CN111592482A - 一种pH可逆激活型光热/光动力/荧光一体化探针分子 - Google Patents
一种pH可逆激活型光热/光动力/荧光一体化探针分子 Download PDFInfo
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- CN111592482A CN111592482A CN202010415029.1A CN202010415029A CN111592482A CN 111592482 A CN111592482 A CN 111592482A CN 202010415029 A CN202010415029 A CN 202010415029A CN 111592482 A CN111592482 A CN 111592482A
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种pH可逆激活型光热/光动力/荧光一体化探针分子,属于生物医药技术领域。本发明以pH响应型不对称菁类结构为核心单元,通过结构修饰及在共轭体系中引入卤素等重原子,构建可被肿瘤弱酸性微环境特异性激活的光热/光动力/荧光一体化探针分子。本发明光热/光动力/荧光一体化探针具有良好的稳定性、光热效果以及优越的产生活性氧的能力,在肿瘤治疗方面具有巨大潜力。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种pH可逆激活型光热/光动力/荧光一体化探针分子。
背景技术
癌症的早期诊治是提高癌症患者治愈率的关键,研发特异性强、灵敏度高的检测技术用于肿瘤早期诊断是十分必要的。光学成像技术具有非侵袭性、实时、灵敏度高、操作简单以及可视性强等诸多优势,已成为肿瘤检测的理想方法。尤其是近红外荧光成像技术,因其吸收和发射波长均处于生物光学成像窗口内,具有组织穿透能力强、生物组织的光吸收及自发荧光干扰小等优势而倍受青睐。但传统的成像探针大多为“always on”型显影剂,一直处于荧光激活状态的显影在体内代谢转运过程中始终显示出荧光信号,易造成自身背景干扰及“假阳性”结果等问题。
光线治疗是一种温和的、局部的、相对安全的治疗模式,它本身无明显的生物毒性,外界光照射下才产生特异性杀伤效果,在肿瘤精准治疗方面展现了良好的应用前景。根据外界光照射光热/光敏剂后将光能转化成热能或活性氧的不同,又可分为光热治疗(Photothermal therapy,PTT)及光动力治疗(Photodynamic therapy,PDT)。近年来,已有将PDT和PTT等新型疗法相结合,实现联合治疗的报道,并取得了比单独治疗更好的治疗效果。但现有研究中光热/光动力治疗所用光敏剂大多为两类不同的材料的组合,且大多为“always on”型光热/光敏剂,因其理化性质的差异,易造成两类材料在体内分布和对靶向区域的选择并不完全相同,因此很难实现真正的精准的联合治疗;亦或需要两种波长的激发光刺激,延长了治疗时间,增加了副作用及操作难度;同时,对正常在组织的非特异性损伤亦无法避免。
发明内容
为了解决以上问题,我们设计合成了肿瘤微环境特异性激活型兼具光热、光动力以及荧光多功能于一体的新型探针分子,并将其用于肿瘤精准成像指引的光热/光动力联合治疗。本发明以pH响应型不对称菁类结构为核心单元,通过结构修饰及在共轭体系中引入卤素等重原子,构建可被肿瘤弱酸性微环境特异性激活的光热/光动力/荧光一体化探针分子,提供一种可被肿瘤弱酸性微环境特异性激活的光热/光动力/荧光一体化探针分子及其制备方法和应用。
包括如下技术方案:
一种用作pH响应型光热/光动力/荧光一体化探针分子的化合物,其结构如式(I)所示:
在本发明的一种实施方式中,所述的pH响应型光热/光动力/荧光一体化探针分子的制备方法,其特征在于:包括以下步骤:
(2)以化合物2为起始物与4-溴甲基苯甲酸甲酯反应得到化合物3:
(3)利用化合物2’、3、4反应得到式(I)化合物:
在本发明的一种实施方式中,所述制备方法具体包括如下步骤:
步骤(1):化合物1与3-甲基-2-丁酮的摩尔比为摩尔比为1:l-2,加热到100℃,反应2h,冷却至室温后,用乙酸乙酯萃取,浓缩,向所得红色液体中加入20mL冰醋酸,回流反应5h,冷却至室温,加入50mL二氯甲烷,用饱和的碳酸钠水溶液中和冰醋酸,二氯甲烷萃取,浓缩,柱层析分离,得到化合物2;
步骤(2):化合物2与4-溴甲基苯甲酸甲酯的摩尔比为1:1-2,100℃下搅拌5h,冷却至室温,再向反应体系中加入30mL NaOH溶液,室温下搅拌1h;用二氯甲烷萃取,旋蒸浓缩,后经洗脱剂先为石油醚,后为石油醚:乙酸乙酷=40:1(V/V)柱层析分离得到化合物3;
步骤(3):化合物2’,3,4的摩尔比为1:1:1,溶剂N,N-二甲基甲酰胺,在氮气保护下,加热至50℃反应5h。冷却到室温后,用水洗涤,二氯甲烷萃取,浓缩,经柱层析分离,洗脱剂先为石油醚:乙酸乙酷=10:1(V/V),后为石油醚:乙酸乙酷=2:1(V/V),得到式(I)化合物。
在本发明的一种实施方式中,所述化合物4的制备方法包括如下过程:先将N,N-二甲基甲酰胺在冰盐浴冷却至0℃以下,滴加三氯氧磷和二氯甲烷的混合液,继续搅拌0.5h;再缓慢滴加环己酮,滴毕撤去冰浴,将反应液加热到80℃,反应4h后,冷却至室温,丙酮洗涤,得到化合物4;其中,三氯氧磷和环己酮的摩尔比为1:(1-2)。
本发明的第二个目的是提供一种pH响应型的光热/光动力/荧光一体化探针分子,所述pH响应型的光热/光动力/荧光一体化探针分子为式(1)所示结构的化合物,其中R1、R2分别为式(1)中定义的取代基。
本发明的第三个目的是将上述的化合物或者pH响应型光热/光动力/荧光一体化探针分子应用于生物成像领域中。
本发明的第四个目的是提供一种活细胞成像或者活体成像的方法,所述方法是利用上述的化合物作为探针。所述的细胞具体可以包括癌细胞如肺癌细胞A549、小鼠鳞状细胞癌细胞SCC-7等。
本发明的第五个目的是将上述的化合物用于制备治疗肿瘤的药物中。
在本发明的一种实施方式中,所述药物的还包括药物载体和/或药用辅料。
在本发明的一种实施方式中,所述药物的剂型包括注射液、注射用冻干粉针、控释注射剂、脂质体注射剂、混悬剂、植入剂、栓塞剂、胶囊剂、片剂、丸剂和口服液。
在本发明的一种实施方式中,所述药物载体包括微囊、微球、纳米粒和脂质体。
在本发明的一种实施方式中,所述药用辅料包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂以及释放阻滞剂。
在本发明的一种实施方式中,所述药用辅料包含微晶纤维素、羟丙基甲基纤维素以及精制卵磷脂。
本发明的显著优点在于:
本发明以具有pH响应性能的不对称菁类结构为核心单元,通过结构修饰及在共轭体系中引入卤素等重原子,构建得到可被肿瘤弱酸性微环境特异性激活的光热/光动力/荧光一体化探针分子。
本发明探针分子化合物可被肿瘤弱酸性微环境特异性激活,提高肿瘤诊断的特异性;兼具有光热/光动力联合治疗效果;目标化合物结构单一,不存在异构体,产物容易纯化。
本发明提供的pH可逆激活的光热/光动力/荧光一体化探针分子结构新颖,具有pH响应性能,弱酸性微环境激活型近红外吸收发射,能有效的避开生物自发荧光和细胞内源性物质的干扰,特异性强、灵敏度高、光学稳定性好、能够作为检测生物成像和光热/光动力治疗的探针。该荧光分子探针在分析化学、生命科学等领域具有较强的实际应用价值。
附图说明
图1是实施例1所得探针分子的1H NMR谱图;
图2是实施例1所得探针分子的质谱谱图;
图3是实施例1所得探针分子在不同pH值下的紫外吸收光谱和荧光发射光谱图;
图4是实施例1所得探针分子的光热效果图;
图5是实施例1所得探针分子的产生单线态氧能力图;
图6是实施例1所得探针分子的细胞毒性图;
图7是实施例1所得探针分子的体外癌细胞杀伤效果图。
具体实施方式
pH响应型光热/光动力/荧光一体化探针分子的制备方法,包括以下步骤:
步骤(1)中,化合物1与3-甲基-2-丁酮的摩尔比为摩尔比为1:l-2,加热到100℃,反应2h,冷却至室温用乙酸乙酯萃取,浓缩,向所得红色液体中加入20mL冰醋酸,回流反应5h,冷却至室温,加入50mL二氯甲烷,用饱和的碳酸钠水溶液中和冰醋酸,二氯甲烷萃取,浓缩,柱层析分离,得到化合物2,产率为78-82%。
步骤(2)的制备过程包括:化合物2与4-溴甲基苯甲酸甲酯的摩尔比为1:1-2,100℃下搅拌5h,冷却至室温,再向反应体系中加入30mL NaOH溶液,室温下搅拌1h。用二氯甲烷萃取,旋蒸浓缩。经柱层析分离,洗脱剂先为石油醚,后为石油醚:乙酸乙酯=40:1(V/V)得到化合物3,产率为70-75%。
步骤(3)的制备过程包括:三氯氧磷和环己酮的摩尔比为1:1-2,先将N,N-二甲基甲酰胺加入250mL两口瓶中磁力搅拌,冰盐浴冷却至0℃以下,滴加三氯氧磷和二氯甲烷的混合液,继续搅拌0.5h。再缓慢滴加环己酮,滴毕撤去冰浴,将反应液加热到80℃,反应4h。冷却至室温,丙酮洗涤,得到化合物4,产率为85-89%。
步骤(4)的制备过程包括:化合物2,3,4的摩尔比为1:1:1,溶剂N,N-二甲基甲酰胺,在氮气保护下,加热至50℃反应5h。冷却到室温,用水洗涤,二氯甲烷萃取,浓缩,经柱层析分离,洗脱剂先为石油醚:乙酸乙酷=10:1(V/V),后为石油醚:乙酸乙酯=2:1(V/V),得到式(I)化合物,产率为33-40%。
pH响应型光热/光动力/荧光一体化探针的应用性能评价,包括以下步骤:
主要研究内容包括探针的光学性质、光热转换能力、产生单线态氧的能力、细胞毒性、细胞杀伤效果评价。
分子的光学性质表征:准确称取探针分子配制浓度为10-3M的乙醇储备液。用溶剂为乙醇:水=1:1(体积比)稀释为10-5M待测液,然后分别用NaOH(1M)和HCl(1M)溶液将待测液pH调为7.4(模拟正常体液)和6.0(模拟肿瘤弱酸性微环境)。分别用紫外分光光度计和荧光光谱仪测定其吸收和发射光谱(激发波长为探针的最大发射波长)。
光热转换能力测定:取0.8mL上述待测液(1×10-5M)(pH分别为6.0和7.4)分别置于1.5mL离心管中,用不同功率(0.2、0.4、0.6、0.8和1.0w cm-2)808nm激光器照射10min,用热成像仪每间隔30s采集一次热成像图片并记录温度,绘制温度随时间的变化曲线。
产单线态氧能力测定:取19.9mL浓度为6×10-5M的DPBF溶液和100uL浓度为10-3M的上述储备液于50mL离心管中混合摇匀。一式两份,并将其pH分别调节至7.4和6.0,用锡箔纸包裹避光保存。将该待测液分装于1.5mL离心管中(每管1mL)。用功率为0.6w cm-2的808nm激光器照射不同时间(0s、10s、20s、30s、40s、50s、1min、1.5min、2min、3min和5min)。用紫外-可见分光光度计测定照射后溶液的吸收光度。
细胞毒性实验(细胞存活率MTT法测定):采用MTT法进行生物相容性测定。简而言之,向96孔孔板中加入1×105cell/mL细胞悬液(100μL/孔)并生长过夜。然后将培养基取出,加入100μL含不同浓度的探针(2×10-6,5×10-6,8×10-6,1×10-5M)的新鲜培养基,每组平行5次,继续孵育24小时。去除培养基,PBS洗涤,加入100μL含0.5mg mL-1MTT的新鲜培养基,再培养4h。吸出孔内液体,PBS洗涤一次,加入100μL DMSO,置于37℃恒温摇床避光震荡30min。用酶标仪测570nm波长下的吸光度(A570)。细胞存活率计算公式如下:细胞存活率=实验组A570/对照组A570×100%。
细胞光热/光动力杀伤效果评价:细胞培养基孵育同上述细胞毒性试验。待细胞与探针孵育后调节培养体系pH为6.0或7.4,用不同功率的808nm激光器照射10min,然后再孵育12h。12h后用PBS洗一次并换上100uL含0.5mg mL-1MTT的新鲜培养基继续培养4h。去除孔内液体,PBS洗涤,加入100uL DMSO,置于37℃恒温摇床避光震荡30min。用酶标仪测570nm波长下的吸光度。
以下几个实施例进一步阐述本发明,但是本发明不仅限于此。
实施例1一种兼具光热/光动力的探针分子的制备(式(I)化合物:R1=Br,R2=NO2)
1)将9.37g(50.12mmol)对溴苯肼加入到100mL圆底烧瓶中,搅拌下缓慢滴加4.41g(51.20mmol)3-甲基-2-丁酮,加热到100℃,反应2h,冷却至室温后,用乙酸乙酯萃取反应液,分去水层,合并有机相,加入无水硫酸镁干燥,过滤,浓缩,得到棕红色液体;向所得红色液体中加入20mL冰醋酸,回流反应5h,冷却至室温,加入50mL二氯甲烷,用饱和碳酸钠水溶液中和冰醋酸,二氯甲烷萃取,浓缩,柱层析分离,得到化合物2(R1=Br)。
化合物2(R1=Br)的结构表征:1H NMR(400MHz,DMSO-d6)δ(ppm):7.68(d,J=1.6Hz,1H),7.45(dd,J1=8.4Hz,J2=2.0Hz,1H),7.37(d,J=8.0Hz,1H),2.20(s,3H),1.26(s,6H).
13C NMR(100MHz,DMSO-d6)δ(ppm):189.17,153.21,149.03,130.66,125.49,121.50,118.30,54.34,22.68,15.52.
MS(ESI Positive),m/z:calculated for C11H12BrN,[M+H+]238.02,found238.10.
2)9.49g(49.94mmol)硝基苯肼和4.39g(51.02mmol)3-甲基-2-丁酮经上述相同步骤得到化合物2’(R2=NO2)。
3)将9.51g(39.96mmol)化合物2(R1=Br)及12.60g(55.04mmol)4-溴甲基苯甲酸甲酯加入50mL单口瓶中,100℃下搅拌5h,冷却至室温,得红色稠状液体。向反应体系中加入30mL NaOH溶液,室温下搅拌1h。二氯甲烷萃取,旋蒸浓缩,柱层析分离(洗脱剂先为石油醚,后为石油醚:乙酸乙酯=40:1(V/V))得到化合物3。
化合物3的结构表征:1H NMR(400MHz,CDCl3)δ(ppm):7.99(d,J=8.4Hz,2H),7.28(d,J=8.4Hz,2H),7.24(d,J=2.0Hz,1H),7.19(dd,J1=8.4Hz,J2=2.0Hz,1H),3.92(s,3H),3.89(s,2H),1.41(s,6H)(Supplementary Fig.6).
13C NMR(400MHz,CDCl3)δ(ppm):166.49,160.73,144.86,142.28,139.74,130.40,130.10,129.28,126.49,125.39,111.17,106.79,75.85,51.97,45.90,44.42,30.07(Supplementary Fig.7).
MS(ESI Positive),m/z:calculated for C20H20BrNO2,[M+H+]386.0750,found386.00
4)将23.1mL(300.0mmol)N,N-二甲基甲酰胺加入250mL两口瓶中磁力搅拌,冰盐浴冷却至0℃以下,滴加13.8mL(150.0mmol)三氯氧磷和5mL二氯甲烷的混合液,继续搅拌0.5h。再缓慢滴加4.90g(50.00mmol)环己酮,滴毕撤去冰浴,将反应液加热到80℃,反应4h后,冷却至室温,丙酮洗涤,得到化合物4。
5)将1.16g(2.99mmol)化合物3、0.52g(3.00mmol)化合物4和0.57g(3.00mmol)化合物2’(R2=NO2)溶于30mL N,N-二甲基甲酰胺中,在氮气保护下,加热至50℃反应5h。冷却到室温,水洗涤,二氯甲烷萃取,浓缩,经柱层析分离,洗脱剂先为石油醚:乙酸乙酯=10:1(V/V),后为石油醚:乙酸乙酯=2:1(V/V),得到式(I)化合物。
式(I)化合物的结构表征:1H NMR(400MHz,CDCl3)δ(ppm):8.03(d,J=8.0Hz,3H),7.43(d,J=8.0Hz,2H),7.31-7.30(m,2H),7.25(dd,J1=8.0Hz,J2=1.6Hz,1H),6.69-6.45(m,3H),6.45(d,J=8.4Hz,1H),5.44(d,J=12.8Hz,1H),3.93(s,3H),2.54(d,J=6.4Hz,2H),2.38(d,J=6.4Hz,2H),2.24(s,1H),2.07(s,1H),1.82-1.76(m,2H),1.72(s,6H),1.48(s,6H)(图谱见图1)。
MS(ESI Positive),m/z:calculated for C39H37BrClN3O4,[M+H+]726.17,found726.20(图谱见图2)。
实施例2探针分子的性能评价
对实施例1所得的式(I)化合物(R1=Br,R2=NO2)的光热/光动力抗癌性能进行了探索。
主要研究内容包括探针的光学性质、光热转换能力、产生单线态氧的能力、细胞毒性、细胞杀伤效果评价。光敏剂的细胞毒性实验采用MTT法测定。
1)光学性质考察:
测定方法:准确称取7.26mg的式(I)化合物(R1=Br,R2=NO2)用乙醇溶解,定容于10mL容量瓶,配制浓度为10-3M的储备液。用溶剂为乙醇:水=1:1(体积比)稀释为10-5M,然后用pH计将溶液分别调为7.4和6.0。用紫外分光光度计测定其吸收光谱和荧光光谱仪扫描发射光谱(激发波长为780nm)。
结果如图3所示,目标探针在pH 7.4条件下在520nm处出现特征吸收峰,并无荧光发射。而当溶液转为弱酸性时原有520nm处特征吸收峰明显降低,780nm处出现很强的特征吸收,同时伴随着溶液颜色由红色向浅绿色的转变。此外,酸性条件下探针的荧光亦被特异性激活,在810nm附近出现很强的近红外荧光发射。更重要的是,当探针溶液由弱酸性转至中性时会发生上述过程的逆过程。该pH特异性可逆响应特性使得所得探针有望实现肿瘤环境呈弱酸性微环境特异性激活的高信噪比近红外荧光成像,同时还为肿瘤特异性治疗提供了可能。
2)光热/光动力性能测试:
光热转换能力测定:实验组:取0.8mL pH为6.0的1×10-5M探针置于1.5mL离心管中,用不同功率(0.2、0.4、0.6、0.8和1.0w cm-2)808nm激光器照射10min,用热成像仪每间隔30s采集一次热成像图片并记录温度,绘制温度随时间的变化曲线。pH为7.4的1×10-5M探针重复上述操作作为对照组。
结果如图4所示,弱酸性条件下,探针溶液呈现出明显的升温效果,且随照射时间的延长和激光功率的增加而加强。与之相反,在pH 7.4条件下,整个照射过程中未表出出明显的温度变化。由此可见,探针仅在弱酸性条件下才具有强的光热转化效果,确保了肿瘤特异性治疗的可能。
单线态氧能力测定:取19.9mL浓度为6×10-5M的DPBF溶液和100uL浓度为10-3M的探针储备液于50mL离心管中混合摇匀,并将溶液等分为两份备用。取一份溶液将其pH调节至6.0。将所得待测液分装于1.5mL离心管中(每管1mL)。用功率为0.6w cm-2的808nm激光器照射不同时间(0s、10s、20s、30s、40s、50s、1min、1.5min、2min、3min和5min),用荧光光谱仪测定照射后溶液的荧光强度的变化。取另一份溶液将其pH调节至7.4,重复上述808nm激光照射过程,测定照射后溶液的荧光光谱。
结果如图5所示,弱酸性条件下,随着光照时间的延长对应DPBF的紫外吸收逐渐降低,说明该过程有单线态氧的产生。而在pH 7.4条件下,整个照射过程中未发现DPBF的荧光强度改变,说明pH 7.4条件下没有单线态氧的产生。该实验结果证实在弱酸性条件下探针才具有产生单线态氧的能力,确保了肿瘤特异性光动力治疗,而不对临近正常组织造成损伤。
3)细胞毒性考察:以NIH-3T3为例,向96孔孔板中加入1×105cell/mL细胞悬液(100μL/孔)并孵育过夜。然后将培养基取出,加入100uL含不同浓度的探针(2×10-6,5×10-6,8×10-6,1×10-5M)的新鲜培养基,每组平行5次,继续孵育24小时。去除培养基,PBS洗涤,加入100μL含0.5mg mL-1MTT的新鲜培养基继续4h。弃去培养液,PBS洗涤,加入100μL DMSO,置于37℃恒温摇床避光震荡30min后用酶标仪测量570nm波长下的吸光度。
结果如图6所示,直到探针浓度高达1.0×10-5M,NIH-3T3细胞的相对存活率仍高于80%,即此浓度下该探针没有明显的细胞毒性,可用于细胞成像研究。
4)光热/光动力杀伤效果评价:细胞培养与孵育同上述细胞毒性试验。待细胞与探针(5×10-5M)孵育后调节培养体系pH为6.0或7.4,用808nm 0.6W cm-2激光器照射10min,然后再孵育12h。12h弃去培养基,PBS洗涤,加入100μL含0.5mg mL-1MTT的新鲜培养基继续培养4h。去除孔内液体,PBS洗涤,加入100μL DMSO,置于37℃恒温摇床避光震荡30min。用酶标仪测570nm波长下的吸光度。
结果如图7所示,探针浓度为5×10-5M时,在pH 6.0条件下能够杀死80%左右的肿瘤细胞,而在正常体液条件下对肿瘤细胞没有杀伤作用。证明了探针只有在弱酸性的肿瘤部位才能发挥作用。
Claims (10)
3.根据权利要求2所述的方法,其特征在于,在步骤(1)中,化合物1与3-甲基-2-丁酮的摩尔比为摩尔比为1:(1-2);反应的温度为80-120℃,反应的时间为2-3h。
4.根据权利要求2所述的方法,其特征在于,在步骤(2)中,化合物2与4-溴甲基苯甲酸甲酯的摩尔比为1:(1-2);反应的温度为80-120℃,反应的时间为4-6h。
5.根据权利要求2所述的方法,其特征在于,步骤(3)的制备过程包括:化合物2,3,4的摩尔比为1:1:1,以N,N-二甲基甲酰胺作为溶剂,在氮气保护下,加热至50℃反应5h。
6.一种pH响应型的光热/光动力/荧光一体化探针分子,其特征在于,所述pH响应型的光热/光动力/荧光一体化探针分子为权利要求1中式(1)所示结构的化合物,其中R1、R2分别为权利要求1中定义的取代基。
7.权利要求1所述化合物或者权利要求6所述pH响应型光热/光动力/荧光一体化探针分子在制备用作光热/光动力/荧光一体化诊疗剂中的应用。
8.权利要求1所述化合物或者权利要求6所述pH响应型光热/光动力/荧光一体化探针分子在非疾病诊断和治疗的生物成像领域中的应用。
9.权利要求1所述化合物或者权利要求6所述pH响应型光热/光动力/荧光一体化探针分子在制备治疗肿瘤的药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述药物的还包括药物载体和/或药用辅料。
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