱性增强而逐渐减弱的。在pH﹤5.6的酸性条件下,磷光变化并不规律,但仍保持在一定强度,在pH﹥5.6的条件下随碱性增强磷光强度规律性地减小。
实施例3配合物的细胞摄取时间依赖试验
研究细胞对配合物的摄取速率,可以给接下来一系列细胞实验配合物孵育时间的确定提供参考。
细胞摄取时间依赖实验步骤:HeLa细胞接种在35mm的共聚焦皿中培养24小时,加入20μM配合物分别孵育不同的时间(1h、2h、4h和6h)后,PBS洗涤两次,立刻用激光共聚焦显微镜观察,采用405nm激发,收集发射波长:620±30nm(Ir-ppy);640±30nm(Ir-thpy)。
由细胞摄取时间依赖的激光共聚焦显微镜成像结果,如图13和图14可知,在Ir-ppy或Ir-thpy处理1h后,HeLa细胞中磷光强度都非常弱。在Ir-ppy处理2h后,可以观察到细胞中已有明显的磷光,而在Ir-thpy处理2h后胞内磷光强度还是较弱的,这种差异性可能来自于它们亲脂性的差距,Ir-ppy对应的lgPo/w为2.71,略高于Ir-thpy对应的2.66,即Ir-ppy亲脂性略高于Ir-thpy。
实施例4配合物的细胞内定位
细胞内定位实验步骤:HeLa细胞接种在35mm的共聚焦皿中培养24小时,将细胞用20μM配合物孵育4小时,再分别用200nM溶酶体探针LTR和100nM线粒体探针MTR孵育30min,PBS洗涤两次,立刻用激光共聚焦显微镜观察。配合物用405nm激发,LTR和MTR用543nm激发,收集发射波长:620±30nm(Ir-ppy);640±30nm(Ir-thpy)590±30nm(LTR);599±30nm(MTR)。
为研究配合物在细胞内中的分布情况,选用商用的线粒体红色探针(MTR)和溶酶体红色探针(LTR)分别对HeLa细胞的线粒体和溶酶体染色,利用共聚焦显微镜观察比较配合物的磷光发射和探针的荧光发射,结果如图15所示。由Image-Pro Plus 6.0软件计算的共定位系数和皮尔森相关系数可知,配合物Ir-ppy和Ir-thpy在线粒体、溶酶体都有分布,Ir-ppy较好地定位于线粒体,Ir-thpy较好地定位于溶酶体。也可以从图中看出两个配合物基本不进入细胞核中,几乎全部分布在细胞质中。
实施例5配合物的细胞摄取机制
细胞摄取机制实验步骤:HeLa细胞接种在35mm的共聚焦皿中培养24小时,将细胞分别在4℃、37℃加入20μM CCCP或37℃加入100μM氯喹中预处理20min,再分别加入20μM配合物孵育4小时,PBS洗涤两次,立刻用激光共聚焦显微镜观察,用405nm激发,收集发射波长:620±30nm(Ir-ppy);640±30nm(Ir-thpy)。
用低的孵育温度(4℃)或使用能损害线粒体功能,进而影响能量供给的CCCP对细胞进行预处理,能明显降低细胞对配合物Ir-ppy和Ir-thpy的摄取效率。用内吞抑制剂氯喹对细胞进行预处理,几乎没有影响细胞对配合物的摄取。结果如图16和图17所示,这表明配合物Ir-ppy和Ir-thpy是通过非内吞的能量依赖的途径进入细胞,即通过主动运输进入细胞。
实施例6配合物水环境中的单线态氧量子产率
单线态氧作为PDT过程中杀伤癌细胞的主要活性氧物种,其量子产率(ΦΔ)是衡量光敏剂性能的重要指标。
单线态氧量子产率测定实验步骤:以ABDA作为单线态氧探针,[Ru(bpy)3]Cl2作为标准参照物,其在空气饱和的水中的单线态氧产率(ΦΔ)为0.18。首先将含待测样品或参照物和ABDA(100μM)的缓冲溶液在黑暗中通气10min使其与空气平衡,然后使用波长为425nm的LED灯(40mW·cm-2)进行光照。每光照2s记录一次ABDA在波长为377nm(pH 3.0)、378nm(pH5.0)和380nm(pH 6.4和7.4)处的紫外吸光度。待测样品和参照物[Ru(bpy)3]Cl2在波长425nm处的紫外吸光度需调节在0.15左右。待测样品的单线态氧量子产率根据式5计算得到:
其中,下标x和std分别代表待测样品和标准参照物[Ru(bpy)3]Cl2。S是ABDA在波长为377nm(pH 3.0)、378nm(pH 5.0)和380nm(pH 6.4和7.4)处的吸光度随时间变化曲线的斜率,F是吸收校正因子(F=1-10-OD,OD表示样品和[Ru(bpy)3]Cl2在425nm处的光密度)。
根据以上方法以ABDA作为单线态氧指示剂,[Ru(bpy)3]Cl2作为标准参照物,测定了配合物Ir-ppy和Ir-thpy在不同pH的磷酸氢二钠-柠檬酸缓冲液中的单线态氧量子产率,结果如图18和表3所示。
表3不同pH条件下Ir-ppy和Ir-thpy的单线态氧量子产率
可以发现,相比于[Ru(bpy)3]Cl2配合物Ir-ppy和Ir-thpy在不同pH条件下产生单线态氧的能力具有非常显著的差距,在酸性条件下有较高的单线态量子产率,且随pH降低ΦΔ值明显升高。其中配合物Ir-ppy在pH 7.4时ΦΔ值只有0.02,到pH 3.0时升高至0.60,即提高至30倍。数据表明同样pH条件下Ir-ppy产生单线态氧的能力比Ir-thpy高。
实施例7配合物的细胞光、暗毒性
细胞毒性实验步骤:细胞毒性采用MTT法测定,当细胞培养传代3、4次后长至对数期时,将细胞用0.25%胰蛋白酶消化成单细胞悬液,用血球计数板对活细胞进行计数,调整活细胞浓度为5×104/mL接种于96孔板,每孔160μL。在细胞贴壁后,分别加入用培养基稀释的不同浓度药物,然后在培养箱中孵育48小时,或在加药12小时后用425nm的LED光源(40mW·cm-2)照射15min(36J·cm-2),再继续培养36小时。在结束前4小时每孔加入20μL的MTT(5mg/mL),4小时吸去培养基,每孔加入150μL的DMSO,于摇床上震荡10分钟左右,然后利用酶标仪测定570nm处的OD值。按照式6计算存活率,同时作图并求得半数杀伤浓度(IC50),以没有加入药物的孔的细胞存活率为100%作为参照,评价药物的细胞毒性。
细胞毒性采用MTT法测定,对人宫颈癌细胞株(HeLa)、人肺癌细胞株(A549)、耐顺铂人肺癌细胞细胞株(A549R)、人乳腺癌细胞株(MCF-7)和人正常肝脏细胞株(LO2)进行了光毒性和暗毒性的测试,IC50结果如表4所示。
表4配合物Ir-ppy和Ir-thpy对不同细胞株的光、暗毒性IC50值
数据显示配合物Ir-ppy和Ir-thpy都具有相对较低的暗毒性和较高的光毒性,对实验细胞都有较高的光毒性指数,而作为对照的顺铂对实验细胞的光暗、毒性都非常接近。特别地,配合物Ir-ppy和Ir-thpy对A549和A549R有更为显著的光毒性,光毒性指数高达几千,其中对Ir-thpyA549的光毒性指数达到了4000以上,有机会发展成为高效的光动力治疗试剂。
实施例8PDT诱导细胞凋亡
Annexin Ⅴ/PI双染检测细胞凋亡实验步骤:本实验使用BeyotimeBiotechnology公司的Annexin Ⅴ-FITC凋亡检测试剂盒,按说明书方法进行实验。HeLa细胞接种在六孔板中,待密度长到70%左右时用不同浓度(0.2、0.4、0.8μM)的配合物处理12小时后,用波长为425nm的LED光源(40mW·cm-2)照射15min(36J·cm-2),继续孵育12小时。用胰酶消化细胞,PBS洗涤两次。用500μL结合缓冲液重悬细胞,细胞用5μL Annexin Ⅴ和10μL PI 37℃避光孵育20min,立即用流式细胞仪检测。
用Annexin Ⅴ/PI双染和流式细胞术检测被不同浓度配合物处理后的HeLa细胞凋亡情况,结果如图19和图20所示。正常情况下,磷脂酰丝氨酸位于细胞膜内侧,而在细胞发生早期凋亡时会外翻到细胞膜的外侧,Annexin Ⅴ对磷脂酰丝氨酸具有很高的亲和能力,可以利用Annexin Ⅴ-FITC染色来检测早期凋亡细胞。PI可以进入晚期凋亡或坏死的细胞中,对其细胞核进行染色,于是用Annexin Ⅴ/PI双染可区分早期凋亡和晚期凋亡或坏死细胞。配合物孵育12小时后用波长为425nm的LED光源(40mW·cm-2)照射15min(36J·cm-2),可以看到大量的凋亡早期和晚期的细胞,并且随浓度的升高细胞存活率有所降低,晚期凋亡细胞明显增多。通过与顺铂做对比,50μM的顺铂诱导细胞凋亡的能力不及光照后0.2μM的配合物Ir-ppy或Ir-thpy,可以证明配合物Ir-ppy和Ir-thpy杀伤细胞的能力比顺铂强很多。
实施例9PDT诱导细胞内活性氧(ROS)水平升高
细胞内活性氧(ROS)水平测定实验步骤:HeLa细胞接种在六孔板中,待密度长到70%左右时用不同浓度(0.4、0.8、1.2μM)的配合物处理6小时后,用波长为425nm的LED光源(40mW·cm-2)照射15min(36J·cm-2)。胰酶消化并收集细胞,加入10μM DCFH-DA的无血清培养基37℃避光孵育20min,离心除去上清液,再用不含血清的培养基洗涤两次,并尽快用流式细胞仪检测绿色荧光强度。488nm激发,测定范围530±15nm。每个样品分析10,000个细胞。数据用FlowJo V10软件分析。
ROS可以杀伤癌细胞,是光动力治疗过程中扮演着重要的角色。DCFH-DA是可以穿透细胞膜的染料,自身不发荧光,细胞内的ROS可将其氧化成具有强荧光的DCFH,于是可以通过检测DCFH的荧光强度评估细胞内的ROS水平。由图21可以看出,用不同浓度配合物处理6小时后,再用波长为425nm的LED光源(40mW·cm-2)照射15min(36J·cm-2),可以显著地提高细胞内ROS的水平,且具有浓度依赖性。配合物Ir-ppy的效果好于配合物Ir-thpy,这与水环境中单线态氧量子产率的结果相吻合。DCFH平均荧光强度(MFI)如下述所示。Lightonly:77.2;Ir-ppy,0.4μM,light:299;Ir-ppy,0.8μM,light:872;Ir-ppy,1.2μM,light:2107(图21左)。Light only:76.2;Ir-thpy,0.8μM,light:254;Ir-thpy,0.4μM,light:581;Ir-thpy,0.8μM,light:1340(图21右)。
实施例9PDT诱导线粒体膜电位(MMP)下降
线粒体膜电位(MMP)测定实验步骤:本实验使用Beyotime Biotechnology公司的线粒体膜电位检测试剂盒,按说明书方法进行实验。HeLa细胞接种在六孔板中,待密度长到70%左右时用不同浓度(0.4、0.8μM)的配合物处理6小时后,用波长为425nm的LED光源(40mW·cm-2)照射15min(36J·cm-2)。继续孵育3小时,胰酶消化并收集细胞,用含有5μg/mLJC-1的稀释好的结合液重悬细胞,37℃染色15min。用不含JC-1的稀释好的结合液洗涤两次,立即用流式细胞仪检测。绿色荧光用488nm激发,测定范围为530±15nm;红色荧光用488nm激发,测定范围为585±20nm。每个样品分析10,000个细胞。数据用FlowJo V10软件分析。
线粒体功膜电位下降与细胞凋亡有很密切的关系,我们用JC-1对细胞进行染色后通过流式细胞术检测线粒体膜电位的变化。JC-1是一种可以特异性标记线粒体膜电位的荧光探针,线粒体的去极化会导致JC-1聚集体减少及单体增加,表现为红色荧光强度降低和绿色荧光强度上升。由图22可见,单独光照和只加入配合物不光照的流式细胞术结果与暗对照基本无差别,而加入配合物且光照的组别红色荧光强度和绿色荧光强度各自有明显降低和上升,这说明配合物Ir-ppy和Ir-thpy能通过PDT诱导线粒体膜电位下降。但在实验浓度(0.4μM和0.8μM)下,配合物均没有表现出浓度依赖的线粒体膜电位变化。平均红色荧光强度与平均绿色荧光强度的比值,即MFI(R)/MFI(G)如后述所示。Control,dark:23.04;Control,light:23.07;Ir-ppy,0.8μM,dark:23.00;Ir-ppy,0.4μM,light:1.52;Ir-ppy,0.8μM,,light:1.58;Ir-thpy,0.8μM,dark:23.53;Ir-thpy,0.4μM,light:1.50;Ir-thpy,0.8μM,light:1.49。
以上所述仅为本申请的实施例而已,并不用于限制本申请。对于本领域技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等,均应包含在本申请的权利要求范围之内。
Claims (10)
2.根据权利要求1所述的金属铱(Ⅲ)配合物,其特征在于,所述金属铱(Ⅲ)配合物的化学式为[Ir(ppy)2(L)]PF6或[Ir(thpy)2(L)]PF6。
3.一种如权利要求1或2所述的金属铱(Ⅲ)配合物的制备方法,其特征在于,所述方法包括如下步骤:
步骤一、将4-二苯氨基苯甲醛和N-溴代琥珀酰亚胺按摩尔比为1:(2-3)的比例混合,室温搅拌反应20-30h,分离获得产物A;
步骤二、将所述产物A和4-吡啶硼酸以摩尔比为1:(3-3.5)的比例混合,加入钯催化剂、碳酸钾和1,4-二氧六环,于100-115℃下反应15-25h,提纯获得产物B;
步骤三、将所述产物B和1,10-邻菲罗啉-5,6-二酮、醋酸铵以摩尔比为1:(1-2):(25-35)的比例溶于冰醋酸中,加热至110-130℃回流3-5h,调pH至5.5-6.5,提纯获得主配体L;
步骤四、将所述主配体L与环金属铱氯桥前体以摩尔比为1:(0.4-0.6)的比例混合溶解,避光通氮气,60-70℃回流搅拌7-9h,冷却后加入六氟磷酸铵搅拌1-2h,抽滤,提纯获得所述金属铱(Ⅲ)配合物。
4.根据权利要求3所述的制备方法,其特征在于,所述环金属铱氯桥前体为[Ir(ppy)2Cl]2或[Ir(thpy)2Cl]2。
5.根据权利要求3所述的制备方法,其特征在于,所述步骤二中,所述钯催化剂、碳酸钾与产物A的摩尔比为(17-19):(2-4):1;和/或,所述1,4-二氧六环的用量为(8-12)mL 1,4-二氧六环:1mmol产物A。
6.根据权利要求3所述的制备方法,其特征在于,所述步骤四中,所述六氟磷酸铵与主配体L的摩尔比为1:0.08。
7.一种光敏剂,包括如权利要求1或2所述的金属铱(Ⅲ)配合物。
8.如权利要求1或2所述的金属铱(Ⅲ)配合物,和/或,如权利要求7所述的光敏剂在制备抗肿瘤药物中的应用;优选的,所述抗肿瘤药物包括适用于光动力疗法的药物。
9.根据权利要求8所述的应用,其特征在于,所述抗肿瘤药物适用的肿瘤细胞包括人宫颈癌细胞株HeLa、人非小细胞肺癌细胞株A549、人非小细胞肺癌细胞株A549R和人乳腺癌细胞株MCF-7。
10.根据权利要求8所述的应用,其特征在于,所述金属铱(Ⅲ)配合物定位于肿瘤细胞的线粒体和/或溶酶体;优选的,式(1)所示的结构式定位于线粒体,式(2)所示的结构式定位于溶酶体。
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