CN110407873B - 一种肿瘤微环境h2o2响应交联型近红外分子探针及其应用 - Google Patents
一种肿瘤微环境h2o2响应交联型近红外分子探针及其应用 Download PDFInfo
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Abstract
本发明公开了肿瘤微环境H2O2响应交联型近红外分子探针及其应用,制备方法包括以下步骤:炔丙胺与N‑叔丁氧羰基‑N`‑芴甲氧羰基‑D‑赖氨酸发生酰胺缩合反应,得到化合物A01‑01;化合物A01‑01脱去保护基团,得到化合物C1‑2;化合物C1‑2与NHS活化后的(3‑羧丙基)三苯基溴化膦反应,得到化合物C1‑3;化合物C1‑3脱去保护基团,得到化合物C1‑4;化合物C1‑4与NHS活化后的3,5‑二氧代环己烷羧酸反应,得到化合物C1‑5;化合物C1‑5与近红外染料反应,得到肿瘤微环境H2O2响应交联型近红外分子探针。探针自身利用肿瘤微环境中的H2O2交联在肿瘤部位,达到长期滞留的目的,进而改善肿瘤成像的效果,为改善近红外分子探针在肿瘤部位长期滞留提供了新的策略与手段。
Description
技术领域
本发明属于近红外染料功能化修饰技术领域,具有涉及肿瘤微环境H2O2响应交联型1,3-环己二酮基团对传统近红外染料Cy5修饰的制备方法,通过该方法制备的分子探针,以及该探针在肿瘤的近红外荧光成像中的应用。
背景技术
在癌症的诊断中,近红外荧光染料检测技术可以实现癌症原位、靶向的无损伤动态监测。作为被美国食品药品监督管理局(Food and Drug Administration,FDA)批准的可用于临床诊断的近红外荧光染料吲哚菁绿(Indocyanine Green, ICG)。大量的文献报道,基于该染料设计了很多近红外荧光探针用于肿瘤的成像,但是由于自身原因容易被生物组织快速***,导致其在体内滞留时间过短,对肿瘤的成像效果影响严重。因此,设计一种能够长时间滞留在肿瘤部位的策略和新方法,对提高肿瘤部位成像效果意义重大。
发明内容
为了克服上述问题,本发明设计了一种肿瘤微环境H2O2响应交联型近红外分子探针。探针自身利用肿瘤微环境中的H2O2交联在肿瘤部位,达到长期滞留的目的,进而改善肿瘤成像的效果。该方法适用于多种近红外染料,为改善近红外分子探针在肿瘤部位长期滞留提供了新的策略与手段。
本发明采用以下技术方案:
一种肿瘤微环境H2O2响应交联型近红外分子探针,其具有以下化学结构式:
上述肿瘤微环境H2O2响应交联型近红外分子探针在制备肿瘤内长时间滞留的探针中的应用。
上述肿瘤微环境H2O2响应交联型近红外分子探针在制备肿瘤诊断或者治疗试剂中的应用,优选的,肿瘤诊断试剂为肿瘤成像诊断试剂;即上述肿瘤微环境H2O2响应交联型近红外分子探针在制备肿瘤成像试剂中的应用。
上述肿瘤微环境H2O2响应交联型近红外分子探针的制备方法,包括以下步骤:
(1)炔丙胺与N-叔丁氧羰基-N`-芴甲氧羰基-D-赖氨酸发生酰胺缩合反应,得到化合物A01-01;
(2)化合物A01-01脱去保护基团,得到化合物C1-2;
(3)化合物C1-2与NHS活化后的(3-羧丙基)三苯基溴化膦反应,得到化合物C1-3;
(4)化合物C1-3脱去保护基团,得到化合物C1-4;
(5)化合物C1-4与NHS活化后的3,5-二氧代环己烷羧酸反应,得到化合物C1-5;
(6)化合物C1-5与近红外染料反应,得到肿瘤微环境H2O2响应交联型近红外分子探针。
一种近红外分子探针在肿瘤内长时间滞留的方法,包括以下步骤:
(1)炔丙胺与N-叔丁氧羰基-N`-芴甲氧羰基-D-赖氨酸发生酰胺缩合反应,得到化合物A01-01;
(2)化合物A01-01脱去保护基团,得到化合物C1-2;
(3)化合物C1-2与NHS活化后的(3-羧丙基)三苯基溴化膦反应,得到化合物C1-3;
(4)化合物C1-3脱去保护基团,得到化合物C1-4;
(5)化合物C1-4与NHS活化后的3,5-二氧代环己烷羧酸反应,得到化合物C1-5;
(6)化合物C1-5与近红外染料反应,得到肿瘤微环境H2O2响应交联型近红外分子探针。
(7)肿瘤微环境H2O2响应交联型近红外分子探针到达肿瘤内,完成近红外分子探针在肿瘤内长时间滞留。
上述方案中,肿瘤微环境H2O2响应交联型近红外分子探针到达肿瘤内的方式为非手术方式,比如常规注射、输液、服用等;肿瘤可以为肿瘤组织也可以为肿瘤细胞。举例为,将肿瘤微环境H2O2响应交联型近红外分子探针溶于PBS(磷酸缓冲液,PH=7.2~7.4)注射液中(浓度为100μM),注射到达肿瘤部位,肿瘤微环境H2O2响应交联型近红外分子探针在肿瘤内部的H2O2作用下交联在肿瘤内部,即可完成近红外分子探针在肿瘤部位长时间滞留的目的;还可先将肿瘤细胞用过氧化氢处理,再将DATC溶于细胞培养基中,加入到肿瘤细胞的培养皿中,放入培养箱培养,在分子探针孵育过程中,其在过氧化氢作用下可与肿瘤细胞内的蛋白等大分子化合物发生交联反应,从而延长其在肿瘤细胞内的滞留时间,有利于实现对肿瘤细胞的长时间追踪。
上述方案中,步骤(1)中,炔丙胺与N-叔丁氧羰基-N`-芴甲氧羰基-D-赖氨酸的摩尔比例为1.2:1;酰胺缩合反应在1-羟基苯并***、苯并***-1-四甲基六氟磷酸酯和二异丙基乙胺存在的条件下进行;酰胺缩合反应为室温反应18~23小时。
上述方案中,步骤(2)中,化合物A01-01脱去保护基团在哌啶/二氯甲烷的混合溶液中进行;哌啶与二氯甲烷的体积比为1:5;脱保护在室温下进行。
上述方案中,步骤(3)中,化合物C1-2与NHS活化后的(3-羧丙基)三苯基溴化膦在二异丙基乙胺存在的条件下进行反应,化合物C1-2、NHS活化后的(3-羧丙基)三苯基溴化膦、二异丙基乙胺的摩尔比为1:1.2:1.2;反应为室温反应3~6小时。
上述方案中,步骤(4)中,化合物C1-3脱去保护基团在三氟乙酸/二氯甲烷的混合溶液中进行;三氟乙酸与二氯甲烷的体积比为1:4;脱保护在室温下进行。
上述方案中,步骤(5)中,化合物C1-4与NHS活化后的3,5-二氧代环己烷羧酸在二异丙基乙胺存在的条件下进行反应,化合物C1-4、NHS活化后的3,5-二氧代环己烷羧酸、二异丙基乙胺的摩尔比为1:1.2:1.2;反应为室温反应3~6小时。
上述方案中,步骤(6)中,化合物C1-5与近红外染料的反应在抗坏血酸钠与硫酸铜存在的条件下进行;化合物C1-5、近红外染料、抗坏血酸钠、硫酸铜的摩尔比为1.2:1:2:1;反应为室温反应8~12小时。
本发明中,化合物A01-01、化合物C1-1、化合物C1-2、化合物C1-3、化合物C1-4、化合物C1-5的化学结构式分别如下:
NHS活化的(3-羧丙基)三苯基溴化膦的化学结构式如下:
NHS活化后的3,5-二氧代环己烷羧酸的化学结构式如下:
近红外染料的化学结构式如下:
具体而言,本发明的技术方案包括如下步骤:
(1)构建、合成H2O2响应型近红外分子探针:
首先炔丙胺与N-叔丁氧羰基-N’-芴甲氧羰基-D-赖氨酸发生酰胺缩合反应,随后用20%的哌啶/二氯甲烷(哌啶:二氯甲烷=1:4,v/v);将中间体化合物的保护基团脱去;接着与用NHS活化的(3-羧丙基)三苯基溴化膦反应,得到中间体化合物再用20%的三氟乙酸/二氯甲烷(三氟乙酸:二氯甲烷=1:4,v/v)脱去保护基团,与NHS活化的3,5-二氧代环己烷羧酸进行酰胺化缩和反应,得到的中间体化合物进一步在抗坏血酸钠和硫酸铜的催化下,与Cy5发生点击化学反应得到最终的近红外分子探针DATC,其结构如下所示:
(2) H2O2响应型近红外分子探针DATC在过氧化氢作用下在细胞内的交联作用:
先将4T1细胞用100μM的过氧化氢处理15分钟后,用PBS清洗两遍后,将DATC溶于细胞培养基中,加入到4T1细胞的培养皿中(探针浓度为5μM),放入培养箱培养8小时。待探针与4T1细胞孵育时间结束,用PBS清洗两遍,加入新鲜的培养基。在分子探针孵育过程中,其在过氧化氢作用下可与肿瘤细胞内的蛋白等大分子化合物发生交联反应,从而延长其在肿瘤细胞内的滞留时间,有利于实现对肿瘤细胞的长时间追踪。
(3) H2O2响应型近红外分子探针DATC在肿瘤内的交联作用:
将DATC溶于PBS注射液中(探针浓度为100μM),注射进荷有4T1乳腺癌的BALB/c/nu磁性裸鼠的肿瘤内,即可开始观察分子探针的长滞留时间。
本发明中,化合物C1-5与近红外染料反应后,使用常规半制备型高效液相色谱分离提纯,得到H2O2响应型近红外分子探针DATC,产物为深蓝色的固体粉末;高效液相色谱分离方法为:C18柱,3.5μm,4.6×100mm;流动相:A相是三氟乙酸:超纯水=1:1000;B相是三氟乙酸:乙腈=1:1000;流速是3mL/min;线性洗脱程序:0min,A:B=95:5;17min,A:B=0:100。
本发明的制备方法通过H2O2触发交联反应,延长近红外分子探针在肿瘤细胞内或者肿瘤组织内的滞留时间。其具有良好的荧光成像性能,能够对肿瘤部位进行有效的荧光成像,克服了可见光穿透深度浅,不能有效的对生物体进行实时荧光成像的缺点。
由于上述技术方案的运用,本发明与现有技术相比较具有如下优点:
(1)本发明首次使用了3,5-二氧代环己烷羧酸功能化修饰近红外染料H2O2触发条件便捷、温和;
(2)H2O2响应交联型近红外分子探针在肿瘤微环境的触发下发生光交联反应不受外界条件的干扰;
(3)当探针进入肿瘤细胞后,在H2O2的作用下,3,5-二氧代环己烷羧酸会与蛋白上的巯基迅速反应,连接在蛋白上,交联反应效率较高。
附图说明
图1为本发明H2O2响应型近红外分子探针应用参考图;
图2为本发明H2O2响应型近红外分子探针DATC的制备流程图;
图3为本发明DATC与TC的化学结构式;
图4为本发明H2O2响应型近红外分子探针DATC的高效液相色谱纯度表征(a)和质谱表征(b);
图5为本发明H2O2响应型近红外分子探针DATC的紫外吸收光谱和荧光吸收光谱情况(a)以及探针的细胞毒性情况(b);
图6为本发明不同浓度探针与4T1细胞孵育相同时间的荧光照片(a)和对应的标准荧光强度图(b);
图7为本发明细胞内探针(浓度为5μM)与细胞内线粒体的共定位情况;
图8为本发明相同浓度过氧化氢处理4T1细胞后,细胞内探针(浓度为5μM)的荧光照片(a)和对应的标准荧光强度图(b);
图9为本发明相同浓度过氧化氢处理4T1细胞后,探针DATC和TC与4T1细胞孵育相同时间,重新培养12小时后,细胞内探针的免疫荧光照片;
图10为本发明瘤内注射探针后,不同时间内老鼠肿瘤部位的近红外荧光照片(a)和对应的标准荧光强度图(b)。
具体实施方式
本发明的肿瘤微环境H2O2响应交联型近红外分子探针在其光谱范围内被检测的生物体和组织的自发荧光干扰较小,且组织穿透深度可达几厘米,在一定程度上有效的提高了成像的准确性和灵敏度;与传统的染料相比,各方面的性能都有所改善,其具有良好的荧光量子产率、较好的耐光性、较低的生物毒性、较强的荧光强度、可塑的分子结构、较好的水溶性以及价格低廉等优点,因此,可以被广泛的应用于近红外生物成像。图1为本发明H2O2响应型近红外分子探针应用参考图。
下文将结合附图和具体实施例来进一步阐述本发明。应当理解的是,这些实施例仅用于解释和说明本发明中的技术方案,而并非旨在限制本发明的范围。此外,除非另有说明,下列实施例中所使用的材料、试剂、仪器等均可通过商业手段获得。
实施例1:H2O2响应型近红外分子探针DATC和对照组探针TC的合成和表征
(1)在100mL圆底烧瓶中加入N-叔丁氧羰基-N’-芴甲氧羰基-D-赖氨酸(2.34g,5mmol)、N,N-二甲基甲酰胺(30mL)、苯并***-1-四甲基六氟磷酸酯(2.28g,6mmol)、1-羟基苯并***(0.81g,6mmol)和二异丙基乙胺(1.02mL,6mmol)冰水浴搅拌15min。随后向反应瓶中加入丙炔胺(330μL,6mmol),继续搅拌下于室温反应15小时。反应结束后,旋蒸除去溶剂,加入150mL乙酸乙酯重新溶解中间体产物。随后把有机相分别用30mL去离子水、饱和碳酸氢钠和氯化钠水溶液各洗一次,用无水氯化钠干燥后,旋干,得到白色粉末状中间体A01-01(其结构如图2a中的化合物A01-01所示)(1.90g,产率:71%)。1H-NMR (400 MHz,Chloroform-d, ppm) δ = 7.73 (s, 2H), 7.56 (s, 2H), 7.37 (s, 2H), 7.29 (s,2H), 4.39 (s, 2H), 4.176-4.126(m, 2H), 4.00 (s, 2H), 3.07 (s, 2H), 2.96 (s,1H), 1.65 (s, 2H), 1.66-1.58 (m, 2H), 1.40 (s, 9H), 1.35-1.24 (m, 2H); 13C-NMR(151 MHz, Chloroform-d, ppm) δ 171.44, 166.24, 143.67, 141.27, 127.72,127.07, 126.02, 119.97, 79.15, 71.73, 67.04, 64.62, 47.12, 39.77, 31.86,29.54, 29.17, 28.41, 22.37; MS (MALDI-TOF): Calc’d for: C29H35N3O5([M+Na]+):528.59,found: 528.34。
(2)在50mL圆底烧瓶中加入8mL的二氯甲烷和2mL的哌啶搅拌均匀。随后将中间体化合物A01-01(0.5g,0.99mmol)加入反应瓶中,是室温搅拌15min。反应结束后,旋蒸除去反应液,用硅胶柱层析纯化(二氯甲烷:甲醇30:1,v/v),得到白色固体中间体C1-2(其结构式如图2a中的化合物C1-2所示)(0.21g,产率:75%)。1H-NMR (400 MHz, Chloroform-d, ppm)δ 4.01 (s, 2H), 3.35 (t, J = 3.2 Hz, 1H), 3.09 (s, 2H), 2.00-1.86 (m, 2H),1.49-1.45(m, 2H), 1.40 (s, 9H), 1.28-1.16 (m, 2H); 13C-NMR (151 MHz,Chloroform-d, ppm) δ 174.63, 156.06, 79.69, 54.81, 40.07, 34.36, 29.82,28.74, 28.39, 22.71; MS (MALDI-TOF) Calc’d for: C14H25N3O3 ([M+K]+): 322.46,found:322.35。
(3)在50mL圆底烧瓶中加入15mL二氯甲烷、NHS活化的(3-羧丙基)三苯基溴化膦(0.182g,0.424mmol)、中间体化合物C1-2(0.1g,0.353mmol)和二异丙基乙胺(0.2mL,0.494mmol),室温反应5小时。反应结束后,有机相用20mL去离子水洗三遍,25mL氯化钠水溶液洗一遍,用无水硫酸钠干燥后旋干。随后用硅胶柱层析纯化(二氯甲烷:甲醇=50:1,v/v),得到白色固体中间体C1-3(其结构如图2a中的化合物C1-3所示)(0.173g,产率:80%)。1H-NMR (400 MHz, Chloroform-d, ppm) δ 7.61 (s, 15H), 3.91 (s, 1H), 3.54 (s, 2H),3.26 (s, 2H), 3.04 (s, 1H), 2.57-2.46 (m, 2H), 2.11 (s, 2H), 1.55-1.44 (m,2H), 1.28-1.17 (m, 4H), 1.08 (s, 9H), 0.98-0.94 (m, 2H); 13C-NMR (151 MHz,Chloroform-d, ppm) δ 172.07, 171.24, 155.98, 135.36, 134.01, 133.95, 130.73,130.64, 119.23, 119.12, 118.67, 118.55, 81.45, 77.77, 73.39, 52.90, 46.03,42.42, 29.65, 28.69, 28.31, 24.40, 23.23, 18.73, 18.52. MS (MALDI-TOF) Calc’dfor: C36H45N3O4p +([M]+): 614.73, found: 614.49。
(4)在50 mL 圆底烧瓶中加入16 mL的二氯甲烷和4 mL的三氟乙酸搅拌均匀。随后将中间体化合物C1-3(0.122g,0.2mmol)加入反应瓶中,室温搅拌2小时。待反应结束后,旋蒸除去反应液,用硅胶柱层析纯化(二氯甲烷:甲醇=10:1,v/v),得到黄色油状中间体C1-4(其结构如图2a中的化合物C1-4所示)(0.092g,产率:90%)。1H-NMR (600 MHz, DMSO-d6,ppm) δ 7.83 – 7.68 (m, 15H), 4.19-4.15 (m, 1H), 3.82 – 3.77 (m, 2H), 3.54-3.48 (m, 2H), 3.03 (s, 1H), 2.73 – 2.69 (m, 2H), 2.42 – 2.32 (m, 2H), 1.72-1.68 (m, 2H), 1.61-1.45 (m, , 4H), 1.30 – 1.19 (m, 2H). 13C-NMR (151 MHz,DMSO-d6,ppm) δ 171.97, 171.36, 135.34, 134.00, 133.93, 130.71, 130.63,119.10, 118.54, 117.35, 81.42, 73.35, 52.76, 40.31, 35.21, 35.09, 31.72,28.34, 27.03, 20.56, 20.22, 18.72; MS (MALDI-TOF) Calc’d for: C31H37N3O2P+([M]+):514.62, found: 514.41。
(5)在50mL圆底烧瓶中加入10mLN,N-二甲基甲酰胺,随后加入中间体化合物C1-4(0.055g,0.107mmol)、NHS活化的3,5-二氧代环己烷羧酸(20mg,0.128mmol)和二异丙基乙胺(25.5μL,0.154mmol),室温反应6小时。反应结束后,旋蒸除去反应液,用硅胶柱层析纯化(二氯甲烷:甲醇=10:1,v/v),得到黄色油状中间体化合物C1-5(其结构如图2a中的化合物C1-5所示)(0.056mg,产率:81%)。1H-NMR (600 MHz, DMSO-d6,ppm) δ 7.88 – 7.71 (m,15H), 4.16 (td, J = 8.4, 5.4 Hz, 1H), 3.80 – 3.79 (m, 2H), 3.51 – 3.47 (m,2H), 3.03 (t, J = 2.4 Hz, 0H), 2.99 – 2.95 (m, 2H), 2.80 (td, J = 11.3, 5.6Hz, 1H), 2.41 – 2.30 (m, 4H), 2.23 (dd, J = 16.9, 4.7 Hz, 2H), 1.73 – 1.66(m, 2H), 1.56 (dt, J = 14.8, 5.7 Hz, 1H), 1.49 – 1.43 (m, 1H), 1.36 – 1.28(m, 2H), 1.28 – 1.03 (m, 4H);13C-NMR (151 MHz, DMSO-d6) δ 172.44, 172.07,171.29, 135.35, 134.00, 133.93, 130.72, 130.64, 119.10, 118.53, 81.45, 73.37,52.83, 29.08, 28.31, 25.64, 23.17, 20.54, 20.20, 18.70; MS (MALDI-TOF) Calc’dfor: C38H43N3O5P+([M]+): 652.74, found: 652.26。
(6)在5mL圆底烧瓶中加入1mLDMSO、中间体化合物C1-5(2.66mg,0.0041mmol)和近红外染料(2.68mg,0.0037mmol,结构式见图3),搅拌均匀。同时将抗坏血酸钠(8.1mg,0.0409mmol)和无水硫酸铜(5.1mg,0.0204mmol)混合溶于1mL去离子水中,再将混合液加入到反应瓶中,室温搅拌反应10小时。反应结束后,使用半制备型高效液相色谱分离提纯(分离方法为:C18柱,3.5μm,4.6×100mm;流动相:A相是三氟乙酸:超纯水=1:1000;B相是三氟乙酸:乙腈=1:1000;流速是3mL/min;线性洗脱程序:0min,A:B=95:5;17min,A:B=0:100),得到分子探针DATC(深蓝色的固体粉末4.51mg,产率:91%)。1H-NMR (600 MHz, DMSO-d6,ppm)δ 8.40-8.52 (m, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.86 (d, J = 7.9 Hz, 3H), 7.80– 7.71 (m, 15H), 7.32-7.23 (m, 1H), 6.37 – 6.17 (m, 1H), 4.30 – 4.13 (m, 4H),4.12 – 4.03 (m, 3H), 3.53-3.47 (m, 4H), 3.39-3.33 (m, 1H), 3.28 (t, J = 6.7Hz, 1H), 3.04 (s, 1H), 3.03 – 2.86 (m, 5H), 2.77 – 2.70 (m, 1H), 2.48 – 2.47(m, 6H), 2.42 – 2.26 (m, 5H), 2.04 – 1.97 (m, 1H), 1.81 – 1.74 (m, 1H), 1.74-1.68 (m, 3H), 1.67 – 1.61 (m, 6H), 1.59-1.54 (m, 2H), 1.52 – 1.39 (m, 3H),1.38 – 1.28 (m, 3H), 1.25-1.14 (m, 5H). 13C-NMR (400 MHz, DMSO) δ 172.46,172.10, 171.31, 162.79, 158.80, 158.45, 135.40, 134.08, 133.98, 130.80,130.67, 119.29, 118.44, 81.50, 73.46, 52.84, 49.40, 48.87, 47.57, 36.25,35.29, 32.06, 30.44, 29.08, 28.34, 27.52, 27.41, 27.16, 25.32, 23.22, 20.64,20.12, 18.74, 12.57.MS (ESI) : m/z Calc’d for: C73H88N9O12P+S2 2+([M+TFA+2Cl]-):1562.57; found, 1562.0。
(7)在5mL圆底烧瓶中加入1mLDMSO、中间体化合物C1-3(4.67mg,0.0076mmol)和近红外染料(5.00mg,0.0069mmol),搅拌均匀。同时将抗坏血酸钠(13.67mg,0.069mmol)和无水硫酸铜(8.61mg,0.0345mmol)混合溶于1mL去离子水中,再将混合液加入到反应瓶中,室温搅拌反应5小时。反应结束后,使用半制备型高效液相色谱分离提纯(分离方法为:C18柱,3.5μm,4.6×100mm;流动相:A相是三氟乙酸:超纯水=1:1000;B相是三氟乙酸:乙腈=1:1000;流速是3mL/min;线性洗脱程序:0min,A:B=95:5;17min,A:B=0:100),得到对照组分子探针TC(深蓝色的固体粉末8.6mg,产率:93%)。1H-NMR (600 MHz, DMSO-d6) δ 8.40 (t, J= 6.0 Hz, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 5.1 Hz, 3H), 7.79 –7.72 (m, 15H), 7.30-7.64 (m, 1H), 6.71 (s, 1H), 6.54 (t, J = 12.0 Hz, 2H),6.27 (dd, J = 13.8, 5.5 Hz, 2H), 4.31 – 4.21 (m, 5H), 4.21 – 4.14 (m, 2H),4.12-4.05 (m, 4H), 2.93 (d, J = 6.0 Hz, 2H), 2.80 (d, J = 6.8 Hz, 2H), 2.38-2.33 (m, 2H), 2.01 (t, J = 7.1 Hz, 2H), 1.80 – 1.76 (m, 2H), 1.64 (d, J = 5.1Hz, 12H), 1.54 – 1.40 (m, 6H), 1.31 (s, 9H), 1.27 (s, 4H), 1.24-1.21 (m, 8H).13C-NMR (400 MHz, DMSO-d6) δ 173.42, 173.15, 172.45, 172.28, 171.36, 158.40,156.02, 145.85, 145.07, 142.53, 141.94, 141.11, 135.41, 134.07, 133.97,130.78, 130.66, 123.29, 120.42, 119.30, 118.44, 77.82, 49.40, 49.33, 47.56,36.11, 35.47, 32.13, 30.46, 29.48, 28.72, 27.52, 27.41, 26.07, 25.31, 23.31,18.79, 12.57。
近红外染料的化学结构式如下:
图2为上述制备DATC的流程图,图3为制备的DATC以及对比用TC的结构式。
实施例2:H2O2响应型近红外分子探针DATC在过氧化氢作用下的交联作用
先用100μM的过氧化氢处理4T1细胞15min,然后将实施例1中制得的H2O2响应型近红外分子探针DATC稀释到细胞培养基中,之后加入到4T1细胞培养皿中共同孵育(探针浓度为5μM)。经过氧化氢处理的4T1细胞内蛋白上的巯基会被氧化成次磺酸,当探针进入肿瘤细胞后,其所带的基团3,5-二氧代环己烷羧酸会与次磺酸交联形成C-S共价键,使探针分子与肿瘤细胞内的大分子蛋白等牢固连接,从而延长分子探针在肿瘤细胞内的滞留时间,改善探针分子的成像效果,技术效果参见图8。
实施例3:H2O2响应型近红外分子探针DATC在高效液相色谱纯度表征和质谱表征
将实施例1中制得的H2O2响应型近红外分子探针用溶剂甲醇稀释到浓度为5μM后,通过质谱对其进行分子量确定,并使用高效液相色谱仪对其进行纯度分析。
如图4a所示,使用Agilent 1260高效液相色谱仪对样品进行分析,探针DATC的保留时间在5.260分钟,进一步对分面积进行积分,计算得到样品中探针浓度高达98%。图4b展示探针DATC的理论m/z:1378.64,实际得到质谱谱图中m/z([M+ TFA+2Cl]-):1562.0,两者相吻合,即为所要的化合物。
实施例4:H2O2响应型近红外分子探针DATC的紫外吸收光谱和荧光光谱情况以及探针的毒性情况
将实施例1中制得的H2O2响应型近红外分子探针用甲醇稀释到浓度为1μM后,分别使用紫外-可见吸收光谱仪和稳态/瞬态荧光光谱仪去测量探针的紫外吸收光谱和荧光光谱;通过MTT(噻唑蓝)比色法考察探针对4T1的细胞毒性。
如图5a所示,通过紫外吸收光谱发现,探针DATC在630nm-660nm的范围内有两个明显的吸收峰,其最大吸收峰在646nm;通过荧光光谱发现,探针DATC在665-690nm范围内有一个发射峰,其最大发射在670nm。
图5b是分子探针DATC的细胞毒性实验,细胞的存活率与探针浓度基本上不存在依赖关系,随着浓度的增加,探针对细胞无杀伤作用。分子探针DACF在浓度为40μM时,细胞存活率仍有90%以上,这个浓度远远高出临床中所使用的探针浓度。
实施例5:不同浓度探针与4T1细胞共孵育相同时间的情况
基于实施例2中记载的方法,将4T1细胞并接种于共聚焦小皿中,每孔1.5×104个细胞,放入培养箱中培养24小时,之后弃培养基。将实施例1中制得的H2O2响应型近红外分子探针用培养基分别稀释到浓度为1μM、2μM和5μM,随后加入到4T1细胞培养皿中,放入细胞培养箱中共同孵育2小时。待探针与4T1细胞孵育时间结束,用Hoechst33342染液染核,使用共聚焦显微镜观察不同浓度探针相同时间内在4T1细胞内的荧光情况。
如图6a所示,在在相同条件下,不同浓度的探针在4T1细胞内的红色荧光强度是不一样的,其中5μM浓度的探针荧光强度最强,而1μM和2μM浓度的探针在4T1细胞内的荧光强度相差不大。因此,之后的实验均选取探针工作浓度为5μM。
实施例6:细胞内探针(浓度为5μM)与细胞内线粒体的共定位情况
基于实施例2中记载的方法,将4T1细胞并接种于共聚焦小皿中,每孔1.5×104个细胞,放入培养箱中培养24小时,之后弃培养基。将实施例1中制得的H2O2响应型近红外分子探针用培养基稀释到浓度为5μM,随后加入到4T1细胞培养皿中,放入细胞培养箱中共同孵育12小时。待探针与4T1细胞孵育时间结束,分别用线粒体染料和Hoechst33342与细胞共孵育,使用共聚焦显微镜观察探针相同时间内在4T1细胞内的与线粒体的共定位情况。如图7所示,探针DATC与线粒体的荧光基本重合。
实施例7:相同浓度过氧化氢处理4T1细胞后,探针在细胞内的滞留情况
基于实施例2中记载的方法,将4T1细胞并接种于共聚焦小皿中,每孔1.5×104个细胞,放入培养箱中培养24小时。将过氧化氢用细胞培养基稀释到100μM后,加入到4T1细胞的培养皿中共孵育15min后,弃去培养基。将实施例1中制得的H2O2响应型近红外分子探针用培养基稀释到浓度为5μM,随后加入到4T1细胞培养皿中,放入细胞培养箱中共同孵育8小时。待探针与4T1细胞孵育时间结束,加入新的细胞培养基至于培养箱中继续培养4h、8h、12h和24h。然后用Hoechst33342染液染核,用激光共聚焦显微镜观察在过氧化氢处理后,不同时间内4T1细胞中的荧光情况。以不含3,5-二氧代环己烷羧酸的荧光探针(TC)为对照,采用统一的实验方法。
如图8a所示,在起始0h的时候,4T1细胞内的红色荧光强度是一样的。洗去探针8h后,4T1细胞内的红色荧光强度明显强于对照组没有交联基团的探针。并且随着时间的延长,没有交联基团对照组荧光减弱的非常迅速,12h之后对照组荧光信号微弱;24h之后,实验组仍可以观察到较强的荧光信号,而对照组几乎没有荧光信号。图8b是对应的荧光定量数据,有交联基团的探针可以在细胞内滞留至少24h,而无交联基团的探针仅仅保持了12h左右。因此,H2O2响应型近红外分子探针DATC能够应用于细胞示踪。
实施例8:相同浓度过氧化氢处理4T1细胞后,探针DATC和TC与4T1细胞孵育相同时间,重新培养12小时后,细胞内探针的免疫荧光情况
基于实施例2中记载的方法,将4T1细胞并接种于共聚焦小皿中,每孔1.5×104个细胞,放入培养箱中培养24小时。将过氧化氢用细胞培养基稀释到100μM后,加入到4T1细胞的培养皿中共孵育15min后,弃去培养基。将实施例1中制得的H2O2响应型近红外分子探针用培养基稀释到浓度为5μM,随后加入到4T1细胞培养皿中,放入细胞培养箱中共同孵育8小时。待探针与4T1细胞孵育时间结束,加入新的细胞培养基至于培养箱中继续培养12小时。随后,用4%的多聚甲醛在室温的条件下,固定细胞15min后,PBS清洗;其次,0.5% Trition-100与4T1细胞在室温条件下共同孵育5min;在室温条件下孵育抗体anticysteinesulfenic acid antibody 1小时后,PBS清洗,随后37℃孵育羊抗兔-Cy3 30min,最后用Hoechst33342染液染核,用激光共聚焦显微镜观察在过氧化氢处理后,4T1细胞内的免疫荧光情况。以不含3,5-二氧代环己烷羧酸的荧光探针(TC)为对照,采用统一的实验方法。
如图9所示,两组细胞经过处理后,细胞中均有蛋白质次磺酸形成。探针DATC在细胞中的含量较多,而探针TC基本上观测不到荧光信号。该实验结果证明DATC在肿瘤中能达到长时间滞留的效果。
实施例9:H2O2响应型近红外分子探针DATC在小鼠肿瘤部位近红外荧光成像情况
将接种了4T1小鼠乳腺癌细胞的BALB/c/nu雌性裸鼠,随机分为两组,每组5只。待何在小鼠右前肢的肿瘤长大后,以瘤内注射的方式将探针DATC和TC分别注入到两组裸鼠肿瘤内,给药浓度为100μM(50μL/每只)。之后将注射探针的小鼠至于小动物IVIS LuminaXRMS活体成像***中,观察探针在裸鼠肿瘤内的荧光强度随时间的变化,并通过IVIS活体成像分析软件计算实验组探针和对照组探针在裸鼠肿瘤部位不同时间点的荧光强度。
如图10a所示,刚在瘤内注射完探针,实验组和对照组探针在肿瘤部位的荧光信号强度基本上是一致的。之后随着时间的延长,探针在荷瘤小鼠体内逐渐代谢,在2h对照组探针信号明显减弱,而实验组只有微弱的降低。在12h时,实验组探针仍具有明显的荧光信号,而对照组荧光信号很微弱。通过图10b肿瘤部位的荧光信号统计,可以清晰看到,实验组肿瘤部位的荧光信号可以持续36h。通过体内荧光成像的结果证明了本发明的光交联探针可以在肿瘤部位进行光交联反应,交联在肿瘤内,减少细胞代谢外排,使探针在肿瘤部位长时间滞留,从而延长荧光成像时间。
H2O2响应型探针应用于交联型标记蛋白方面的研究已经取得了令人瞩目的发展。为了克服传统近红外分子探针的缺点,本发明构建一种H2O2响应型交联近红外分子探针,提高探针在肿瘤部位的富集量和延长其滞留时间,进而有效的改善肿瘤的成像效果。它具有以下几个优点:首先,其触发条件简单;第二,交联反应受肿瘤部位H2O2的影响,肿瘤部位pH等基本上对交联反应无影响;第三,当探针进入肿瘤细胞后,在内部H2O2的影响下,会迅速与肿瘤部位含有巯基的蛋白交联,达到长期滞留的目的。因而使近红外分子探针在生物等领域有进一步的应用。
Claims (7)
2.一种近红外分子探针在肿瘤内长时间滞留的方法,其特点在于,包括以下步骤:
(1)炔丙胺与N-叔丁氧羰基-N`-芴甲氧羰基-D-赖氨酸发生酰胺缩合反应,得到化合物A01-01;
(2)化合物A01-01脱去保护基团,得到化合物C1-2;
(3)化合物C1-2与NHS活化后的(3-羧丙基)三苯基溴化膦反应,得到化合物C1-3;
(4)化合物C1-3脱去保护基团,得到化合物C1-4;
(5)化合物C1-4与NHS活化后的3,5-二氧代环己烷羧酸反应,得到化合物C1-5;
(6)化合物C1-5与近红外染料反应,得到肿瘤微环境H2O2响应交联型近红外分子探针;
(7)肿瘤微环境H2O2响应交联型近红外分子探针到达肿瘤部位,完成近红外分子探针在肿瘤内长时间的滞留。
3. 根据权利要求2所述近红外分子探针在肿瘤内长时间滞留的方法,其特点在于,炔丙胺与N-叔丁氧羰基-N`-芴甲氧羰基-D-赖氨酸的摩尔比为1.2 : 1;化合物C1-2与NHS活化的(3-羧丙基)三苯基溴化膦的摩尔比为1 : 1.2;化合物C1-4与NHS活化的3,5-二氧代环己烷羧酸的摩尔比为1: 1.2;化合物C1-5、近红外染料的摩尔比为1 : 1.1。
4. 根据权利要求2所述近红外分子探针在肿瘤内长时间滞留的方法,其特点在于,化合物A01-01在哌啶/二氯甲烷混合溶液中脱去保护基团;化合物C1-3在三氟乙酸/二氯甲烷混合溶剂中脱去保护基团;化合物C1-5与近红外染料的反应在抗坏血酸钠和硫酸铜存在下进行;化合物C1-5、抗坏血酸钠和硫酸铜的摩尔比为1: 5: 2.5。
5.权利要求1所述肿瘤微环境H2O2响应交联型近红外分子探针在制备肿瘤内长时间滞留的探针中的应用。
6.权利要求1所述肿瘤微环境H2O2响应交联型近红外分子探针在制备肿瘤诊断或者治疗试剂中的应用。
7.根据权利要求6所述的应用,其特点在于,肿瘤诊断试剂为肿瘤成像诊断试剂。
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