CN112076319B - 青蒿素及其衍生物在制备热动力治疗敏化剂中的应用 - Google Patents
青蒿素及其衍生物在制备热动力治疗敏化剂中的应用 Download PDFInfo
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Abstract
本发明提供了一种青蒿素及其衍生物的新用途,即将青蒿素或其衍生物直接作为热动力治疗的敏化剂,用于肿瘤的热动力治疗;或将其与吲哚菁绿制成的脂质体纳米复合物作为热动力治疗的敏化剂,用于肿瘤的热动力治疗和/或光热治疗。将青蒿素或其衍生物及脂质体纳米复合物用于肿瘤的热动力治疗,不仅显示出优异的肿瘤靶向性,而且具有极高的协同光热治疗和热动力治疗抗肿瘤活性,作为抗癌药物具有显著的优势。
Description
技术领域
本发明属于功能材料以及药物领域,具体涉及青蒿素衍生物、青蒿素衍生物与吲哚菁绿脂质体纳米材料在抗肿瘤中的应用。
背景技术
光动力疗法(PDT)是一种针对癌症的治疗方法,它利用光激发光敏剂,然后在氧气存在下进行光化学反应,以生成活性氧(ROS),诱导癌细胞和组织损伤。PDT具有微创性和非耐药性的优点,因此得到了广泛的开发。最近,出现了一种新的方法,即热动力治疗(TDT)。它使用热作为能源来激活敏化剂,然后产生ROS或其他自由基用于癌症治疗。相对于光,可以通过更多方式提供热,例如化学反应、光、超声、辐射和微波。因此,TDT是一种新颖且很有前景的癌症治疗方法。此外,在TDT中,加热后可直接从热力学敏化剂中获得活性物质,没有氧依赖性,因此TDT可以克服PDT对缺氧肿瘤疗效有限的不足。但是,目前相关报道仍罕见,少数热不稳定的偶氮二异丁腈衍生物可在热活化后分解并导致自由基的产生,因而作为用于癌症治疗的化学热敏剂。
吲哚菁绿(ICG)是目前唯一被美国食品药物管理局(FDA)批准用于临床的近红外成像试剂。ICG是一种三碳花菁染料,最大发射波长在795~845 nm之间,是一种具有两亲性结构的药物分子。ICG能够吸收光能并将其转化为热能或产生单线态氧,可用于光热治疗(PTT)或光动力治疗(PDT)。但它在光照环境中易分解,这给药物的保存和后续应用带来了困难。并且ICG在水溶液中表现出不稳定性,在生物组织中,例如血浆中的清除率较快(其中半衰期:2~4 min)限制了其在诊断及治疗方面的应用。而通过纳米进行修饰改造,则能够有效提高其光稳定性和热稳定性,页能有效的改进其水稳定性。
青蒿素是一种含有内过氧化物桥的倍半萜烯内酯,是一种著名的抗疟药。青蒿素作为一种抗疟药,已显示出较高的安全性。由于其独特的结构,除抗疟疾外,青蒿素及其衍生物还具有其他各种用途。最近报道,青蒿素过氧化物的过氧化物桥可以被Fe2+离子激活产生用于癌症治疗的活性自由基。不同于PDT过程中产生的ROS,青蒿素产生的ROS不依赖于周围环境的氧含量,这对于低氧肿瘤组织的治疗是特别有利。众所周知,由于血红蛋白的存在,血液中Fe2+离子的存储量相对较高。然而,它在肿瘤组织中未显示出特异性分布,并且肿瘤组织中的Fe2+离子水平太低而无法激活青蒿素,从而限制了青蒿素在临床应用中的发展。目前利用青蒿素的热敏感性产生活性物质用于肿瘤治疗仍未见报道。将青蒿琥酯和吲哚菁绿利用脂质体包载制备纳米材料用于肿瘤的光热治疗和热动力治疗也尚未见报道。
发明内容
本发明的目的在于提供了一种青蒿素及其衍生物在制备肿瘤热动力治疗敏化剂中的应用,其不仅显示优异的肿瘤靶向性,而且具有极高的协同光热治疗和热动力治疗的抗肿瘤活性,作为抗癌药物具有显著的优势。
为实现上述目的,本发明采用如下技术方案:
一种青蒿素及其衍生物在制备肿瘤热动力治疗敏化剂中的应用,其是利用青蒿素及其衍生物具有热敏性及能在热效应条件下产生活性氧的特性,将青蒿素或其衍生物直接作为热动力治疗敏化剂,用于肿瘤的热动力治疗;或将青蒿素或其衍生物与吲哚菁绿制成的脂质体纳米复合物作为热动力治疗敏化剂,用于肿瘤的热动力治疗和/或光热治疗;
所述青蒿素衍生物包括蒿甲醚、双氢青蒿素、青蒿琥酯中的任意一种;
所述热效应条件是以直接或间接的加热技术,使温度升至≥48℃;所述加热技术包括加热、激光、超声、微波等。
具体地,当采用青蒿琥酯作为青蒿素衍生物,可与吲哚菁绿包封于类脂质双分子层内形成微型泡囊的脂质体纳米复合物,其制备方法包括如下步骤:
1)将二棕榈酰磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、胆固醇半琥珀酸酯、青蒿琥酯和吲哚菁绿按摩尔比12:1.5:9:3:13加入到三氯甲烷与甲醇的混合溶液(1:1,v/v),使其完全溶解,再超声处理1-5min;
2)超声完后,将溶液旋转蒸干,然后用去离子(DI)水脱壁形成脂质体悬浮液;
3)采用超声细胞破碎机在0℃~20℃下将脂质体悬浮液粉碎5~10分钟,再在4℃~25℃下用去离子水透析(膜截滤分子量为10000)12~48小时,以除去游离的吲哚菁绿和青蒿琥酯,得到所述纳米复合物,其平均粒度约为100-200 nm。
所得吲哚菁绿-青蒿琥酯脂质体纳米材料在抗肿瘤治疗中使用的激光波长为808nm,强度为0.3~0.8W∙cm-2。
本发明的有益效果和突出优势在于:
(1)青蒿素衍生物是临床上治疗疟疾病的主要药物,安全性较高,其人体安全性和药代动力学性质已得到广泛的评价。本发明提供了一种青蒿素类衍生物的新用途,即将其作为热敏剂(其中,青蒿琥酯具有显著的热敏活性),因为该药物是较安全的临床应用药物,因此更利于临床使用和推广。
(2)吲哚菁绿是美国食品药物管理局(FDA)批准用于临床的试剂,安全性高,且具有较强的光动力和光热治疗效果。本发明还提供了一种青蒿素衍生物与吲哚菁绿构成的复合纳米材料,其直接利用吲哚菁绿的光热效应为青蒿琥酯提供原位热源,无需外加热源,整体设计巧妙。
(3)本发明提供的吲哚菁绿-青蒿琥酯脂质体纳米材料在808nm激光照射下,吲哚菁绿可产生良好的光热效应,且青蒿琥酯可产生显著的活性氧,故该纳米材料同时具有良好的光热和热动力效果,可作为热动力治疗及光热治疗的新型抗肿瘤药物,具有良好的生物相容性和较高的人体安全性,且脂质体具有优异的生物相容性(可降解性、无毒、无免疫原性),至今已有多种药物的脂质体制剂在国内外上市。
(4)本发明提供的吲哚菁绿-青蒿琥酯脂质体纳米材料具有优异的肿瘤靶向性。对荷瘤小鼠肿瘤静脉给药后,仅在小鼠的肿瘤部分观察到明显的荧光信号和光声信号。因此,该纳米材料也可用于肿瘤的多模式诊断。
(5)本发明提供的吲哚菁绿-青蒿琥酯脂质体纳米材料具有优异的光热和热动力协同抗肿瘤效果。在治疗的第10天肿瘤基本消失,抑瘤率达100%,且直到14天均无复发情况发生。表明该纳米材料具有优异的抗肿瘤疗效,是一种极具应用前景的抗肿瘤药物。
附图说明
图1是在不同比例的ARS和ICG的混合溶液(ICG固定为100μM)中、808nm激光(强度0.3 W∙cm-2,10分钟)下DCFH的荧光光谱图。
图2是在激光辐射的情况下,用不同纳米药物处理人肝癌细胞HepG2的细胞毒性对比图(其中ICG@NPs,ICG-ARS@NPs是以ICG的含量为指标,ARS@NPs是以ARS的含量为指标)。
图3是对荷瘤H22小鼠在14天内进行不同处理的肿瘤生长曲线对比图。
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。
实施例中所用吲哚菁绿、青蒿素衍生物和制备脂质体的原料均可市购获得。
实施例1 青蒿素衍生物作为热动力治疗敏化剂
以2',7'-二氯荧光黄双乙酸盐(简称DCFH-DA)水解产物2',7'-二氯荧光黄双乙酸(简称DCFH)作为活性氧的探针,该探针激发波长为488nm,发射波长检测范围为500-600nm。DCFH-DA的水解水溶液的制备参考现有论文(《J. Immunol. Methods》1993,159 (1-2),131-138开展)。通过观察DCFH(524nm处左右)在含有待测样品溶液中随激光照射时间的荧光情况,测定样品热敏产生ROS的能力,DCFH产生的荧光强度越强,说明热敏产生ROS的能力越强。
以青蒿琥酯(ARS)为例进行测定,其具体测定方法是称量适量青蒿琥酯,将其溶解在DMF中,得到2 mM的母溶液。测试时,取2',7'-二氯荧光黄双乙酸100μL与青蒿琥酯100μL加入到1.8 mL的去离子水中,得到混合溶液(其中青蒿琥酯最终浓度为100μM,2',7'-二氯荧光素二乙酸最终浓度为10μM),然后在60℃的水浴中加热溶液共30 min,期间每隔一定时间取出,测试其在500-600 nm处的荧光强度,根据荧光强度探究青蒿琥酯受热情况下产生活性氧的能力确定其热敏化性能。使用相同的方法,测量双氢青蒿素、蒿甲醚和青蒿素的活性氧生成。
通过结果分析发现,相对于单纯探针的对照组的荧光强度,青蒿琥酯组的荧光强度是对照组的6.475倍,双氢青蒿素组是对照组的2.813倍,蒿甲醚组是对照组的0.773倍,青蒿素组是对照组的1.164倍。由上述测试结果发现,青蒿琥酯与双氢青蒿素具有较明显的热敏化效果,可以用于热敏剂纳米药物的制备与研究。
实施例2
通过调研文献及实验发现,吲哚菁绿浓度达到100μM时,其所在的水溶液能升温至50℃,可以满足青蒿琥酯受热产生活性氧的要求,接着通过探究青蒿琥酯与吲哚菁绿的比例发现,青蒿琥酯与吲哚菁绿的比例在一定范围内会表现出不一样的性质,如图1所示,青蒿琥酯:吲哚菁绿=1:1与青蒿琥酯:吲哚菁绿=2:1产生活性氧能力最强且强度相当,因此选用吲哚菁绿作为激光照射药物产生热源,并将青蒿琥酯:吲哚菁绿=1:1作为最优的药物比例形式。
实施例3 包载有吲哚菁绿与青蒿琥酯的脂质体纳米药物
按摩尔比12:1.5:9:3:13分别称量二棕榈酰磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、胆固醇半琥珀酸酯、青蒿琥酯和吲哚菁绿,加入三氯甲烷(10 mL)和甲醇(10 mL),使其溶解在混合溶剂中,再超声处理5min。然后将溶液旋蒸干燥,进一步用去离子(DI)水脱壁形成脂质体悬浮液。再通过超声细胞破碎机在10℃下将脂质体悬浮液粉碎10分钟,在25℃下用去离子水透析(膜截滤分子量为10000)48小时以除去游离的吲哚菁绿和青蒿琥酯,得到ICG-ARS@NPs。
电镜分析表明,所获得的脂质体纳米复合物为较均匀的球状或近球状小囊泡,粒径约150 nm。纳米粒度分析仪测定也表明所获得的青蒿琥酯脂质体的粒径约为90-160 nm之间。通过HPLC分析了脂质体中青蒿琥酯的包封率,显示吲哚菁绿和青蒿琥酯包封率分别为81.4%和69.0%。
对比例1 包载有吲哚菁绿的脂质体纳米药物
按照实施例3的方法制备只添加吲哚菁绿的脂质体纳米药物ICG@NPs(粒径约为90-160 nm)。
对比例2 包载有青蒿琥酯的脂质体纳米药物
按照实施例3的方法制备只添加青蒿琥酯的脂质体纳米药物ARS@NPs(粒径约为90-160 nm)。
实施例4
将ICG-ARS@NPs置于透析袋(MW10000)中,室温下于摇床培养箱中、在PBS溶液(pH6.5和pH 7.4)中以120 rpm搅拌。在不同的时间点(0小时,6小时,12小时,24小时,48小时和72小时)取出透析液进行UV-Vis光谱测量。通过比较780 nm处的吸光度与校准曲线来计算释放的ICG浓度。使用以下公式获得药物释放:
药物释放(%)=释放的ICG/纳米药物中的总ICG。
通过使用UV-Vis光谱法检测ICG的释放百分比,在不同pH值的PBS溶液中评估ICG-ARS@NPs的pH敏感效率。结果表明,在pH 6.5的PBS溶液中,ICG在24小时内迅速释放,在48 h达到稳定水平,释放百分比约为80%。但在pH 7.4的PBS溶液中,ICG仅有少量释放,48小时内释放约20%。
实施例5
本发明所述青蒿琥酯与吲哚菁绿脂质体纳米材料在激光照射下能有效地产生热效应。在水溶液中808nm激光照射10分钟(激光强度为0.3~0.8W∙cm-2),溶液温度可达50℃。
光照产生的热效应可诱导青蒿琥酯产生活性氧。将青蒿琥酯与吲哚菁绿脂质体纳米材料进行激光照射,所采用的激光波长为808nm,激光强度为0.3W∙cm-2。样品每光照1min测其电子吸收光谱,扫描范围500~600 nm。实验结果表明,青蒿琥酯与吲哚菁绿脂质体在激光激活下能有效地产生荧光。
实施例6
首先,将复合纳米药物ICG-ARS@NPs、ICG@NPs和ARS@NPs分别用水或生理盐水或磷酸盐缓冲溶液稀释为1mM溶液,再将此混合溶液用培养基稀释为100μm的含药培养基。96孔板中当生长细胞铺展面积占培养瓶底面积75%以上时可供上药。吸出旧培养基,每孔中加入60、50、40、30μL含药培养基,并补充培养基至每孔最终体积为100μL,并设空白对照组(不加药光照和不加药不光照),培养2h。将96孔板放在808nm激光下光照(功率0.3W∙cm-2),每孔光照5min。光照后将药吸出,换新鲜培养基。培养24h后,每孔加入5g/L的MTT 10μL,再培养4h。倒尽板中的培养液,各孔加150μL DMSO溶液,振荡10min,使结晶物充分溶解,用酶标仪(波长492nm)测定各孔的吸光度(A)值,以每组n个孔A的平均值作为各组的平均A值。根据下列公式计算抑制率:
抑制率(%)=(1-实验组A/对照组A)×100%。
实验结果如图2所示。实验结果表明,单独光照作用,或单独纳米药物,对癌细胞没有明显的杀伤作用。但是,在光照作用下,当复合纳米药物ICG-ARS@NPs中ICG的浓度为40μM时,它对癌细胞的抑制率可达到75%,说明复合纳米药物具有显著的热动力抗癌效应。
实施例7
将H22荷瘤ICR小鼠随机分为7组:(I)空白(生理盐水),(II)单独激光,(III)ARS@NPs +激光,(IV)ICG-ARS@NPs,(V)游离ICG+激光,(VI)ICG@NPs+激光,(VII)ICG-ARS@NPs +激光。分别给小鼠静脉注射生理盐水,游离ICG,ICG@NPs,ICG-ARS@NPs和ARS@NPs的剂量分别为10 mg·kg-1的ICG或7.5 mg·kg-1的ARS。注射后8小时,将肿瘤暴露于808 nm激光(0.8W∙cm-2)10分钟。在照射期间,通过红外热成像相机(TiX520,Fluke,USA)记录红外热图像。每2天测量小鼠的体重和肿瘤体积,总共14天。
如图3所示,激光照射后,ICG@NPs处理的小鼠的肿瘤生长部分受到阻滞,其肿瘤抑制率为75%,这主要是由于光热效应引起的;而辐照后的ARS@NPs几乎没有抑制这种作用,肿瘤的生长速度与空白组(仅盐水或激光)一样快。而激光照射后的ICG-ARS@NPs组,肿瘤在第10天逐渐缩小,直至最后消失,直到14天也没有继续生长,这证明了ICG-ARS@NPs具有非常有效的PTT和TDT的协同作用。
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 (4)
1.一种青蒿素衍生物在制备肿瘤热动力治疗敏化剂中的应用,其特征在于:利用青蒿素衍生物具有热敏性及能在热效应条件下产生活性氧的特性,将青蒿素衍生物直接作为热动力治疗敏化剂,用于肿瘤的热动力治疗;或将青蒿素衍生物与吲哚菁绿制成的脂质体纳米复合物作为热动力治疗的敏化剂,用于肿瘤的热动力治疗和/或光热治疗;
所述青蒿素衍生物为青蒿琥酯;
所述热效应条件是以直接或间接的加热技术,使温度升至≥48℃;所述加热技术包括激光、超声、微波。
2.根据权利要求1所述的青蒿素衍生物在制备肿瘤热动力治疗敏化剂中的应用,其特征在于:采用青蒿琥酯与吲哚菁绿为原料制备脂质体纳米复合物的方法包括如下步骤:
1)将二棕榈酰磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、胆固醇半琥珀酸酯、青蒿琥酯和吲哚菁绿按比例加入到三氯甲烷与甲醇的混合溶液,使其完全溶解,再超声处理1-5 min;
2)超声完后,将溶液旋转蒸干,然后用去离子水脱壁形成脂质体悬浮液;
3)采用超声细胞破碎机在0℃~20℃下将脂质体悬浮液粉碎5~10分钟,再在4℃~25℃下用去离子水透析12~48小时,以除去游离的吲哚菁绿和青蒿琥酯,得到所述纳米复合物。
3.根据权利要求2所述的青蒿素衍生物在制备肿瘤热动力治疗敏化剂中的应用,其特征在于:步骤1)中所用二棕榈酰磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000、胆固醇半琥珀酸酯、青蒿琥酯和吲哚菁绿的摩尔比为12:1.5:9:3:13;
所述三氯甲烷与甲醇的混合溶液中,两者的体积比为1:1。
4.根据权利要求2所述的青蒿素衍生物在制备肿瘤热动力治疗敏化剂中的应用,其特征在于:步骤3)所述透析采用截滤分子量为10000的滤膜。
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