CN118078778A - 一种肿瘤细胞和线粒体双级靶向雷公藤红素脂质体及其制备和应用 - Google Patents
一种肿瘤细胞和线粒体双级靶向雷公藤红素脂质体及其制备和应用 Download PDFInfo
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Abstract
本发明公开了一种雷公藤红素纳米脂质体,它由脂质体内核与外层包裹材料组成,所述脂质体内核含有脂质材料、TPP‑TPGS与雷公藤红素,所述包裹材料为透明质酸,所述透明质酸可主动靶向肿瘤细胞表面过表达CD44受体,提高所载药物在肿瘤部位的蓄积,所述TPP‑TPGS可将药物进一步递送至线粒体,提高肿瘤细胞活性氧含量,降低线粒体膜电位,破坏线粒体功能,进一步引起细胞凋亡。因此,该脂质体是一种肿瘤细胞和线粒体双级靶向脂质体,有利于降低雷公藤红素的毒性,提高药物抗肝内胆管癌的疗效。
Description
技术领域
本发明属于药物制剂领域,涉及一种肿瘤细胞和线粒体双级靶向雷公藤红素纳米脂质体的制备方法以及应用。
背景技术
肝内胆管癌(Intrahepatic Cholangiocarcinoma,ICC)是起源于二级及以上肝内胆管分支的胆道上皮细胞恶性肿瘤,其发病率仅次于肝细胞癌,约占原发性肝脏恶性肿瘤的10%~15%,并呈逐渐上升趋势。ICC病因复杂,高度侵袭、预后不良,目前根治性外科手术仍是治愈肝内胆管癌的主要手段,但大部分肝内胆管癌患者因发病隐匿,初诊时已处于晚期而失去手术机会。病理分子学研究显示,ICC发病机制的复杂性和显著的异质性阻碍了有效的临床治疗,非手术治疗病人能否接受有效抗癌药物,对当前的ICC科研状况仍是巨大挑战。因此,开发安全且高效的新型靶向药物及治疗策略对控制ICC的发生发展具有重要意义。
雷公藤红素(Celastrol,Cela)是一种从传统中药雷公藤中提取的三萜类奎宁甲基化物,作为一种具有天然生物活性的化合物,具有抗炎症、影响代谢通路、诱导细胞自噬或凋亡以及抗肿瘤等功能。研究表明,雷公藤红素可以通过调控糖代谢重编程抑制肝内胆管癌的发生。具体来说,雷公藤红素可以通过提高肿瘤细胞内活性氧(ROS)水平,影响线粒体功能,抑制肿瘤细胞的迁移、侵袭和增殖发挥抗肿瘤作用。
然而,雷公藤红素溶解度低、渗透性差、生物利用度低,并且存在较大的肝、肾、心脏和生殖毒性,而且对器官、组织选择性与较低,需要高剂量才能在病变部位达到有效浓度。总之,Cela的低溶解度和潜在的脱靶毒性限制其在临床的应用。
纳米脂质体可以提高难溶性药物的溶解度,促进药物吸收。并且脂质体具有缓释功能可以保护药物在到达病灶部位前不被酶降解。进一步还可以依赖于被动增强的渗透性和保留(EPR效应)来提高药物在靶点部位的累积,减小系统毒性,目前已有大量研究证明其对现代药物制剂研究的重要意义。三苯基膦-D-α-生育酚聚乙二醇1000琥珀酸酯(TPP-TPGS)是一种阳离子聚合物,可以通过静电作用与带负电荷的透明质酸(hyaluronicacid,HA)或线粒体膜相互作用。用TPP-TPGS修饰的脂质体具有线粒体靶向作用,且外层可以被HA包覆。而HA可以与肿瘤细胞表面过表达的CD44受体发生特异性结合,因此用HA修饰载药纳米粒子外层后,可通过受体介导作用靶向传递药物,提高肿瘤细胞对载药粒子的摄取效率,增加抗肿瘤疗效。
基于上述背景,申请人设计了一种具有肿瘤细胞和线粒体双级靶向的雷公藤红素纳米脂质体递送系统,并证明了该药物递送系统对ICC具有更好的疗效。
发明内容
本发明的目的是提供一种具有肿瘤细胞和线粒体双级靶向的雷公藤红素纳米脂质体及其制备方法和应用,旨在提高抗癌药物雷公藤红素在治疗肝内胆管癌方面的疗效。
为了实现上述目的,本发明提供了以下技术方案:
一种雷公藤红素纳米脂质体,它由脂质体内核与外层包裹材料组成,所述脂质体内核含有脂质材料、TPP-TPGS与雷公藤红素,所述脂质材料由磷脂和胆固醇组成,所述包裹材料为透明质酸,所述透明质酸可主动靶向肿瘤细胞表面过表达CD44受体,提高所载药物在肿瘤部位的蓄积,所述TPP-TPGS可将药物进一步递送至线粒体,提高肿瘤细胞活性氧含量,降低线粒体膜电位,破坏线粒体功能,进一步引起细胞凋亡,该脂质体是一种肿瘤细胞和线粒体双级靶向脂质体。
其中,所述磷脂与TPP-TPGS的质量比为5-15:1,优选为6-12:1,最佳为10:1。
其中,所述磷脂与胆固醇的质量比为10-20:1,优选为12-15:1,最佳为14:1。
其中,所述磷脂与雷公藤红素的质量比为10-30:1,优选为15-20:1,最佳为15:1。
其中,所述透明质酸与TPP-TPGS的质量比为1-5:1,优选为2-4:1,最佳为2:1。
根据本发明的一个具体实施例,所述纳米脂质体的最佳原料配比为:所述磷脂与TPP-TPGS的质量比为10:1;所述磷脂与胆固醇的质量比为14:1;所述磷脂与雷公藤红素的质量比为15:1;所述透明质酸与TPP-TPGS的质量比为2:1。该较佳实施例具有药物包封率高,载药量大,粒径分布均匀等优点。
本发明进一步提供一种制备所述雷公藤红素纳米脂质体的方法:首先使用常规的薄膜分散法制备含脂质材料、TPP-TPGS与雷公藤红素的脂质体内核,然后使用超声法在脂质体内核的外层包裹透明质酸。
本发明还提供了所述的雷公藤红素纳米脂质体在制备治疗肝内胆管癌药物中的用途,所述纳米脂质体双级靶向肿瘤细胞和线粒体,促进胆管癌细胞凋亡,发挥对肝内胆管癌更好的疗效。
本发明的有益效果是:
本发明使用透明质酸对脂质体进行包裹,一方面可以掩盖内核脂质体的阳离子电荷,提高脂质体体内安全性;另一方面透明质酸可主动靶向肿瘤细胞表面过表达CD44受体,提高所载药物在肿瘤部位的蓄积,而减少在其他器官的蓄积,大大降低了雷公红素的系统性毒性,尤其是对心脏和肾的毒性。进一步地,TPP-TPGS可将雷公藤红素进一步递送至线粒体,并显著提高肿瘤细胞活性氧含量,降低线粒体膜电位,破坏线粒体功能,从而加速肿瘤细胞凋亡,同时,TPGS又含有聚乙二醇亲水长链,因而具有较好的表面活性及性质和水溶性,能显著增加难溶药物的吸收,改善脂质体的稳定性和缓释效果。事实证明,本发明所制备的肿瘤细胞和线粒体双级靶向雷公藤红素纳米脂质体具有更好的抗肝内胆管癌效果。
本发明使用薄膜分散法结合超声法制备双级靶向脂质体,并根据所载药物雷公藤红素的理化性质对工艺参数条件进行了筛选,所制备的脂质体具有包封率高,载药量大,粒径分布均匀等优点,并且药物缓释效果和质量稳定性好,生物相容性高。
附图说明
图1是HCTL的聚酯比筛选的粒径和电位变化图。
图2是HCTL与Cela原料药以及载体的紫外吸收光谱。
图3是HCTL和两个对比药物CL、CTL的扫描电镜图。
图4是HCTL和两个对比药物CL、CTL以及Cela原料药的体外释放曲线图。
图5是HCTL和两个对比药物CL、CTL的溶血实验结果。
图6是HCTL和两个对比药物CL、CTL的低温储藏稳定性以及血清稳定性结果。
图7是HCTL和两个对比药物CL、CTL以及Cela原料药的HUCCT1细胞摄取图。
图8是HCTL和两个对比药物CL、CTL的HUCCT1的线粒体共定位图、荧光共定位线扫描图以及皮尔逊系数图。
图9是HCTL和两个对比药物CL、CTL以及DiR原料药的活体成像图。
图10是HCTL和两个对比药物CL、CTL以及Cela原料药的周期实验结果。
图11是HCTL和两个对比药物CL、CTL以及Cela原料药的凋亡实验图。
图12是HCTL和对比药物CTL以及Cela原料药的AKT/YAP肝内胆管癌模型体内药效体重变化图。
图13为HCTL和对比药物CTL以及Cela原料药的AKT/YAP肝内胆管癌模型体内药效肝脏质量图和肝重比图。
图14为HCTL和对比药物CTL以及Cela原料药的AKT/YAP肝内胆管癌模型药效鼠肝脏形态学及病理学的变化(肝脏图片、HE染色和CK19免疫组化)。
图15为HCTL和对比药物CTL以及Cela原料药的AKT/YAP肝内胆管癌模型药效血清肝脏损伤生化指标AST、ALT含量图。
图16为HCTL和对比药物CTL以及Cela原料药的AKT/YAP肝内胆管癌模型药效心脏、肾脏、脾脏和肺的HE切片图。
图17为HCTL和对比药物CTL以及Cela原料药的AKT/YAP肝内胆管癌模型药效的心脏损伤血清生化指标LDH、CK图。
图18为HCTL和对比药物CTL以及Cela原料药的AKT/YAP肝内胆管癌模型药效的心脏损伤血清生化指标CRE、BNU图。
具体实施方式
以下结合实施例对本发明做进一步详细说明,但是本发明的保护范围并不限于这些实施例,实施例仅用于解释本发明,而不用于限制本发明的范围。基于以下实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明所需TPP-TPGS为实验室自制,TPP-TPGS是离域亲脂性阳离子三苯基膦(TPP)与生育酚聚乙二醇琥珀酸酯(TPGS)的缀和物。三苯基磷(TPP)是一种亲脂性阳离子,具有较强的亲脂性,可优先在细胞线粒体内聚集,并可作为线粒体靶向配体。生育酚聚乙二醇琥珀酸酯(TPGS)中存在“生育酚琥珀酸酯”部分。生育酚琥珀酸酯可通过置换酶的内源性配体泛醌来改变线粒体复合物II(CII)的功能,泛醌产生过量的活性氧(ROS)并诱导细胞死亡。因此TPGS与TPP+(TPP-TPGS)的缀合物也可以引发类似的线粒体定向凋亡作用。并且TPGS又含有聚乙二醇亲水长链,因而具有较好的表面活性及性质和水溶性,能显著增加难溶药物的吸收,改善脂质体的稳定性和缓释效果。
其余药剂为常规药剂,采购自市售渠道,未提及的实验方法为常规实验方法。
实施例1肿瘤细胞和线粒体双级靶向的雷公藤红素纳米脂质体(HCTL)的制备
制备方法:称取处方量的雷公藤红素,大豆磷脂,胆固醇、TPP-TPGS溶于三氯甲烷中,超声溶解,旋转蒸发除去有机溶剂,使脂质在器壁上形成一层均匀薄膜,然后加入超纯水,水化薄膜,37℃水浴1h使薄膜充分溶胀,接着超声,经0.22μm微孔滤膜过滤得到脂质体内核,最后加入HA溶液包裹脂质体,经超声,过0.22μm微孔滤膜过滤得到HCTL。
(1)聚脂比的筛选:
按照上述方法制备HCTL脂质体,固定胆脂比(胆固醇:磷脂)为1:10、药脂比(雷公藤红素:磷脂)为1:30、透聚比(透明质酸:TPP-TPGS)为3:1,改变聚脂比(TPP-TPGS:磷脂)为1:2,1:4,1:6,1:8和1:10,以粒径和Zeta电位为指标,筛选对HCTL脂质体粒径及电位影响较大的聚脂比。
结果如1A图所示,随着聚脂比的降低,脂质体中阳离子聚合物TPP-TPGS含量的降低,通过静电作用吸附的荷负电的HA也相应减少,伴随着脂质体粒径的显著减小。当聚脂比为1:6,1:8,1:10时脂质体的粒径和Zeta电位变化趋于平台期,推测为HA正好全面覆盖脂质体表面的比例范围(图1A-B)。为获得粒径较小的荷负电的脂质体和出于成本考虑选择最佳聚脂比为1:10。
(2)透聚比、胆脂比和药脂比的筛选:
固定聚脂比为1:10,选择对HCTL脂质体的包封率及载药量影响较大的透聚比、胆脂比和药脂比为考察因素,以为综合筛选指标,采用正交试验表L9(34)进行实验及处方工艺优化。正交因素选择、正交试验结果、方差分析结果见表1、表2和表3。
表1正交试验因素水平表
表2正交试验结果
表3方差分析表
因素 | 偏差平方和 | 自由度 | F比 | F临界值 | 显著性 |
A | 5.402 | 2 | 216.080 | 19.000 | * |
B | 0.444 | 2 | 17.760 | 19.000 | |
C | 33.610 | 2 | 1344.400 | 19.000 | * |
误差 | 0.03 | 2 |
结果表明,直观分析和方差分析结果表明聚脂比和药脂比对脂质体的包封率及载药量有显著性影响(P<0.05),各因素的影响程度是C>A>B,因此HCTL的最佳处方为A1B3C1,即透聚比的质量比为2:1、胆脂比的质量比为1:14、药脂比的质量比为1:15。
(3)最优处方平行验证试验
对正交试验所得HCTL的最优处方进行了3组平行验证试验,试验结果如表4所示。
表4平行验证试验结果
结果表明,当大豆磷脂(SPC):胆固醇(Chol):雷公藤红素(Cela):TPP-TPGS:HA的质量比为15:1.1:1.0:1.5:3.0时,综合筛选指标为最高值(20.92),即成功负载的Cela占脂质体总质量比例最高。验证结果为3批HCTL样品包封率为97.25%、98.57%、95.85%,RSD为1.36%;载药量为4.50%、4.57%、4.44%,RSD为0.07%;粒径分别为170.2、171.4、171.1nm,RSD为0.4%;电位分别为-25.2mv、-25.4mv、-25.3mv,RSD为0.4%。试验重现性良好。
根据以上试验,最终确定HCTL的制备工艺为:称取2.0mg雷公藤红素,30.0mg大豆磷脂,2.1mg胆固醇、3.0mg TPP-TPGS溶于三氯甲烷中,超声溶解,旋转蒸发除去有机溶剂,使脂质在器壁上形成一层均匀薄膜,然后加入10mL超纯水,水化薄膜,37℃水浴1h使薄膜充分溶胀,接着超声6min,后经0.22μm微孔滤膜过滤4次得到脂质体内核,最后加入透明质酸溶液(含透明质酸6.0mg)包裹脂质体,超声10min,过0.22μm微孔滤膜过滤8次得到HCTL。
图2紫外吸收光谱显示,HCTL脂质体成功负载了雷公藤红素,并且所有辅料在雷公藤红素最大吸收波长425nm处无干扰吸收,不会影响雷公藤红素的含量测定。
实施例2HCTL的药剂学检测
对比药物1——普通雷公藤红素脂质体(CL)的制备:方法同实施例1,区别在于,不使用TPP-TPGS和HA。
对比药物2——线粒体靶向雷公藤红素脂质体(CTL)的制备:方法同实施例1,区别在于,不使用HA。
(1)粒径分布与Zeta电位
将制备脂质体适当稀释,用马尔文粒度仪测定脂质体的粒径及Zeta电位,测得结果如下:
Size(nm) | PDI | Zeta potionial(mv) | |
CL | 126.9±1.3 | 0.266±0.010 | -19.2±0.3 |
CTL | 92.6±0.3 | 0.207±0.002 | 15.2±0.1 |
HCTL | 172.3±0.6 | 0.280±0.005 | -29.6±0.1 |
由结果分析可知,与未添加靶向材料的雷公藤红素脂质体CL的粒径相比,添加了TPP-TPGS的CTL组粒径有所降低,这是由于其既含有维生素E亲酯基团,又含有聚乙二醇亲水长链,因此具有较好的表面活性剂性质和水溶性更有利于脂质体的形成。HCTL组在CTL组结构外层又静电吸附了一层带负电荷的透明质酸多糖骨架,其粒径有所增大,并且实现电位逆转,有利于提高脂质体纳米体的生物相容性。
(2)形貌表征
将制备的脂质体经5%磷钨酸染色,透射电镜对他们的形貌进行表征,结果如图3所示。
结果表明,HCTL、CL和CTL脂质体在透射电镜中均为球状纳米颗粒,尺寸分散范围窄,边缘规则。表面覆盖了HA的HCTL脂质体与阳离子脂质体CTL相比,透明质酸外壳切面呈现为同心环形状,结构清晰可见,表明透明质酸成功通过静电吸附到CTL表面,所获得的HCTL脂质体为多层脂质体。
(3)体外药物释放
采用透析袋扩散法研究Cela原料药、HCTL、CL和CTL脂质体在pH7.4的磷酸盐缓冲溶液中的体外释放情况。将1mL Cela原料药、HCTL、CL和CTL脂质体溶液转移到透析袋中,然后在37℃下浸入20毫升含有1%(重量百分比)吐温80的PBS(pH7.4)中。在预定的时间间隔内取出2毫升的PBS释放介质,并用2毫升的新鲜介质代替。采用紫外分光光度法(UV)测定药物浓度,并计算累计药物释放量。
各组药物释放情况如图4所示,游离雷公藤红素释放迅速,在最初的2h内释放率达到28.2%,18h内全部释放完毕。72h时CL、CTL、HCTL的累计释放率分别为64.0%、60.6%、49.4%。表明HCTL表面的HA覆盖层能阻塞药物释放通道,实现药物更好的缓释效果。
(4)安全性评价
采用溶血实验对CL、CTL和HCTL脂质体进行静脉注射安全性评价。将不同浓度的脂质体悬液与10%红细胞悬液混合,37℃孵育2h,用xMark分光光度计(Bio-Rad,USA)测定540nm处的OD值,计算溶血率并进行安全性评价。
实验结果表明,在对三种脂质体的血液相容性进行安全性评价时,CL与HCTL具有较低的溶血率,表明出良好的生物相容性(图5)。
(5)稳定性考察
对HCTL、CL和CTL脂质体进行稳定性考察,连续测量储存在4℃冰箱条件下的脂质体的粒径,比较三组脂质体的低温稳定性;将含药脂质体悬液与含10%胎牛血清的培养基孵育不同时间点后,测定脂质体的粒径分布考察血清稳定性。
如图6A所示储存在4℃冰箱中的HCTL在半个月里粒径仅增大2.8%,相比于CL与CTL的3.7%、9.9%,HCTL表现出更好的稳定性。同样的,HCTL相比于另外两组脂质体在与10%胎牛血清的培养基共孵育过程中,其粒径的多分散系数(PDI)均表现出较小的波动(图6B)。总之,HCTL表现出更好低温储藏稳定性和血清稳定性。
实施例3HCTL的靶向性考察及其对ICC的疗效评价
(1)细胞摄取实验
将带有绿色荧光的香豆素6(C6)按相同的制备方法载入脂质体(分别命名为C6@CL、C6@CTL和C6@HCTL),以代替雷公藤红素,用来表征药物在细胞中的摄取情况。首先将胆管癌细胞HUCCT 1在6孔板中培养24h。用含有C6@CL、C6@CTL和C6@HCTL的新培养基代替原培养基。每孔的香豆素6浓度为500ng/mL。培养4h后用荧光倒置显微镜观察每组细胞摄取量。此外,为了证实细胞对HCTL的摄取与CD44受体介导的内吞作用有关,在孔中加入3mg过量透明质酸,然后再加入HCTL。6h后,按上述方法处理细胞。
如图7所示,在给药后第4h,与普通脂质体C6@CL相比,CTL的荧光强度显著增强,这可能是带正电荷的TPP部分可以与带负电荷的肿瘤细胞膜相互吸引。值得注意的是,经HA修饰后HCTL具有最高的摄取量,并且在透明质酸提前预饱和处理细胞后,再用相同量的HCTL处理,结果导致细胞内绿色荧光强度显著下降。这说明HCTL的HA外壳可以与肿瘤细胞表面过表达的CD44受体发生特异性结合,从而显著促进细胞对HCTL的摄取。同时,游离HA可以对这种结合产生竞争性抑制。
(2)线粒体靶向性试验
同样的用载香豆素6的脂质体代替雷公藤红素脂质体,考察HCTL的线粒体靶向性。取处于对数生长期的HUCCT 1细胞,接种于六孔板中,培养24h后,分别加入含有相同浓度的含药培养基培养。4h后去除含药培养基,用Mitotracker-red(50nM)于37℃下对细胞线粒体染色30min。然后经4%多聚甲醛固定后用荧光染料Hoechst33342(10μg/mL)在室温下避光染色10min。用PBS清洗后,用激光共聚焦显微镜(Nikon C2;Japan)观察线粒体共定位情况。使用Image J软件分析共聚焦图像,进行line scanning。数据导入GraphPad Prism 9.0得到相应的荧光空间位置的line scanning图。
如图8(a)-(c)所示,C6@CL无靶向性,进入细胞后在细胞质中散在分布,与线粒体重合度低。然而,C6@CTL与C6@HCTL组中,红、绿色荧光大部分重合,叠加后显示出黄色荧光,这表明它们均具有更好的线粒体靶向能力。同样地,各组组合图对应的线扫描图也表明C6@CTL与C6@HCTL组的红绿荧光信号的位置和强度的同步性更高。此外,通过计算Pearson相关系数(Pearson's coefficient)进行定量分析,结果表明C6@CTL、C6@HCTL的P值分别比C6@CL高2.3倍和2.8倍(图8(d))。上述结果说明TPP基团具有线粒体靶向的能力。
(3)体内肿瘤靶向试验
为了进一步验证HA包膜的肿瘤靶向性,将HUCCT1细胞(1×107个细胞,含100μLRPMI 1640)皮下注射于4周龄雌性BALB/c裸鼠,制备异种移植模型。当荷瘤裸鼠荷瘤体积达到100~200mm3时,每只裸鼠尾静脉注射不同的近红外荧光染料(DiR)制剂(游离DiR、DiR@CL、DiR@CTL和DiR@HCTL),每只小鼠DiR注射剂量为0.2mg/kg。小鼠在指定时间点被异氟醚麻醉,用小动物在体成像系统在EM530/635nm荧光强度下快速拍摄。注射24h后,用异氟醚麻醉后处死小鼠。随后采集肿瘤和主要器官(心、肝、脾、肺、肾)并拍照。
如图9所示,HCTL借助HA外壳的主动靶向肿瘤能力,在给药1h后就能在肿瘤部位观察到DiR荧光的富集,主动靶向转运速度明显高于另外两种单纯依靠EPR效应被动转运的纳米脂质体。游离药组靶向效果最差,给药24h内无明显肿瘤蓄积趋势。此外,给药后24h,HCTL组肿瘤部位仍能观察到强烈荧光,与离体结果一致,进一步说明了HCTL具有稳定的肿瘤靶向能力。
(4)体外药效学评价
①周期实验
采用碘化丙啶(PI)DNA染色法观察各组制剂对细胞周期的影响。将HUCCT1细胞接种于小皿中,培养过夜。加入相同浓度的雷公藤制剂作用于细胞。培养24h后收集细胞,用70%预冷的乙醇固定。离心后将细胞用PBS洗涤,并在0.5mL PI/RNase染色液中室温黑暗条件下染色15分钟。最后,细胞样品立即进行流式细胞术分析。
抗肿瘤药物主要在细胞周期的三个阶段(即G0/G1、S和G2/M)阻断肿瘤细胞增殖。从图10中可以看出与对照组相比,当含相同浓度雷公藤红素的CL、CTL和HCTL分别处理HUCCT 1细胞24h后,增加了细胞周期中G0/G1期的百分比,这种现象同时伴随着S和G2/M期细胞周期占比减少。具体来说,与对照组(35.7%)相比,三组脂质体CL、CTL和HCTL组细胞的G0/G1期百分比分别显着增加至41.8%、44.4%和46.6%,其中HCTL对细胞周期影响最显著。表明HCTL能将细胞周期阻滞在G1期,影响DNA复制,促进细胞凋亡。
②凋亡实验
采用Annexin V-EGFP细胞凋亡检测试剂盒检测各给药组的细胞凋亡率,比较几种脂质体的促凋亡效果。将HUCCT 1细胞接种于六孔板中,使细胞充分贴壁。然后将细胞置于含药培养基中处理24h,收集细胞。然后,用荧光染料即Annexin V-EGFP染色凋亡早期细胞,并且用Propidium Iodide(PI)标记坏死细胞和处于凋亡晚期的细胞。随后采用流式细胞术分析。
如图11所示,对照组和四种制剂的总凋亡率(早期凋亡率和晚期凋亡率之和)分别为7.0%、13.8%、31.5%、35.3%和39.6%。相比于游离雷公藤红素组,HCTL组细胞凋亡率提高了2.9倍,HCTL能够显著提高雷公藤红素对HUCCT1细胞的凋亡率,这与HCTL更高的摄取量和线粒体靶向效率有关。
(5)体内药效学评价
本实验部分我们采用了高压尾静脉转染技术构建小鼠ICC原位瘤模型,此方法具有造模时间短,成模稳定,且模型的分子治疗靶点较为明确等优点。通过高压尾静脉转染技术,将携带活化的致癌基因(AKT/Yap)质粒转入小鼠体内,使其在肝脏内稳定表达并快速形成ICC。通过在ICC发生初期给药,来考察HCTL对ICC的靶向作用及作用机制。简而言之,就是将含有AKT、Yap和SB三种质粒的2mL生理盐水溶液快速注射到小鼠尾静脉中。三种质粒的质量比为10:15:1。随后,将转染成功的小鼠随机分为四组(n=8),未转染的小鼠作为WT对照。将所有小鼠以标准饮食饲养3周。第四周开始,经尾静脉以2mg/kg的雷公藤红素剂量每隔一天向转染小鼠注射约0.2mL的PBS、游离雷公藤红素、CTL和HCTL溶液。连续注射两周,并且纪录小鼠体重。给药周期结束后,收集小鼠眼眶血并处死小鼠。解剖小鼠,并且分离心、肝、脾、肺、肾等脏器,拍照称重。取部分组织置于装有组织固定液4%多聚甲醛的离心管中固定,用于后续苏木精和伊红染色(H&E)和免疫组织化学分析。剩余各脏器组织置于-80℃保存备用。测定血清中的谷草转氨酶(ALT)和谷氨酸转氨酶(AST)、肌酐(CRE)、尿素氮(BUN)、激酸激酶(CK)和乳酸脱氢酶(LDH)等指标,来评估不同制剂对肝、肾、心脏等主要脏器的损伤,考察HCTL的肿瘤靶向性和减毒增效情况。
如图12所示,比正常组(WT)相比,由于肿瘤的快速生长,AKT/Yap组(即用PBS处理的AKT/Yap转染小鼠)的体重显著快速增加(P<0.001)。然而,三个治疗组的体重在治疗开始时略有下降,然后逐渐增加。轻微的体重减轻可能与雷公藤红素作为瘦素受体敏化剂引起的小鼠食欲不振有关。和WT组相比,三组的体重下降均在10%以内。图13A中肝脏质量结果显示,AKT/Yap转染后伴随着肿瘤的恶性病变,肝脏质量显著增加,三种制剂给药后小鼠的肝脏质量均有减轻,其中HCTL治疗后肝脏质量最低。从图13B中肝脏与体重质量比的结果可知,AKT/Yap造模后的体重增加主要是由于肝脏质量增加,三种制剂给药后这一指标均有改善,其中HCTL的疗效最优。因此,除了药物的减肥作用外,HCTL引起体重减轻的原因主要是通过抑制肿瘤生长而减轻肝脏的质量。
图14A是各组小鼠的肝脏形貌,相比于WT组,模型组小鼠肝脏体积明显较大,泛灰白色无光泽,表面凹凸不平,触之有颗粒感,内部被肿瘤结块覆盖,HCTL脂质体治疗后该组肝脏形貌明显改善。图14B、C中,各组的肝脏HE染色组织学分析显示肿瘤具有管状表型,胆管细胞特异性标志CK19阳性率升高,这与人ICC组织病理特征相似。以上形态学和病理学的变化提示原发性ICC的发生。并且,经HCTL脂质体治疗后不仅肝脏质量降低为模型组的1/2,并且肿瘤体积和数量明显缩小,肝脏纤维化程度降低,CK19阳性率降低,并且血清中肝损伤标志物AST、ALT含量下调最接近正常值(图15)。我们进一步考察了双级靶向制剂HCTL降低雷公藤红素毒性的能力。图16是各组的心、脾、肺、肾HE染色切片,从心脏切片中可以观察到,游离Cela能引起一定的心脏损伤,诸如心肌纤维断裂、出现大量无序空隙、肌原纤维结构紊乱和萎缩、肌纤维上有空洞和炎症细胞的聚集等现象。此外,游离Cela的肾脏切片中也可见部分病理学改变,具体表现为部分肾小球体积的中度、重度萎缩,肾小球上皮细胞部分坏死和空泡的形成,以及炎症细胞的聚集。与游离Cela相比,由于HA与CD44受体介导的主动靶向,HCTL脂质体可以选择性在肿瘤部位聚集,从而减轻药物对其他器官的损伤。此外,未见Celastrol对肺和脾脏有明显损伤。除了HE染色切片之外,我们还测定了血清中多种心肌酶如肌酸激酶(CK)、乳酸脱氢酶(LDH),以及肾功能指标如肌酐(CRE)、尿素氮(BUN)等。当心肌细胞被损伤时,CK、LDH会被释放到血液中,导致血液中水平升高;当发生肾损伤时,CRE、BUN会因无法排出体外而升高。从图17、图18中可以看到,三种制剂治疗后,游离Cela会导致上述四个指标升高,而HCTL可以改善药物对心脏和肾脏的损伤,这与HE切片中的结果是一致的。
Claims (8)
1.一种雷公藤红素纳米脂质体,它由脂质体内核与外层包裹材料组成,所述脂质体内核含有脂质材料、TPP-TPGS与雷公藤红素,所述脂质材料由磷脂和胆固醇组成,所述包裹材料为透明质酸,所述透明质酸可主动靶向肿瘤细胞表面过表达CD44受体,提高所载药物在肿瘤部位的蓄积,所述TPP-TPGS可将药物进一步递送至线粒体,提高肿瘤细胞活性氧含量,降低线粒体膜电位,破坏线粒体功能,进一步引起细胞凋亡,该脂质体是一种肿瘤细胞和线粒体双级靶向脂质体。
2.如权利要求1所述的雷公藤红素纳米脂质体,其特征在于:所述磷脂与TPP-TPGS的质量比为5-15:1。
3.如权利要求1所述的雷公藤红素纳米脂质体,其特征在于:所述磷脂与胆固醇的质量比为10-20:1。
4.如权利要求1所述的雷公藤红素纳米脂质体,其特征在于:所述磷脂与雷公藤红素的质量比为10-30:1。
5.如权利要求1所述的雷公藤红素纳米脂质体,其特征在于:所述透明质酸与TPP-TPGS的质量比为1-5:1。
6.如权利要求1所述的雷公藤红素纳米脂质体,其特征在于:所述磷脂与TPP-TPGS的质量比为10:1;所述磷脂与胆固醇的质量比为14:1;所述磷脂与雷公藤红素的质量比为15:1;所述透明质酸与TPP-TPGS的质量比为2:1。
7.一种制备权利要求1-6任一项所述雷公藤红素纳米脂质体的方法,其特征在于:首先使用常规的薄膜分散法制备含脂质材料、TPP-TPGS与雷公藤红素的脂质体内核,然后使用超声法在脂质体内核的外层包裹透明质酸。
8.权利要求1-6任一项所述的雷公藤红素纳米脂质体在制备治疗肝内胆管癌药物中的用途,所述纳米脂质体双级靶向肿瘤细胞和线粒体,促进胆管癌细胞凋亡,发挥对肝内胆管癌更好的疗效。
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