CN117414424A - 基于萘二酐小分子的光敏剂制备方法 - Google Patents
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Abstract
本发明公开了一种基于萘二酐小分子的光敏剂制备方法,包括以下步骤:A.将四溴取代萘四甲酸酐与1,2‑苯二胺在N,N‑二甲基甲酰胺混合物加热生成NDI‑1,将NDI‑1与PbO2在二氯甲烷中搅拌纯化得到NDI‑AB。B.CaCl2·2H2O、槲皮素以及NDI‑AB加入含有乙醇的平底烧杯中,在另一个平底烧杯中加入NH4HCO3,在40℃下反应,离心收集产物。C.将该产物的乙醇溶液在磁力搅拌器上缓慢加入到DSPE‑mPEG2000的混合水溶液中,通过旋蒸去除溶液中的乙醇和超净水。重新加入超净水溶解后得到PQCN。本发明所制得的光敏剂可实现荧光/光声成像引导下的钙超载治疗肝癌。
Description
技术领域
本发明涉及癌症光诊疗试剂技术领域,尤其是一种基于萘二酐小分子的光敏剂制备方法。
背景技术
肝癌是一种非常常见的恶性肿瘤,在癌症引起的死亡中占有非常高的比例。由于早期肝癌没有明显的症状,并且不具备典型的影像学特征,所以患者不能得到早期的诊断和治疗。现有的传统治疗方法有经导管动脉化疗栓塞、消融、肝切除、肝移植等,但是治疗效果并不尽如人意。与此同时,肿瘤部位的血管损伤、肿瘤微环境的微酸性以及肿瘤的耐药性都给肿瘤的治疗以及现有药物的靶向带来了难度。随着近年来纳米生物技术的发展,纳米材料已经成为肿瘤治疗的有效手段。研究表明,纳米颗粒独有的体积效应和表面效应等物理性质,在血液循环中能通过“EPR”效应在肿瘤部位有效的累积,并且与各种传统医疗手段相结合实现更加有效副作用更小的诊疗。因此,纳米技术为肿瘤治疗提供了新的希望。
随着分子成像技术的快速发展,分子成像技术已成为研究生命过程、疾病机理和药物设计的重要工具。在体内,生物分子具有独特的理化性质和空间分布特征,这些性质和空间分布特征可以用来表征生物系统。在过去的几十年里,基于活体分子成像技术的发展,已建立了许多用于研究生物过程、疾病机理和药物设计的成像平台。
长久以来,肿瘤被研究者认为是孤立的细胞群,在器官特定的部位独立存在。近年来,人们逐步意识到,肿瘤与ECM、血管、结缔组织和免疫细胞之间存在着某种交互作用,被称为“肿瘤微环境”(tumor microenvironment,TME)。癌症的发生与其所处的环境有着紧密的联系。肿瘤可以将其向细胞外释放出的信号因子,从而对肿瘤血管的产生起到加速作用,引发周围癌细胞的免疫耐受性,从而对肿瘤的生长、侵袭以及转移产生积极的影响。同时,研究人员通过研究了免疫细胞与肿瘤微环境之间的相互作用,从而了解免疫细胞如何通过改变肿瘤微环境来影响癌细胞的生长和演化。他们发现,免疫细胞可以通过直接或间接影响肿瘤微环境来改变癌细胞的生物学特性。例如,当免疫细胞被困在肿瘤周围时,它们可以进入癌细胞并抑制其增殖或促进其凋亡。此外,它们还可以刺激癌细胞对放化疗产生耐药性。这样不断变化、相互影响的微妙的肿瘤与微环境的关系,给肿瘤的诊断、药物的设计以及肿瘤的治疗方面提出了严峻的挑战。面对以上情况,在治疗的过程中,针对TME与正常组织的不同设计出TME响应性的分子探针可以有效得应对肿瘤发展过程中复杂的生化反应。
光热治疗(PTT)是一种高效的,而且具有比较低的侵入性的治疗方法,它是依靠光热制剂(PTAs)在受到近红外激光(NIR)照射时,使局部快速升温,从而导致肿瘤细胞凋亡和坏死。PTT治疗的步骤有两个:一是将光敏剂输送到肿瘤,二是使用特殊的激光对其进行照射。光敏剂经过激光照射之后,将吸收的光能转化为热能。这种热可以增加肿瘤的温度,对其造成不可逆的细胞损伤至肿瘤消融清除。肿瘤的升温程度取决于光敏剂的光热转换效率,肿瘤内富集的光敏剂浓度以及照射激光的功率。同时,恶性肿瘤与人体正常组织的血管形成方式有所不同,恶性肿瘤的血管通常是不规则的,血液在其中流通的时候会发生回流不畅的情况,所以在光热治疗的时候,肿瘤组织局部温度往往比正常组织温度要高一些。然而,光热治疗也有其无法避免的缺陷,例如,PTA在肿瘤中的传递效果比较差,肿瘤区域过热会造成对正常组织的不必要损伤,而且有些肿瘤中热激蛋白的过表达会降低PTT的疗效等。为提高PTT对肿瘤的疗效,研究人员曾作过许多尝试,如:确定药物进入体内后的最优吸收时机;通过调控纳米粒的形态、尺寸、表面修饰等,增强PTA的靶向性等。通常情况下,现在的科学研究会与其他的治疗方法相结合。这种疗法,不是简单的每种疗法单独作用的简单加和,而是协同作用。这个治疗,并不是单纯的两种治疗方法的叠加,而是两种治疗方法的协同。光热治疗(PTT)是一种可以提高载药系统的载药能力、促进药物的快速释放、调节肿瘤微环境等,从而实现对肿瘤细胞的直接杀伤和提高其它治疗方法的疗效。
线粒体是细胞内重要的细胞器,是进行生物氧化和能量交换的主要场所,也是细胞中非常重要的钙库。线粒体的功能发生障碍是诱导细胞发生凋亡最主要的原因之一。正常机体在代谢过程中,细胞内外钙离子浓度之间存在着一定的确定的浓度差。在此过程中,钙离子的浓度差的形成有赖于某些跨膜运输机制,使其保持在一个较低的水平,我们称之为钙稳态。正常的钙离子平衡是维持机体生理功能和Ca2+功能的前提。外源性的刺激能够导致细胞内Ca2+浓度的增高,从而导致细胞处于一种钙超载的状态,由于线粒体钙超载而导致的线粒体外膜渗透性的提高,这可能是激活线粒体凋亡通路的一个关键原因。由于线粒体钙超载,会让ROS过剩产生,进而引发膜脂质过氧化,对线粒体膜及线粒体的结构造成破坏,还会激发内源性核酸酶等多种酶,会造成线粒体膜电位稳态被破坏、通透性提高,将线粒体膜内的促凋亡因子释放到胞中,最终造成细胞凋亡。
瞬时受体电位蛋白(Transient receptor potential,TRP)家族是存在于细胞膜或胞内细胞器上的一种超家族离子通道蛋白,是细胞Ca2+转运的重要通道。目前,TRPV1是人们关注的焦点,它也被称为辣椒素受体,能够被多种因素选择性地活化,比如:capsaicin辣椒素、伤害性热刺激、酸刺激等。TRPV1通道在机体中参与疼痛感知,体温的维持以及渗透压调控等重要的生理功能。在一些肿瘤的发生过程中,伤害性感受神经的TRPV1通道的表达量上调,同时这种通道对Ca2+具有高通道性,而当TRPV1通道激活,Ca2+由外部大量流入细胞内部之后,能够减弱线粒体的膜电位,抑制线粒体的生物活性,显著提高受损肿瘤细胞的凋亡率。
热休克蛋白(Heat Shock Proteins,HSPs)是机体中的一种高度保护蛋白,目前,细胞凋亡最主要的凋亡方式主要分为外在的死亡受体介导的凋亡途径以及内在的线粒体凋亡,均由半胱氨酸蛋白酶(Caspase)家族介导,两种途径的信号传导都依赖Caspase-3水平。热休克蛋白(HSP)是一种很好的抗肿瘤药物设计切入点,可转变成其它生物因子的分子伴侣。这种功能是指在正确的情况下,通过对蛋白进行再折叠或者将其降解。热刺激诱导HSP的表达和积累,证明了死亡刺激可以在细胞中引起保护性反应,HSP具有重要的抗细胞凋亡特性。HSP70是一个具有决定作用的负向调控蛋白,它可以通过对应激引起的信号进行抑制,从而在线粒体凋亡的不同时期,阻止细胞的凋亡。由一氧化氮和热应激刺激的凋亡级联Bax从细胞质转移至线粒体,其被HSP70的过表达抑制,从而抑制了细胞的凋亡。近年来的研究中,槲皮素常常被作为HSP的抑制剂来使用。研究发现,染料木素、山奈酚和黄酮在热胁迫下对HSP70、HSP110和HSP40的抑制程度可降低到无热激发的正常状态。在以往的研究中,槲皮素表现出来的热休克蛋白抑制机制与其他抑制剂不尽相同。格尔德霉素(Geldanamycin,GA)可以和ATP竞争结合位点,干扰与HSP90招募蛋白的结合,抑制HSP90的功能。而槲皮素则表现出抑制HSP转录或者翻译的活性。
萘二酰亚胺(NDIs)是一种快速发展的芳香族化合物,具有优异的电子、光谱和自组装性质,在有机、生物超分子化学、生物医学和材料科学等领域具有重要的应用价值。研究者们通过酰亚胺氮或通过核取代(萘核上的取代)的功能化产生改变NDIs的电学、光学以及氧化还原性质,产生类似物。本申请的实施将为制备高稳定性、高发光效率的发光纳米粒子提供一条有效途径,并为其在生命科学领域的发展提供一条新的思路。
发明内容
本发明针对现有技术的不足,提供了一种基于萘二酐小分子的光敏剂制备方法。
为解决上述技术问题,本发明所采取的技术方案如下
一种基于萘二酐小分子的光敏剂制备方法,包括以下步骤:
A.将四溴取代萘四甲酸酐与1,2-苯二胺在N,N-二甲基甲酰胺混合物加热生成NDI-1;
纯化后将NDI-1与PbO2在二氯甲烷中搅拌纯化得到NDI-AB。
B.将CaCl2·2H2O、槲皮素以及NDI-AB加入含有乙醇的平底烧杯A中,在另一个平底烧杯B中加入NH4HCO3,将A置于B中;待颗粒负载后,收集CaCO3@Quercetin@NDI-AB。
C.将CaCO3@Quercetin@NDI-AB的乙醇溶液加入到DSPE-mPEG2000的混合水溶液中。通过旋蒸去除溶液中的乙醇和超净水。
重新加入超净水溶解后得到PQCN(DSPE-mPEG2000@Quercetin@CaCO3@NDI-AB)。
作为优选,以下份数均为质量数:
A.将12~20份四溴取代萘四甲酸酐与1~4份1,2-苯二胺在N,N-二甲基甲酰胺混合物加热生成NDI-1,在通过TLC监测到4Br-NDI耗尽后,用石油醚稀释反应混合物,然后用超纯水洗涤除去DMF。有机溶液用Na2SO4干燥,然后真空旋蒸除去溶剂。粗产物用硅胶柱色谱纯化,用二氯甲烷/石油醚洗脱,得到NDI-1,为深紫色固体。
纯化后将2~4份NDI-1与5~10份PbO2在二氯甲烷中搅拌,当NDI-1耗尽时,通过TLC监测,将溶液冷却并过滤以除去不溶物质。溶液用无水Na2SO4干燥,然后真空旋蒸除去溶剂,得到NDI-AB。
B.40~60份CaCl2·2H2O、1~4份槲皮素以及1~4份NDI-AB加入含有乙醇的平底烧杯A中,在另一个平底烧杯B中加入1000~2000份NH4HCO3;
然后,将平底烧杯A置于平底烧杯B中,后将两个平底烧杯放在密封容器中,反应在40℃下进行72小时;通过离心收集CaCO3@Quercetin@NDI-AB,并用乙醇洗涤。
C.将1~4份CaCO3@Quercetin@NDI-AB的乙醇溶液加入到1~4份DSPE-mPEG2000的混合水溶液中。通过旋蒸去除溶液中的乙醇和超净水。重新加入超净水溶解后得到PQCN(DSPE-mPEG2000@Quercetin@CaCO3@NDI-AB)。
作为优选,以下份数均为质量数:
A.将15.21份四溴取代萘四甲酸酐与2.16份1,2-苯二胺在N,N-二甲基甲酰胺混合物130℃下加热1小时,在通过TLC监测到4Br-NDI耗尽后,用石油醚(50mL)稀释反应混合物,然后用超纯水洗涤三次以除去DMF。有机溶液用Na2SO4干燥,然后真空旋蒸除去溶剂。粗产物用硅胶柱色谱纯化,用二氯甲烷/石油醚(DCM/PE=1/4,v/v)洗脱,得到NDI-1,为深紫色。
将2.66份NDI-1与10.0份PbO2在二氯甲烷(4mL)中在50℃下搅拌24小时,溶液的颜色从紫色变成黄色。当NDI-1耗尽时,通过TLC监测,将溶液冷却并过滤以除去不溶物质。溶液用无水Na2SO4干燥,然后真空旋蒸除去溶剂,得到NDI-AB,为黄色固体。
B.50.0份CaCl2·2H2O、1.33份槲皮素以及1.33份NDI-AB加入含有乙醇的平底烧杯A中,并用带孔的塑料包装覆盖平底烧杯;在另一个平底烧杯B中加入1666.67份NH4HCO3。
然后,将平底烧杯A置于平底烧杯B中,后将两个平底烧杯放在密封容器中。反应在40℃下进行72小时。通过以10,000rpm重复离心收集CaCO3@Quercetin@NDI-AB,并用乙醇洗涤两次。
C.将1.33份CaCO3@Quercetin@NDI-AB的乙醇溶液在磁力搅拌器(900r/min)上缓慢加入到1.33份DSPE-mPEG2000的混合水溶液中。在超声环境中充分震荡,通过旋蒸去除溶液中的乙醇和超净水。
重新加入超净水溶解后得到PQCN(DSPE-mPEG2000@Quercetin@CaCO3@NDI-AB)。
作为优选,所述NDI-AB的分子结构式为:
本发明还提供了一种基于萘二酐小分子的光敏剂在钙超载治疗中的应用。
作为优选,所述的钙超载治疗为荧光成像/光声成像双模态的成像引导下的钙超载治疗。
采用上述方法所带来的有益效果为:
(1)通过亲核试剂取代1,4,5,8-萘二酰亚胺(NDIs)萘核上的卤素原子,有易于调节的HOMO/LUMO水。
(2)通过将较大的基团连接到核心来修饰NDIs染料,限制分子间的p-相互作用并阻碍分子间的电子耦合,解决荧光猝灭的问题,使得核心发色团增加光发射的量子效率。
(3)基于“EPR”效应,PQCN纳米颗粒可以实现对肿瘤组织的被动靶向,使其长时间滞留在肿瘤之中,提高疗效。
(4)PQCN纳米颗粒具有有效的生物组织穿透力,可以实现荧光成像和光声成像的实时监测。
(5)具有pH响应的特性,只有在肿瘤微酸环境中,可以释放出NDI-AB小分子、槲皮素、Ca2+,降低了治疗的副作用。
(6)在外源性激光激发下,利用小分子的光热性能,可以有效激发肿瘤细胞膜上的TRPV1通道,使得钙离子大量内流,实现钙超载治疗,大大提升了治疗效率。
本发明对提高肝癌治疗效率,全面评估与分析肝癌及其他肿瘤诊疗体系具有非常重要的理论研究价值和临床使用价值。
附图说明
图1是本发明NDI-AB、DSPE-mPEG2000@NDI-AB、PCN与PQCN纳米颗粒的吸收光谱;
图2是本发明PQCN纳米颗粒的透射电镜图;
图3是本发明不同浓度PQCN纳米颗粒水溶液的(由左至右0,25,50,75,100微克/毫升)的光声成像对比图;
图4是本发明PQCN纳米颗粒在HepG2肝癌细胞中的MTT实验结果图,其中a为pH=7.4时不同浓度PQCN纳米颗粒的MTT毒性分析图;b为pH=6.5时不同浓度PQCN纳米颗粒的MTT毒性分析图;
图5是本发明DSPE-mPEG2000@NDI-AB、PCN与PQCN纳米颗粒在长有HepG2肿瘤的裸鼠体内进行治疗的肿瘤体积大小变化图。
具体实施方式
实施例1
将15.21份四溴取代萘四甲酸酐与2.16份1,2-苯二胺在N,N-二甲基甲酰胺混合物130℃下加热1小时,在通过TLC监测到4Br-NDI耗尽后,用石油醚(50mL)稀释反应混合物,然后用超纯水洗涤三次以除去DMF。有机溶液用Na2SO4干燥,然后真空旋蒸除去溶剂。粗产物用硅胶柱色谱纯化,用二氯甲烷/石油醚(DCM/PE=1/4,v/v)洗脱,得到NDI-1,为深紫色。将2.66份NDI-1与10.0份PbO2在二氯甲烷(4mL)中在50℃下搅拌24小时,溶液的颜色从紫色变成黄色。当NDI-1耗尽时,通过TLC监测,将溶液冷却并过滤以除去不溶物质。溶液用无水Na2SO4干燥,然后真空旋蒸除去溶剂,得到NDI-AB,为黄色固体。
50.0份CaCl2·2H2O、1.33份槲皮素以及1.33份NDI-AB加入含有乙醇的平底烧杯A中,并用刺有几个孔的塑料包装覆盖平底烧杯;在另一个平底烧杯B中加入1666.67份NH4HCO3。然后,将A置于B中,后将两个平底烧杯放在密封容器中。反应在40℃下进行72小时。通过以10,000rpm重复离心收集CaCO3@Quercetin@NDI-AB,并用乙醇洗涤两次。
将1.33份CaCO3@Quercetin@NDI-AB的乙醇溶液在磁力搅拌器(900r/min)上缓慢加入到1.33份DSPE-mPEG2000的混合水溶液中。在超声环境中充分震荡,通过旋蒸去除溶液中的乙醇和超净水。重新加入超净水溶解后得到PQCN(DSPE-mPEG2000@Quercetin@CaCO3@NDI-AB)。
所得到光敏剂可以实现荧光成像/光声成像双模态的成像引导下的钙超载治疗,用于肝癌的诊疗过程中的监测与治疗。
对比例1
15.21份四溴取代萘四甲酸酐与2.16份1,2-苯二胺在N,N-二甲基甲酰胺混合物130℃下加热1小时,在通过TLC监测到4Br-NDI耗尽后,用石油醚(50mL)稀释反应混合物,然后用超纯水洗涤三次以除去DMF。有机溶液用Na2SO4干燥,然后真空旋蒸除去溶剂。粗产物用硅胶柱色谱纯化,用二氯甲烷/石油醚(DCM/PE=1/4,v/v)洗脱,得到NDI-1,为深紫色。将2.66份NDI-1与10.0份PbO2在二氯甲烷(4mL)中在50℃下搅拌24小时,溶液的颜色从紫色变成黄色。当NDI-1耗尽时,通过TLC监测,将溶液冷却并过滤以除去不溶物质。溶液用无水Na2SO4干燥,然后真空旋蒸除去溶剂,得到NDI-AB,为黄色固体。
将1.33份NDI-AB的乙醇溶液在磁力搅拌器(900r/min)上缓慢加入到1.33份DSPE-mPEG2000的混合水溶液中。在超声环境中充分震荡,通过旋蒸去除溶液中的乙醇和超净水。重新加入超净水溶解后得到DSPE-mPEG2000@NDI-AB。
对比例2
15.21份四溴取代萘四甲酸酐与2.16份1,2-苯二胺在N,N-二甲基甲酰胺混合物130℃下加热1小时,在通过TLC监测到4Br-NDI耗尽后,用石油醚(50mL)稀释反应混合物,然后用超纯水洗涤三次以除去DMF。有机溶液用Na2SO4干燥,然后真空旋蒸除去溶剂。粗产物用硅胶柱色谱纯化,用二氯甲烷/石油醚(DCM/PE=1/4,v/v)洗脱,得到NDI-1,为深紫色。将2.66份NDI-1与10.0份PbO2在二氯甲烷(4mL)中在50℃下搅拌24小时,溶液的颜色从紫色变成黄色。当NDI-1耗尽时,通过TLC监测,将溶液冷却并过滤以除去不溶物质。溶液用无水Na2SO4干燥,然后真空旋蒸除去溶剂,得到NDI-AB,为黄色固体。
50.0份CaCl2·2H2O、以及1.33份NDI-AB加入含有乙醇的平底烧杯A中,并用刺有几个孔的塑料包装覆盖平底烧杯;在另一个平底烧杯B中加入1666.67份NH4HCO3。然后,将A置于B中,后将两个平底烧杯放在密封容器中。反应在40℃下进行72小时。通过以10,000rpm重复离心收集CaCO3@NDI-AB,并用乙醇洗涤两次。
将1.33份CaCO3@NDI-AB的乙醇溶液在磁力搅拌器(900r/min)上缓慢加入到1.33份DSPE-mPEG2000的混合水溶液中。在超声环境中充分震荡,通过旋蒸去除溶液中的乙醇和超净水。重新加入超净水溶解后得到PCN(DSPE-mPEG2000@CaCO3@NDI-AB)。
NDI-AB、DSPE-mPEG2000@NDI-AB、PCN与PQCN纳米颗粒的吸收光谱测试实验:
精密称取10mg的NDI-AB、DSPE-mPEG2000@NDI-AB、PCN与PQCN,以CHCl2和超纯水为溶剂,以CHCl2空白溶剂作为参比,使用紫外分光光度计分别扫描不同溶液在300-1000nm范围内的紫外吸收图谱,并记录吸光度值。如图1所示。NDI-AB分子的紫外吸收峰在647nm,在外层修饰DSPE-mPEG2000后,吸收峰有了25nm的红移,并且吸收峰变成了两个宽泛峰;合成PCN之后,颗粒的紫外吸收峰趋于不明显,没有明显的峰位;PQCN纳米颗粒组装完毕之后,整个颗粒的紫外吸收峰在571nm。
PQCN的透射电镜测试
分别取预先制备好的PQCN纳米颗粒,在透射电镜专用铜网上稀释,孵育2分钟,多次滴后,用滤纸小心吸收残留液体。空气干燥后,用透射电镜观察纳米颗粒的形貌。
如图2所示通过透射电镜(TEM)表征了PQCN纳米颗粒(2-6b),通过测试可以看出粒径在143.3nm,呈球形,这原因在于DLS所测得为水合粒径而TEM表征的时候纳米颗粒处于干燥状态。
NDO-AB的光声成像:
在毛细管里分别加入5组不同浓度的探针NDI-AB溶液:0μg/mL、25μg/mL、50μg/mL、100μg/mL、200μg/mL,然后用TOMOWAVE分别进行光声成像并对信号强度进行定量分析。激发波长为660nm。效果如图3所示,在660nm近红外(NIR)激发下,随着PQCN纳米颗粒浓度的升高,光声信号也逐渐增强,从而证明了其优秀的优异的光声性能。也因此,可以将其应用于肿瘤处的诊疗探针。
HepG2肝癌细胞体外光热治疗实验:
实验选取HepG2肝癌细胞进行光热治疗,测试其暗毒性,普通光毒性以及激光毒性。
将HepG2细胞消化后重悬,均匀的铺在96孔板上,每孔100μL细胞(1×104个),同时设置6个复孔,将其放置在37℃恒温CO2培养箱中孵育。用培养基稀释PQCN溶液分别至0μg/mL、25μg/mL、50μg/mL、75μg/mL,除去原本培养基后,每孔加入含材料的培养基100μL。同时使用HCl调节DMEM培养基pH至6.5,使用相同的方法稀释各组溶液并用其替换原本的DMEM培养基。将前述96孔平板置于培养器中进行24小时的温育。将细胞拿出来,在每孔添加20uL过膜无菌5mg/mL MTT溶液,持续进行4h,在吸走上清液后(避免吸走孔板底部的紫色晶体),在每孔添加100uL DMSO,静置孔板10min,就可以观察到底部晶体全部溶液,并且溶液变成了紫色。采用酶标仪,在490nm进行荧光吸收测定,将0μg/mL的药量的吸收值作为对照组(细胞成活率为100%),并计算出各个试验组的细胞成活率。
结果如图4所示,在激光激活后,PQCN的细胞毒性被大幅度增强,即使中性环境下,细胞存活率也仅有26%。微酸环境下(模拟肿瘤微环境),由于药物的摄取量更多,激光激活的纳米颗粒更多,细胞毒性也就更大,细胞存活率不足5%。
肿瘤体内光热治疗实验:
将6-8周的BALB/C裸鼠购入后再SPF级动物房养殖一周之后。将密度达90%的HepG2细胞消化,900rpm离心3min,PBS重悬清洗细胞,重复两次除去残余培养基。通过细胞计数器计数后,将细胞浓度配至5×107个/mL,装至1.5mL离心管中,保存至冰盒中备用。裸鼠的右腿后方用酒精擦拭消毒,用无菌胰岛素注射器将100μL细胞经皮下注射接种至裸鼠右腿后方。荷瘤小鼠随机分为5组,实验组:(1)NDI-AB组;(2)PCN组;(3)PQCN组;(4)PQCN组(外加激光治疗组)。对照组:PBS组。每组6只小鼠,5组小鼠皆通过尾静脉注射100μL稀释后的各组药物以及PBS。给药9h后,PQCN(外加激光治疗组)使用660nm激光在1W/cm2功率下对裸鼠肿瘤部位照射5min。每2天监控一次肿瘤的体积以及小鼠的体重。
图5是PQCN纳米光敏剂在长有HepG2肿瘤的裸鼠体内进行治疗的肿瘤体积大小的变化,17天实验结束后,对比其他两组,光热治疗组对肿瘤起到明显的治疗作用。通过每三天一次的荧光监测可以清晰地察到肿瘤在治疗过程中的大小变化,对照组和NDI-AB、PCN、PQCN组的肿瘤都在逐渐稳定增长,表明未经激光照射的药物抗肿瘤功效弱,并不能有效抑制肿瘤生长。在治疗两次之后,光疗组的荷瘤小鼠的肿瘤被理想得消除,显示出了在光热治疗,钙超载治疗以及抑制热休克蛋白协同治疗下,纳米颗粒的卓越抗肿瘤特性。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (6)
1.基于萘二酐小分子的光敏剂制备方法,其特征在于包括以下步骤:
A.将四溴取代萘四甲酸酐与1,2-苯二胺在N,N-二甲基甲酰胺混合物加热生成NDI-1;
纯化后将NDI-1与PbO2在二氯甲烷中搅拌纯化得到NDI-AB;
B.将CaCl2·2H2O、槲皮素以及NDI-AB加入含有乙醇的平底烧杯A中,在另一个平底烧杯B中加入NH4HCO3,将A烧杯置于B烧杯中;
待颗粒负载后,收集CaCO3@Quercetin@NDI-AB;
C.将CaCO3@Quercetin@NDI-AB的乙醇溶液加入到DSPE-mPEG2000的混合水溶液中,通过旋蒸去除溶液中的乙醇和超净水;
重新加入超净水溶解后得到PQCN(DSPE-mPEG2000@Quercetin@CaCO3@NDI-AB)。
2.根据权利要求1所述的基于萘二酐小分子的光敏剂制备方法,其特征在于包括以下步骤:
以下份数均为质量数:
A.将12~20份四溴取代萘四甲酸酐与1~4份1,2-苯二胺在N,N-二甲基甲酰胺混合物加热生成NDI-1,在通过TLC监测到4Br-NDI耗尽后,用石油醚稀释反应混合物,用超纯水洗涤除去DMF;有机溶液用Na2SO4干燥,然后真空旋蒸除去溶剂;粗产物用硅胶柱色谱纯化,用二氯甲烷/石油醚洗脱,得到NDI-1,为深紫色固体;
将2~4份纯化后NDI-1与5~10份PbO2在二氯甲烷中搅拌,当NDI-1耗尽时,通过TLC监测,将溶液冷却并过滤以除去不溶物质,溶液用无水Na2SO4干燥,然后真空旋蒸除去溶剂,得到NDI-AB;
B.将40~60份CaCl2·2H2O、1~4份槲皮素以及1~4份NDI-AB加入含有乙醇的平底烧杯A中,在另一个平底烧杯B中加入1000~2000份NH4HCO3;
将平底烧杯A置于平底烧杯B中,后将两个平底烧杯放在密封容器中,反应在40℃下进行72小时,通过离心收集CaCO3@Quercetin@NDI-AB,并用乙醇洗涤;
C.将1~4份CaCO3@Quercetin@NDI-AB的乙醇溶液加入到1~4份DSPE-mPEG2000的混合水溶液中,通过旋蒸去除溶液中的乙醇和超净水;
重新加入超净水溶解后得到PQCN(DSPE-mPEG2000@Quercetin@CaCO3@NDI-AB)。
3.根据权利要求2所述的基于萘二酐小分子的光敏剂制备方法,其特征在于包括以下步骤:
A.将15.21份四溴取代萘四甲酸酐与2.16份1,2-苯二胺在N,N-二甲基甲酰胺混合物130℃下加热1小时,在通过TLC监测到4Br-NDI耗尽后,用石油醚稀释反应混合物,用超纯水洗涤三次以除去DMF;有机溶液用Na2SO4干燥,然后真空旋蒸除去溶剂;粗产物用硅胶柱色谱纯化,用二氯甲烷/石油醚洗脱,得到NDI-1;
将2.66份NDI-1与10.0份PbO2在二氯甲烷中在50℃下搅拌24小时,溶液的颜色从紫色变成黄色;当NDI-1耗尽时,通过TLC监测,将溶液冷却并过滤以除去不溶物质;溶液用无水Na2SO4干燥,然后真空旋蒸除去溶剂,得到NDI-AB,为黄色固体;
B.50.0份CaCl2·2H2O、1.33份槲皮素以及1.33份NDI-AB加入含有乙醇的平底烧杯A中,并用带孔的塑料包装覆盖平底烧杯;在另一个平底烧杯B中加入1666.67份NH4HCO3;
将平底烧杯A置于平底烧杯B中,后将两个平底烧杯放在密封容器中,反应在40℃下进行72小时,通过以10,000rpm重复离心收集CaCO3@Quercetin@NDI-AB,并用乙醇洗涤两次;
C.将1.33份CaCO3@Quercetin@NDI-AB的乙醇溶液在磁力搅拌器上缓慢加入到1.33份DSPE-mPEG2000的混合水溶液中,在超声环境中充分震荡,通过旋蒸去除溶液中的乙醇和超净水;
重新加入超净水溶解后得到PQCN(DSPE-mPEG2000@Quercetin@CaCO3@NDI-AB)。
4.根据权利要求3所述的基于萘二酐小分子的光敏剂合成及制备方法,其特征在于包括以下:所述NDI-AB的分子结构为:
5.一种基于萘二酐小分子的光敏剂在钙超载治疗中的应用。
6.根据权利要求6所述的应用,其特征在于:所述的钙超载治疗为荧光成像/光声成像双模态的成像引导下的钙超载治疗。
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