CN109289048A - 一种肿瘤血管阻断协同光治疗试剂及其合成方法与应用 - Google Patents
一种肿瘤血管阻断协同光治疗试剂及其合成方法与应用 Download PDFInfo
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- CN109289048A CN109289048A CN201810899039.XA CN201810899039A CN109289048A CN 109289048 A CN109289048 A CN 109289048A CN 201810899039 A CN201810899039 A CN 201810899039A CN 109289048 A CN109289048 A CN 109289048A
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- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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- Medicinal Preparation (AREA)
Abstract
本发明公开了一种肿瘤血管阻断协同光治疗试剂,由烷基化呋喃吡咯并吡咯二酮、具有微酸刺激响应的二乙基苯胺基和血管阻断剂2,5‑己酮可可碱共轭键接组成。本发明还公开了该肿瘤血管阻断协同光治疗试剂在制备荧光、光热成像介导的肿瘤血管阻断协同光治疗药物中的应用。本发明肿瘤血管阻断协同光治疗试剂除了具有肿瘤微酸刺激响应的光动力、光热性能以及靶向阻断肿瘤血管的能力以外,还具备如下特点:1)优异的水分散性;2)兼具主动和被动靶向性,可实现血管阻断剂在血管内皮细胞处的酸性刺激释放,提高纳米药物到达肿瘤部位的精准度;3)可实现荧光、光热成像介导的光热/光动力多模式肿瘤治疗,有效杀死肿瘤细胞,抑制肿瘤细胞再生和转移。
Description
技术领域
本发明属于材料和生物医药领域,具体涉及一种肿瘤血管阻断协同光治疗试剂及其合成方法、以及在制备荧光、光热成像介导的多模式肿瘤治疗药物中的应用。
背景技术
2017年国家癌症中心发布的中国最新癌症数据结果表明,全国每天约1万人确诊癌症,肿瘤发病率多年持续上升,已成为一个必须高度重视的公共卫生问题乃至社会问题,肿瘤问题将会是人类社会发展的一个巨大障碍,因而需要采取有效的措施来抑制这一疾病。临床病例表明,癌症I期患者的五年生存率超过90%,如果病灶在更早期(癌变前阶段)被发现并得到治疗,患者通常可完全治愈。开发新的肿瘤治疗药物,探究新的治疗方法是解决癌症这一难题的有效途径。
目前,两种主要的非侵入性肿瘤治疗方法为光动力疗法(PDT)和光热疗法(PTT),与传统的癌症治疗方法相比,上述两种方法副作用低、选择性和效率高。PDT和PTT通常是由近红外(NIR)光激发递送到肿瘤内的纳米药物产生的活性氧(ROS)或热量来杀死肿瘤细胞。然而,这两种治疗方法仍然存在一些不足,如:PDT存在肿瘤内乏氧、单线态氧半衰期短(<40ns)、单线态氧扩散半径低(<20纳米)等问题;而PTT存在治疗过程中热休克蛋白表达的上调、难以控制肿瘤细胞转移和复发等。这些关键问题严重降低了肿瘤光治疗的疗效,同时阻碍了其临床应用。
人体肿瘤的生长依赖于血管供应氧气和营养物质。血管阻断剂(VDAs)利用肿瘤血管表面的生长因子、多肽、抗体与正常血管之间的病理差异能够快速且选择性地靶向已有肿瘤血管,通过直接凋亡维持细胞骨架的微管蛋白的内皮细胞,抑制血流,导致肿瘤中心区域出血性坏死。目前,已进入Ⅲ期临床试验的小分子血管阻断剂DMXAA(2,5-己酮可可碱,5,6-dimethylxanthenone-4-aceticacid)的抗肿瘤作用分为直接作用和间接作用两方面:既可直接作用于肿瘤血管,诱导内皮细胞凋亡;又可通过核转录因子的向上调节间接诱导大量细胞因子和化学因子的产生。但由于DMXAA极性偏大,其脂溶性和透膜能力较差,给药后最大血药浓度低,不足以产生抗肿瘤作用,导致其治疗效果不理想。因此,根据前药原理,期望通过将其结构中的羧基官能团酯化,引入亲脂性基团,使药物易于透过脂质膜,改变药物的理化性质,减少不良反应,并通过提高脂水分配系数,使其能够顺利地进入到肿瘤血管内皮细胞内并通过酸性水解释放出原药诱导肿瘤血管内皮细胞凋亡。由于VDAs主要作用于肿瘤的核心区域,而传统的PDT&PTT治疗试剂仅仅通过enhanced permeability andretention(EPR)效应很难抵达该区域,因此VDAs与PDT&PTT互为补充,在抗癌过程中达到双重效果。此外,由于肿瘤内乏氧的共性引起瘤内糖酵解的上调,导致肿瘤的酸微环境(pH值5-6.8),因此研发具有微酸环境刺激响应的治疗试剂越来越受关注,例如具有酸性刺激响应性的二甲基苯胺基、二乙基苯胺基通常被键接到母体肿瘤治疗药物上。
大多数无机纳米材料体内生物相容性差且存在长期毒性,限制了其发展和临床应用。有机小分子吡咯并吡咯二酮(DPP)衍生物是一种具有易修饰、高耐热、耐光、颜色明亮及摩尔吸光系数高的染料,在有机电子器件以及生物探针领域有广泛应用。然而,在生物医药领域,DPP的水溶性和靶向性仍然是一个巨大的挑战。为了克服这些缺点,亲水基团的表面修饰及制备纳米颗粒等方法被广泛研究,在这些方法中,再沉淀自组装的方法具有操作简单,不需要亲水基团即可形成水分散性良好的有机纳米颗粒等优点,引发了广泛的研究。
发明内容
本发明的目的是解决现有肿瘤治疗试剂生物相容性差、无特定靶向性、组织渗透性低、治疗不彻底等缺陷,合成了一种肿瘤血管阻断协同光治疗的试剂,可以作为一种新型的肿瘤精准、高效治疗纳米试剂。
本发明的目的通过以下技术方案实现的:
一种新型肿瘤血管阻断协同光治疗试剂(表达式为DAA),其结构如式Ⅰ所示:
如式Ⅰ所示的DAA的化学名称为:3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)-1,4-二氧吡咯并[3,4-C]吡咯-2,5(1H,4H)-二)双(己烷-6-1-二酰基)双(2-(5-6-二甲基-9-氧代-9H-黄烷-4-基)乙酸酯。
本发明所述的肿瘤血管阻断协同光治疗试剂主要是由烷基化的呋喃吡咯并吡咯二酮(FDPP)与具有微酸刺激响应的二乙基苯胺基以及血管阻断剂2,5-己酮可可碱(DMXAA)共轭键接形成。
本发明的另一个目的是提供所述的肿瘤血管阻断协同光治疗试剂的合成方法,包含以下步骤:
步骤(1)、氮气保护下,双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮(记为DPP-1)、N,N-二乙基-4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)苯胺、三(二亚苄基丙酮)二钯、三(邻甲基苯基)磷、四丁基溴化铵(相转移催化剂)、碳酸钾加入到去离子水和甲苯中,加热搅拌,除去溶剂后得到粗产品,粗产品分离后得到2,5-双(6-溴己基)-3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)吡咯并[3,4-C]吡咯-1,4(2H,5H)-二酮(记为DPP-2);
步骤(2)、2,5-双(6-溴己基)-3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)吡咯并[3,4-C]吡咯-1,4(2H,5H)-二酮、DMXAA、碳酸钾在N,N-二甲基甲酰胺和四氢呋喃的混合溶剂中,加热搅拌,除去溶剂后得到粗产品,粗产品分离纯化得到如式Ⅰ所示的DAA;
步骤(3)、为了得到在磷酸盐缓冲溶液中均匀分散的DAA纳米颗粒,将DAA溶于四氢呋喃中,慢慢滴加到搅拌的磷酸盐缓冲溶液中,继续搅拌5-10分钟后,由DAA分子自组装形成DAA纳米颗粒,氮气充分鼓吹溶液中的四氢呋喃,离心,取上清液,得到肿瘤血管阻断协同光治疗试剂的DAA纳米粒子(记为DAA NPs)。
步骤(1)中,所述的DPP-1与N,N-二乙基-4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)苯胺的摩尔比是1:2-10;DPP-1、三(二亚苄基丙酮)二钯、三(邻甲基苯基)磷、四丁基溴化铵与碳酸钾的摩尔比是1:0.01-0.1:0.1-0.5:0.1-0.5:1-20。
步骤(1)中,所述的加热搅拌的温度为80-120℃,加热搅拌的时间为8-30小时;优选的,所述的加热搅拌的温度为90-120℃,加热搅拌的时间为12-24小时。
所述的去离子水和甲苯的体积比为1:6-10,以增加反应物的溶解性。
所述的粗产品经硅胶色谱柱分离纯化得到DPP-2,洗脱剂为石油醚:二氯甲烷=1:5V/V。
步骤(2)中,所述的DPP-2与DMXAA的摩尔比是1:1-15;DPP-2与碳酸钾的摩尔比是1:1-10。
步骤(2)中,所述的加热搅拌的温度为25-120℃,加热搅拌的时间为8-36小时;优选的,加热搅拌的温度为90-100℃,加热搅拌的时间为12-24小时。
步骤(2)中,为了提高反应物的溶解度,混合溶剂中N,N-二甲基甲酰胺和四氢呋喃的体积比为1-2:1;优选的,N,N-二甲基甲酰胺和四氢呋喃的体积比是1:1。
步骤(2)中,所述的粗产品分离纯化方法包括硅胶色谱柱分离及重结晶法分离。所述的硅胶色谱柱的洗脱剂为石油醚:二氯甲烷=1:5V/V;重结晶采用氯仿和甲醇混合溶剂,氯仿:甲醇=1:1V/V,在室温下重结晶。
步骤(3)中,DAA在四氢呋喃中浓度为1-5mg/mL,滴加速度为1-30滴/分钟,搅拌的转速为500-2000转/分钟;最终使磷酸盐缓冲溶液(上清液)中DAA纳米颗粒的浓度是100-200μg/mL。
所述的磷酸盐缓冲溶液的pH值为5.0~7.4,优选为5.0~6.8。
本发明的另一个目的是提供所述的有机小分子肿瘤血管阻断协同光治疗试剂在制备荧光、光热成像介导的肿瘤血管阻断协同光治疗药物中的应用。
与现有技术相比,本发明的有益效果:
本发明通过自组装的方法得到吸收波长在近红外区的纳米颗粒,该纳米颗粒具有肿瘤微酸刺激响应的光动力、光热性能以及靶向阻断肿瘤血管的能力,具体表现为:
(1)、本发明制备的肿瘤治疗试剂结构明确、产率高、合成工艺简单;
(2)、本发明制备的肿瘤治疗试剂具有优异的水分散性、精准的主、被动肿瘤靶向性,可以实现血管阻断剂在血管内皮细胞处的酸性刺激释放,提高纳米药物到达肿瘤部位的精准度;
(3)、本发明制备的肿瘤治疗试剂具有肿瘤血管阻断行为、微酸刺激响应的光动力/光热治疗能力,有效杀死肿瘤细胞,抑制肿瘤细胞再生和转移,实现多模式肿瘤治疗,应用前景广阔。
附图说明
图1为本发明肿瘤血管阻断及协同光疗的试剂DAA的核磁1H-NMR图(400MHz,CDCl3),横坐标为化学位移,纵坐标为强度。
图2为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒的紫外可见光吸收光谱。
图3为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒的扫描电子显微镜图。
图4为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒在溶液中(pH7.4)的单线态氧的检测,横坐标为化学位移,纵坐标为荧光强度。
图5为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒在溶液中的光热转换效率的检测,其中,去离子水空白检测作为对比,横坐标为时间,纵坐标为温差。
图6为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒在不同pH值的PBS溶液中血管阻断剂DMXAA的释放比例,横坐标为时间,纵坐标为释放比例。
图7为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒通过流式细胞仪分析不同浓度(0μM,4.5μM,9μM,18μM)下细胞凋亡机理图。
图8为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒(pH 7.4)通过荧光共聚焦显微镜观察细胞其内吞作用。
图9为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒通过人脐静脉内皮细胞检测的血管阻断实验。
图10为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒通过活体成像仪实时观察裸鼠尾静脉注射药物后的荧光变化。
图11为本发明纳米肿瘤血管阻断及协同光疗的试剂DAA纳米颗粒的裸鼠治疗瘤体积变化过程。
具体实施方式
下面通过实施例来进一步说明本发明的技术方案,以便较好的理解本发明的内容。
实施例1
(1)DPP-2的合成
DPP-1(0.80g,1.06mmol)、N,N-二乙基-4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)苯胺(0.65g,2.36mmol)、三(二亚苄基丙酮)二钯(48.70mg,0.05mmol)、三(邻甲基苯基)磷(32.37mg,0.11mmol)、四丁基溴化铵(34.29mg,0.11mmol)、碳酸钾(1.47g,10.60mmol)溶于去离子水(3mL)和甲苯(20mL)中,在氮气氛围中90℃反应24小时,然后去除溶剂,粗产品用硅胶色谱柱(石油醚:二氯甲烷=1:5)进行分离提纯,得到DPP-2(0.41g,产量:43%),化学名称为:2,5-双(6-溴己基)-3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)吡咯并[3,4-C]吡咯-1,4(2H,5H)-二酮。
1H-NMR(400MHz,CDCl3)δ:8.38(s,2H),7.56(d,J=8.7Hz,4H),6.70(d,J=8.9Hz,6H),4.22(t,J=7.3Hz,4H),3.44–3.36(m,12H),1.89–1.82(m,8H),1.51(s,8H),1.21(t,J=7.0Hz,12H).
(2)DAA的合成
DPP-2(0.44g、0.50mmol)、DMXAA(0.31g、1.10mmol)和碳酸钾(0.14g、1mmol)加入到四氢呋喃(10mL)和N,N-二甲基甲酰胺(10mL)的混合溶剂中,在90℃下反应24小时,然后去除溶剂,粗产品用硅胶色谱柱(二氯甲烷:乙酸乙酯=1:5V/V)分离后,再用氯仿和甲醇混合溶剂(氯仿:甲醇=1:1V/V)在室温下重结晶,得到DAA(0.45g,产率:70%)。
1H-NMR(400MHz,CDCl3)δ8.35(s,2H),8.21(dd,J=8.0,1.5Hz,2H),8.02(d,J=8.1Hz,2H),7.58–7.55(m,2H),7.51(d,J=8.7Hz,4H),7.27(d,J=6.1Hz,1H),7.24(s,1H),7.12(d,J=8.1Hz,2H),6.66(d,J=8.8Hz,6H),4.12(dt,J=13.0,6.7Hz,8H),3.92(s,4H),3.37(d,J=6.8Hz,8H),2.35(d,J=2.3Hz,12H),1.75(dd,J=14.5,7.3Hz,4H),1.64(dd,J=11.7,4.5Hz,3H),1.60(s,1H),1.50–1.42(m,4H),1.41–1.34(m,4H),1.18(t,J=7.0Hz,12H)。
实施例2
DAA纳米颗粒(DAA NPs)的制备
1mg DAA固体药物溶于500μL THF,以20滴/分钟的滴加速度慢慢滴加到快速搅拌(转速为1000转/分钟)的磷酸盐缓冲溶液(磷酸盐缓冲溶液的pH分别为7.4,6.5和5.0)中,最终使磷酸盐缓冲溶液中DAA纳米颗粒的浓度是200μg/mL。继续搅拌5分钟后,氮气充分鼓吹溶液中的四氢呋喃,然后离心,取上清液,即得肿瘤血管阻断及协同光疗试剂DAA纳米粒子。
如图2,在DAA纳米粒子(pH 7.4)的紫外可见光吸收光谱中,在535nm-850nm之间有很宽的吸收峰,说明最终的DAA纳米颗粒药物对光具有很强的吸收及利用率。图3为DAA纳米粒子(pH 7.4)的扫描电子显微镜形貌(固态样),纳米粒子的平均尺寸约55nm。
采用pH6.5和5.0的磷酸盐缓冲溶液制备得到的DAA纳米粒子,在535nm-850nm之间同样有很宽的吸收峰,纳米粒子的平均尺寸约为55nm。
实施例3
DAA纳米颗粒的单线态氧及光热转换效率检测
通过对单线态氧荧光探针(SOSG)长达20分钟的氧化反应,检测出DAA纳米颗粒(pH7.4)的单线态氧生成能力。如图4,随着光照时间的增长,SOSG在531nm处的荧光强度逐渐增强,说明DAA纳米颗粒具有良好的单线态氧生成能力。
DAA纳米颗粒磷酸盐缓冲溶液于660nm激光(0.8w/cm2)下照射10分钟,如图5,当温度达到平衡时,室温冷却;温度的大幅度上升表明DAA纳米颗粒具有极强的光热转换能力。相同条件下检测去离子水作为空白对照。
实施例4
DMXAA的释放
将DAA纳米颗粒磷酸盐缓冲溶液置于截留分子量为500的透析袋中进行透析,记录不同pH值的磷酸盐缓冲溶液外渗液的DMXAA的吸收值随时间的变化,由图6可见,DMXAA从前体药物的释放具有低pH值依赖性,在pH 5.0时释放最多。这对于DMXAA在肿瘤微酸环境的释放具有很好的指导意义。
实施例5
DAA纳米颗粒对肿瘤细胞体外定量毒性、内吞作用检测及体外血管破坏作用的检测
选取HeLa肿瘤细胞(购于GIBCO)进行体外定量毒性实验,测试其激光毒性。具体实验步骤如下:DAA纳米颗粒溶解在PBS溶液(pH 7.0)中,然后用DMEM稀释成不同浓度(0μM,4.5μM,9μM,18μM)。HeLa细胞被接种在6孔培养板,37℃下培养使其贴壁生长24小时,避光加药(500μL),且避光培养24小时后,由660nm激光器分别照射各孔4分钟(0.8W/cm2),继续培养12小时,然后用Annexin VFITC和propidium iodide(PI)进行染色。使用流式细胞分析仪计算绝对的细胞存活率,如图7所示,细胞存活率存在反浓度依赖性,给药浓度越大细胞存活率越低,IC50值约为9μM。
HeLa细胞被接种在共聚焦小皿中,37℃下培养使其贴壁生长24小时,避光加药(1mL,IC50浓度)培养24小时后,用激光扫描倒置荧光显微镜观察细胞的内的荧光强度,如图8所示,图8A表示HeLa肿瘤细胞形态良好,边界分明;图8B表示纳米药物强烈的荧光信号;图8C为荧光信号与肿瘤细胞的重合情况,说明DAA纳米药物在肿瘤细胞内有良好的荧光成像性能,对活体肿瘤治疗具有指导意义。
人脐静脉内皮细胞(购于GIBCO)接种在解冻的人工基底膜基质胶(购于GIBCO)上,3小时待血管形成后加药培养4小时,用激光扫描倒置荧光显微镜观察血管状态并用钙黄绿素(calcein)进行染色,如图9所示,图9A是血管的明场,图9B是空白组血管的Calcein(钙黄绿苏)染色,图9C是给药组血管的Calcein染色,说明DAA纳米药物对已有血管存在理想的破坏作用。
实施例6
DAA纳米颗粒体内荧光检测及肿瘤治疗实验
通过活体荧光成像仪观察纳米药物进入荷瘤(HeLa细胞)裸鼠体内后荧光从无到有的过程(时间点分别为0h,2h,4h,6h,12h,24h)。处理组:通过尾静脉注射法将pH 7.4的DAA纳米颗粒磷酸盐缓冲溶液(100μg/mL,100μL)注入荷瘤鼠体内,利用活体荧光成像仪观测不同时间点下肿瘤内及最终各器官药物的荧光变化情况。如图10所示,空白组(左侧,不注射DAA纳米颗粒磷酸盐缓冲溶液)肿瘤部位的温度在激光(660nm,0.8W/cm2)照射8min后仅升高3.7℃,处理组(右侧)在相同条件下升高了20.8℃,说明DAA纳米颗粒在体内仍然具有良好的光热性能。
选择将HeLa肿瘤细胞注入腋下的裸鼠作为肿瘤模型,24只裸鼠随机分为4组。当肿瘤体积约为100mm3时,第一组(Saline)通过尾静脉注射生理盐水,第二组(DAA Only)小鼠通过尾静脉注射DAA纳米颗粒(100μg/mL,100μL,pH 7.4)、第三组(DPP-2Only)小鼠通过尾静脉注射DPP-2NPs(制备方法同DAA纳米颗粒的制备,100μg/mL,100μL,pH 7.4)、第四组(DAA+Laser)小鼠通过尾静脉注射以及DAA纳米颗粒溶液(100μg/mL,100μL,pH7.4)。12小时后,第三、四组小鼠的肿瘤在660nm(0.8W/cm2)激光器各照射4分钟,其他两组不进行光照。上述过程重复了18天,每2天测量一次肿瘤大小。如图11,第四组在第六天就基本消除掉了肿瘤组织,由于相比第三组,第四组不仅具有pH响应的光热及光动力治疗效果同时具备了肿瘤血管阻断的效果,可以有效地彻底消除肿瘤。
Claims (10)
1.一种肿瘤血管阻断协同光治疗试剂,其结构如式Ⅰ所示:
2.一种权利要求1所述的肿瘤血管阻断协同光治疗试剂的合成方法,其特征在于包括以下步骤:
步骤(1)、氮气保护下,双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮、N,N-二乙基-4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)苯胺、三(二亚苄基丙酮)二钯、三(邻甲基苯基)磷、四丁基溴化铵、碳酸钾加入到去离子水和甲苯中,加热搅拌,除去溶剂后得到粗产品,粗产品分离纯化得到2,5-双(6-溴己基)-3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)吡咯并[3,4-C]吡咯-1,4(2H,5H)-二酮;
步骤(2)、2,5-双(6-溴己基)-3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)吡咯并[3,4-C]吡咯-1,4(2H,5H)-二酮、DMXAA、碳酸钾在N,N-二甲基甲酰胺和四氢呋喃的混合溶剂中,加热搅拌,除去溶剂后得到粗产品,粗产品分离后得到如式Ⅰ所示的DAA;
步骤(3)、DAA溶于四氢呋喃中,滴加到搅拌的磷酸盐缓冲溶液中,继续搅拌5-10分钟后,氮气充分鼓吹溶液中的四氢呋喃,离心,取上清液,得到肿瘤血管阻断协同光治疗试剂的DAA纳米粒子。
3.根据权利要求2所述的肿瘤血管阻断协同光治疗试剂的合成方法,其特征在于步骤(1)中,所述的双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮与N,N-二乙基-4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)苯胺的摩尔比是1:2-10;双(3-5-溴呋喃-2-基)-5-二(6-4-溴己基)吡咯并吡咯(2,5)-1,4-二酮、三(二亚苄基丙酮)二钯、三(邻甲基苯基)磷、四丁基溴化铵与碳酸钾的摩尔比是1:0.01-0.1:0.1-0.5:0.1-0.5:1-20。
4.根据权利要求2所述的肿瘤血管阻断协同光治疗试剂的合成方法,其特征在于步骤(1)中,所述的加热搅拌的温度为80-120℃,加热搅拌的时间为8-30小时;优选的,所述的加热搅拌的温度为90-120℃,加热搅拌的时间为12-24小时。
5.根据权利要求2所述的肿瘤血管阻断协同光治疗试剂的合成方法,其特征在于步骤(2)中,所述的2,5-双(6-溴己基)-3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)吡咯并[3,4-C]吡咯-1,4(2H,5H)-二酮与DMXAA的摩尔比是1:1-15;2,5-双(6-溴己基)-3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)吡咯并[3,4-C]吡咯-1,4(2H,5H)-二酮与碳酸钾的摩尔比是1:1-10。
6.根据权利要求2所述的肿瘤血管阻断协同光治疗试剂的合成方法,其特征在于步骤(2)中,所述的加热搅拌的温度为25-120℃,加热搅拌的时间为8-36小时;优选的,加热搅拌的温度为90-100℃,加热搅拌的时间为12-24小时。
7.根据权利要求2所述的肿瘤血管阻断协同光治疗试剂的合成方法,其特征在于步骤(1)所述的去离子水和甲苯的体积比为1:6-10;
步骤(2)中,所述的混合溶剂中N,N-二甲基甲酰胺和四氢呋喃的体积比为1:1-10;优选的,N,N-二甲基甲酰胺和四氢呋喃的体积比是1:1-4。
8.根据权利要求2所述的肿瘤血管阻断协同光治疗试剂的合成方法,其特征在于步骤(1)中,所述的粗产品经硅胶色谱柱分离纯化得到2,5-双(6-溴己基)-3,6-双(5-(4-(二乙基氨基)苯基)呋喃-2-基)吡咯并[3,4-C]吡咯-1,4(2H,5H)-二酮,洗脱剂为石油醚:二氯甲烷=1:5V/V;
步骤(2)中,所述的粗产品分离纯化方法包括硅胶色谱柱分离及重结晶法分离;所述的硅胶色谱柱的洗脱剂为石油醚:二氯甲烷=1:5V/V;重结晶采用氯仿和甲醇混合溶剂,氯仿:甲醇=1:1V/V。
9.根据权利要求2所述的肿瘤血管阻断协同光治疗试剂的合成方法,其特征在于步骤(3)中,所述DAA在四氢呋喃中浓度为1-5mg/mL,滴加速度为1-30滴/分钟,搅拌的转速为500-2000转/分钟;磷酸盐缓冲溶液中DAA纳米粒子的终浓度是100-200μg/mL;
所述的磷酸盐缓冲溶液的pH值为5.0~7.4,优选为5.0~6.8。
10.根据权利要求1所述的肿瘤血管阻断协同光治疗试剂在制备荧光、光热成像介导的肿瘤血管阻断协同光治疗药物中的应用。
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