CN113384698A - 一种协同化疗/声-光动力治疗的自组装纳米药物及其应用 - Google Patents
一种协同化疗/声-光动力治疗的自组装纳米药物及其应用 Download PDFInfo
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Abstract
本发明公开了一种协同化疗/声‑光动力治疗的自组装纳米药物及其应用,其具体是将齐墩果酸(OA)和二氢卟吩e6(Ce6)通过π−π堆积和疏水相互作用自组装成无载体纳米药物,其不仅可避免使用载体带来的毒性,且所得纳米药物可发挥化疗和声‑光动力治疗的协同效果,因而在抗肿瘤治疗中具有良好的应用前景。
Description
技术领域
本发明属于生物医药领域,具体涉及一种由齐墩果酸(OA)和二氢卟吩e6(Ce6)自组装形成的纳米药物及应用。
背景技术
癌症是世界上威胁人类健康的主要疾病之一。目前除了化疗、放疗等传统癌症治疗方式外,还有一些新的治疗策略,包括光动力(PDT)、声动力(SDT)疗法。但由于肿瘤的生理复杂性,单药治疗模式往往不能达到令人满意的治疗效果。因此为了提高癌症患者的治疗效果,寻找新的治疗方式是至关重要的。而联合疗法形式进行抗肿瘤治疗能够利用不同机制和不同途径达到更好的治疗效果。
PDT和SDT已被广泛用于癌症治疗。先前的研究已经探索了PDT与化学疗法的结合或SDT与化学疗法的结合,已显示出显著的治疗效果。声-光动力疗法(SPDT)是PDT和SDT的组合,与单药疗法相比,通过协同治疗可以减少药物和超声/光剂量,从而发挥更好的抗癌作用。SPDT依靠光和超声激活敏化剂产生的活性氧(ROS)最终导致细胞死亡。迄今为止,许多已开发的敏化剂均已显示出光动力和声动力效应,可用于SPDT。然而,很少探讨将SPDT与化疗结合用于癌症治疗。
有机敏化剂二氢卟吩e6(Ce6)可以被光和超声激活,从而发挥PDT/SDT的作用。最近,已经开发了Ce6介导的SPDT用于癌症治疗。与在单独光照或者超声刺激下相比,Ce6在光动力治疗和声动力治疗的联合疗法下可降低乳腺癌细胞系的细胞活力,并增强小鼠乳腺癌4T1异种移植模型的抗肿瘤功效。进一步研究表明,Ce6介导的SPDT可以抑制MDA-MB-231细胞迁移并诱导细胞凋亡。另一项研究表明Ce6介导的SPDT会导致线粒体膜电位(MMP)丧失,诱导线粒体依赖性细胞凋亡以及4T1细胞的自噬。齐墩果酸(OA)是一种五环三萜类化合物,具有抗氧化剂和抗炎作用等多种生物活性。更重要的是,低毒性OA具有明显的抗癌作用,已被用于治疗各种癌症。OA可以诱导人胰腺癌细胞线粒体去极化,细胞凋亡并阻止细胞周期。OA还可以通过caspase-3途径抑制颅内神经胶质瘤中的细胞迁移和侵袭。但是,Ce6和OA的水溶性较差未能获得令人满意的癌症治疗结果。
迄今为止,已经开发了不同的纳米载体将疏水性药物递送至肿瘤部位。但是,大多数载体的制备过程复杂、具有潜在的毒性、代谢不清楚等问题。因此,有必要开发一种可以将化学疗法和SPDT相结合的简单、安全、有效的纳米药物输送系统。由纯药物分子通过自组装技术形成的无载体纳米颗粒已被开发用于药物递送和肿瘤治疗。疏水性抗癌药物10-羟基喜树碱(HCPT)和Ce6形成的自组装纳米药物具有化疗光动力双重疗法,在体内外具有良好的协同抗肿瘤作用。利用亲水和疏水自组装技术形成的无载体纳米药物可用于光声成像引导的协同化疗-声动力癌症治疗。另一项研究则制备了一种自组装集诊断、监测和治疗于一体纳米药物,该自组装的纳米粒子可用于成像,实时自我监测不同药物分子的释放和分布,并在体内外实现高效的化疗-光动力联合癌症治疗。自组装的无载体纳米药物不仅可以避免载体的毒性,而且可以提高药物的水溶性,增强抗肿瘤治疗的效果。因此,通过自组装策略制备的无载体纳米药物具有广阔的应用前景。
基于上述背景,本发明利用天然产物OA和水不溶性药物Ce6制备了无载体纳米药物OC,不仅改善了药物的水溶性,而且还协同了SPDT和化学疗法的作用,提高了对肿瘤细胞的杀伤效果。
发明内容
本发明的目的在于提供一种自组装无载体纳米药物及其应用,所制得的纳米药物既可避免载体的毒性,又具有良好的化疗和声-光动力治疗协同效果,可用于肿瘤的治疗。
为实现上述目的,本发明采用如下技术方案:
本发明的目的之一在于保护一种可协同化疗/声-光动力治疗的自组装纳米药物,其是由齐墩果酸(OA)和二氢卟吩e6(Ce6)自组装形成;其中,Ce6的装载率为13.21%~24.60%,包封率为10.55%~57.97%。
本发明的目的之二在于保护所述自组装纳米药物的制备方法,其是将OA和Ce6用有机溶剂溶解后,将所得混合液在搅拌条件下缓慢滴入到水或磷酸盐缓冲液中并搅拌过夜,然后将反应液转移到透析袋中进行透析,最后进行冻干并于-20℃保存。
所用OA和Ce6的质量比为1:1~5:1。
所用有机溶剂为二甲亚砜(DMSO)、甲醇或乙醇。
滴入水或磷酸盐缓冲液后,溶液中OA的浓度为0.2~1 mg/mL,Ce6的浓度为0.2 mg/mL。
所用透析袋的截留分子量为3500。
本发明的目的之三在于保护所述自组装纳米药物的应用,即用于制备抗肿瘤的化疗/声-光动力治疗药物。
本发明的显著优点在于:
本发明开发了一种简单、安全、有效的纳米药物输送系统,其通过自组装技术制备得到无载体纳米药物,避免了载体的毒性,且所得纳米药物可发挥化疗和声-光动力协同治疗的效果,可有效提高肿瘤治疗效果。
附图说明
图1为实施例3所制备自组装纳米药物OC5的粒径分布图。
图2为OA、Ce6和自组装纳米药物OC5的紫外光谱图(A)和荧光扫描图(B)。
图3为自组装纳米OC5的在不同温度(A)及不同环境(B)中的稳定性试验结果图。
图4为自组装纳米OC5在细胞中生成ROS的情况图。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例1
将OA和Ce6分别用DMSO溶解,配成浓度为10 mg/mL和2 mg/mL的两种溶液,然后取OA溶液0.1 mL、Ce6溶液0.5 mL混合在一起,再缓慢滴入正在搅拌的5 mL水中并搅拌过夜(OA/Ce6重量比为1:1)。反应完成后,将反应液转移到透析袋(MWCO3500Da)中,用双蒸馏水(ddH2O)透析3天,最后经冻干得到纳米药物OC1。其中,Ce6的装载率为19.35%,包封率为14.51%。
实施例2
将OA和Ce6分别用DMSO溶解,配成浓度分别为10 mg/mL和2 mg/mL的两种溶液,然后取OA溶液0.25 mL、Ce6溶液0.5 mL混合在一起,再缓慢滴入正在搅拌的5 mL水中并搅拌过夜(OA/Ce6重量比为2.5:1)。反应完成后,将反应液转移到透析袋(MWCO3500Da)中,用双蒸馏水(ddH2O)透析3天,最后经冻干得到纳米药物OC2.5。其中,Ce6的装载率为15.53%,包封率为33.46%。
实施例3
将OA和Ce6分别用DMSO溶解,配成浓度分别为10 mg/mL和2 mg/mL的两种溶液,然后取OA溶液0.5 mL、Ce6溶液0.5 mL混合在一起,再缓慢滴入正在搅拌的5 mL水中并搅拌过夜(OA/Ce6重量比为5:1)。反应完成后,将反应液转移到透析袋(MWCO3500Da)中,用双蒸馏水(ddH2O)透析3天,最后经冻干得到纳米药物OC5。其中,Ce6的装载率为13.87%,包封率为55.26%。
由于肿瘤的特殊生理结构特征(血管、渗漏等),粒径小于200 nm的纳米颗粒基于增强的通透性和保留(EPR)效应有利于在肿瘤部位积累。图1为所得OC5的粒径分布图。由图中可见,所得自组装水合纳米药物OC5的粒径大约为100 nm,故适合在肿瘤部位积累。
实施例4
分别测定OA、Ce6和OC5纳米粒的紫外吸收和荧光性能,结果如图2所示。
由图2可见,OC5在405 nm和665 nm处有紫外特征吸收峰;而荧光光谱表明OC5发出来自Ce6的红色荧光发射,证明Ce6已成功负载到OC5纳米粒中。同时,与单体Ce6相比,自组装纳米粒子内部Ce6的荧光发射峰强度降低,表明纳米粒子的形成可能是通过π-π堆积和疏水相互作用。
实施例5
取OC5纳米粒分散到水溶液中,然后在4℃和常温(25℃)下分别放置7天,每两天取样,测其粒径变化。另取OC5纳米粒分散到PBS、1640、含10%胎牛血清的1640培养基中,分别放置7天,并在1、3、5、7天取样,测其粒径变化,结果见图3。
如图3所示,在4℃和25℃下OC5纳米粒的粒径未发现发生明显变化;且当OC5纳米粒分散在PBS或RPMI-1640中时,尺寸分布没有明显变化,而当OC分散在含10%FBS的RPMI-1640中时,粒径有所增加,这可能是由于高血清浓度影响了OA和Ce6之间的相互作用。但其仍小于200 nm,不会对药物的被动肿瘤靶向产生太大影响。
实施例6
1. 肿瘤细胞的杀伤效果评估,步骤如下:
(1)培养细胞:将PC9细胞(人肺癌细胞)在含10% FBS的1640培养基中于5%CO2、37℃的条件下培养。
(2)检测OC5纳米粒子对细胞的杀伤效果:实验可分为以下几组:(a)OA、Ce6、O+C(OA和Ce6的简单混合)、OC5,(b)Ce6+激光照射、O+C+激光照射、OC5+激光照射(Ce6&L、O+C&L、OC5&L),(c)Ce6+超声处理、O+C+超声处理、OC5+超声处理(Ce6&U、O+C&U、OC5&U),(d)Ce6+激光和超声处理、O+C+激光和超声处理、OC5+激光和超声处理(Ce6&L+U、O+C&L+U、OC5&L+U),按设置将药物分别加入相应12孔板中,4 h以后,分别按设定进行激光照射(680 nm,400 mW/cm2,2 min)或/和超声(0.1 W/cm2,10 s)中。
(3)评价:继续培养48 h以后,将12孔板中的培养基吸出,加入500 μL的MTT(500 μg/mL);4 h以后,吸去MTT,加入800 μL二甲基亚砜,摇晃均匀,在570 nm下用酶标仪检测吸收值,并计算不同药物对细胞生长抑制50%所需的浓度,结果见表1。
表1 不同药物对肿瘤细胞的抑制情况
由表1结果表明,与化疗药OA、敏化剂Ce6及OA和Ce6简单混合相比,自组装形成的OC5纳米粒子具有协同治疗效果,而且在激光和超声照射下具有显著的光-声抗肿瘤效果,当使用光-声协同杀伤癌细胞时,其效果好于单独使用激光或超声治疗。
2. 细胞内ROS测定:
将PC9细胞接种在12孔板中,培养24 h。然后将实验分为五组:(a)空白对照,(b)OC5(1 μg/ mL Ce6),(c)OC5&L,(d)OC5&U,(e)OC5&L+U。处理4 h后,将细胞用PBS洗涤两次,并将无血清RPMI-1640培养基稀释的DCFH-DA染料(10 μM)转移到孔中。再将细胞按设定暴露于激光(680 nm,400 mW/cm2,2 min)或/和超声(0.1 W/cm2,10 s)中,并在37℃的培养箱中培养30 min。最终收集细胞并进行流式细胞仪分析。
OC5细胞内生成ROS的结果如图4所示。从图4中可以看出,OC5&L+U处理组细胞内ROS的生成量比OC5&L和OC5&U处理组都高得多,表明OC5在光照和超声刺激下通过增加细胞内ROS的产生量,能够显著发挥化疗/SPDT抗癌效果。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (6)
1.一种协同化疗/声-光动力治疗的自组装纳米药物,其特征在于:其是由齐墩果酸和二氢卟吩e6自组装形成,其中,二氢卟吩e6的装载率为13.21%~24.60%,包封率为10.55%~57.97%。
2.根据权利要求1所述的自组装纳米药物,其特征在于:其制备方法是将齐墩果酸和二氢卟吩e6用有机溶剂溶解后,将所得混合液在搅拌条件下缓慢滴入到水或磷酸盐缓冲液中并搅拌过夜,然后将反应液转移到透析袋中进行透析,最后进行冻干即得。
3.根据权利要求2所述的自组装纳米药物,其特征在于:所用齐墩果酸和二氢卟吩e6的质量比为1:1~5:1。
4.根据权利要求2所述的自组装纳米药物,其特征在于:所用有机溶剂为二甲亚砜、甲醇或乙醇。
5.根据权利要求2所述的自组装纳米药物,其特征在于:所用透析袋的截留分子量为3500。
6.一种如权利要求1所述的自组装纳米药物在制备抗肿瘤用化疗/声-光动力治疗药物中的应用。
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