CN107812008B - 一种近红外荧光成像小分子抗癌纳米药物的制备方法 - Google Patents
一种近红外荧光成像小分子抗癌纳米药物的制备方法 Download PDFInfo
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Abstract
本发明提供一种近红外荧光成像小分子抗癌纳米药物的制备方法,具体做法为用疏水性药物UA和PTX制备成纳米胶束,再以此纳米胶束为载体与亲水性的ICG共组装成纳米药物。本发明的目的在于通过具有抗癌活性的UA、PTX和光敏剂ICG通过π‑π堆积静电力相互作用制备近红外荧光成像小分子抗癌纳米药物的制备方法,以解决现有技术中人工合成的载体纳米体系复杂、质控困难、作用机制不明确和代谢不清楚等问题,以达到光疗和化疗协同治疗肿瘤的目的。
Description
技术领域
本发明涉及生物医药技术领域,特别涉及一种具有抗癌活性的紫杉醇,一种具有抗癌活性的熊果酸和一种光敏剂吲哚菁绿共组装成纳米胶束,制备成一种近红外荧光成像小分子抗癌纳米药物的方法。
背景技术
肿瘤是当今严重威胁人类健康的三大疾病之一,但在世界范围内癌症的总体发病率及死亡率仍在不断上升,癌症治疗对于提高患者的生存质量、减轻患者的痛苦、降低死亡率具有十分重要的意义。传统的化疗方法存在毒副作用大等缺点,对患者身心造成极大地损伤。随着科技技术的迅速发展,抗肿瘤药物的研究正日益受到人们的普遍关注和重视。开发新材料和新药物用于靶向治疗肿瘤是日前亟待解决的问题。
纳米技术是近年来迅速发展起来的前沿科技领域,并已在各学科的研究中产生了巨大的影响,纳米技术在制药领域的应用:纳米粒径化能增加药物吸收程度;纳米控释系统可以改善药物性质;纳米粒径级载体能够替代病毒载体;纳米机械装置可以辅助设计药物等,因此,将大大推动新药创制和医药健康领域的发展。在纳米技术的推动下光治疗也得到了飞速的发展,光治疗包括光动力治疗和光热治疗,把纳米技术与光治疗结合用于癌症的诊断与治疗也逐渐受到广泛的重视。纳米输送系统能提高光敏剂的光稳定性、水稳定性和热稳定性,同时能够调节光敏剂在体内的循环和分布,纳米技术的快速发展为光敏剂的开发和应用提供了新材料和新思路。但是纳米药物载体本身也存在一些不足,例如载体的生物相容性和细胞毒性,而无载体的纳米药物输送系统既解决了纳米载体体系复杂、质控困难、作用机制不明确和代谢不清楚等问题又能提高光敏剂的光稳定性、水稳定性和热稳定性。同时无载体纳米输送系统既避免了引入载体对人体带来的毒副作用,同时又减轻额外的代谢负担。无载体纳米药物输送系统将是未来发展的热点。
熊果酸(Ursolic acid,UA),又名乌索酸,乌苏酸,属α-香树脂醇(α-amyrin)型五环三萜类化合物。熊果酸以游离或糖苷的形式广泛分布在枇杷叶、冬凌草、夏枯草、山楂、熊果等大自然界的药用植物中。熊果酸也具有来源丰富、价格低廉等优点,因此,熊果酸在生物医药领域中具有良好的研发前景和应用价值,有望成为一种高效低毒的多用途药物。它具有化学预防作用、抗肿瘤活性作用、保肝、抗肝炎作用和抗菌、抗病毒作用。但是熊果酸的水溶性不好,没有靶向性,且生物利用度低等限制因素,使得其在国内外将其作为抗癌药物在临床上的开发应用受到较大阻碍。
紫杉醇(PTX)是从紫杉树皮中分离提取出的一种四环二萜类次生代谢产物,通过促进微管聚合和稳定已聚合微管抑制多种肿瘤细胞的繁殖和转移并触发明显的细胞凋亡,是近几年来被广泛应用于临床的新型广谱抗癌药,可广泛的应用于肺癌、卵巢癌、乳腺癌、胸腺癌、非小细胞肺癌、食道癌、头颈癌、睾丸胚胎癌、淋巴瘤的治疗。尤其是晚期卵巢癌的治疗。紫杉醇在水中的溶解度小,口服生物利用度差,使其临床使用受到了很大限制。为了降低不良反应,提高其疗效,国内外学者对紫杉醇的给药方式和剂型进行了广泛的研究。
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。近红外光在组织中的穿透深度较大,且受生物组织本底的影响较小,由于ICG具有近红外吸收和发射荧光特性,可作为一种优良的体内组织穿透剂。同时,ICG在水溶液中的不稳定性及在血浆中的快速清除率(半衰期:2~4 min)限制了其在诊断及治疗方面的应用。纳米技术的快速发展为ICG的进一步开发应用提供了新材料和新思路。纳米传输系统能够提高ICG的光稳定性、水稳定性和热稳定性,可有效避免ICG的分解及体内清除,同时能够调节ICG的体内循环和分布,使其在生物医学、疾病诊断及治疗方面的应用越来越广泛。
本专利基于溶剂交换法制备共组装的近红外荧光成像小分子抗癌纳米药物输送系统,该纳米药物输送系统具有体内荧光成像、肿瘤微环境响应性和被动靶向性的功能,其可通过EPR效应(enhanced permeability and retention effect) 靶向介导ICG@UA/PTX纳米药物,实现肿瘤诊疗一体化,从而发挥其抗肿瘤效果。
发明内容
本发明的目的在于通过具有抗癌活性的UA、PTX和光敏剂ICG通过π-π堆积静电力相互作用制备近红外荧光成像小分子抗癌纳米药物的制备方法,以解决现有技术中人工合成的载体纳米体系复杂、质控困难、作用机制不明确和代谢不清楚等问题,以达到光疗和化疗协同治疗肿瘤的目的。
本发明的具体做法为用疏水性药物UA和PTX制备成纳米胶束,再以此纳米胶束为载体与亲水性的ICG共组装成纳米药物以达到更好的癌症治疗效果。
具体实现方式为:
1、将药物UA溶于良性溶剂中,为溶液A,熊果酸浓度范围为100 μM-2000 μM;其中良性溶剂为二氯甲烷、氯仿、乙醚、乙酸乙酯、乙酸甲酯、丙酮、正丙醇、甲醇、吡啶、乙酸、二甲基亚砜中一种或多种的混合;
2、将溶液A缓慢滴入搅拌中的不良溶剂中,搅拌一定的时间后得溶液B,药物UA在溶液B中的浓度范围为31.25μM-1000 μM;其中不良溶剂为磷酸盐缓冲液、水、生理盐水、葡萄糖溶液中一种或多种的混合;
3、将药物PTX溶于良性溶剂中,为溶液C,紫杉醇浓度范围为100 μM-2000 μM;其中良性溶剂为二氯甲烷、氯仿、乙醚、乙酸乙酯、乙酸甲酯、丙酮、正丙醇、甲醇、吡啶、乙酸、二甲基亚砜中一种或多种的混合;
4、将溶液C缓慢滴入搅拌中的不良溶剂中,搅拌一定的时间后得溶液D,药物PTX在溶液D中的浓度范围为31.25μM-1000 μM;其中不良溶剂为磷酸盐缓冲液、水、生理盐水、葡萄糖溶液中一种或多种的混合;
5、将溶液B、D混合,搅拌一定的时间后超声、离心,取上清液即得到具抗癌活性的无载体的纳米胶束UA/PTX,其中搅拌的时间为1.5h-2h。
6、取溶液A和溶液C,混合,逐渐滴加到UA和PTX的不良性溶剂中,搅拌一段时间后再缓慢滴加ICG的良性溶液,继续搅拌,超声、离心,取上清液即得近红外荧光成像小分子抗癌纳米药物ICG@UA/PTX。
上述所述的UA的结构式如式Ⅰ所示,PTX的结构式如式Ⅱ所示,ICG的结构式如式Ⅲ所示。
式Ⅰ
式Ⅱ
式Ⅲ
本发明的优点在于:
1、本发明所制备的无载体近红外荧光成像小分子抗癌纳米药物中所用的UA既具有良好的抗癌效果又可以作为载体;
2、本发明所制备的无载体近红外荧光成像小分子抗癌纳米药物中的UA在水中可以通过溶剂交换法自组装成为纳米粒,并且可以做为抗癌药物PTX的载体,有效地解决了抗癌药物UA和PTX的水溶性和生物利用度的问题;
3、本发明所制备的无载体近红外荧光成像小分子抗癌纳米药物的制备过程简单,方便,可以解决传统纳米载体在体内代谢不明确,体系复杂等问题,消除了人工合成载体带来的临床安全性问题,并能为以后新药研发和制备提供新的思路。
4、本发明近红外荧光成像小分子抗癌纳米药物能够提高ICG的光稳定性,增加ICG近红外荧光成像的长循环,实现光化疗的协同作用,并可解决现有纳米载体的诸多问题。
5、本发明近红外荧光成像小分子抗癌纳米药物通过EPR效应提高纳米药物在肿瘤部位的集聚作用。
本发明制备的近红外荧光成像小分子抗癌纳米药物中熊果酸作为纳米载体与之共组装的抗癌药物包括但不限于以下:甲苯磺酸索拉非尼、阿柔比星、阿柔比星B、伊达比星、吡柔比星、紫杉醇、多西他赛、福美坦、雷公藤内酯醇、米非司酮、多西紫杉醇、喜树碱、10-羟基喜树碱、长春新碱、甲氨蝶呤、他莫西芬、盐酸柔红霉素、盐酸表阿霉素、盐酸佐柔比星、替尼泊苷、6-巯基嘌呤和5-氟尿嘧啶;
光敏剂为吲哚菁绿、二氢卟吩e6、酞菁类中一种或多种。
附图说明
图1为实施例5中ICG@UA/PTX纳米胶束粒径分布图;
图2为实施例5中ICG@UA/PTX纳米胶束电势图;
图3为实施例6中ICG@UA/PTX纳米胶束的紫外吸收;
图4为实施例7中ICG@UA/PTX纳米胶束的荧光检测图;
图5为实施例8中ICG@UA/PTX纳米胶束的pH响应结果;
图6为实施例9中ICG@UA/PTX纳米胶束在不同溶液中粒径测定结果;
图7为实施例9中ICG@UA/PTX纳米胶束在不同溶液中电势测定结果;
图8为实施例10中ICG@UA/PTX纳米对HepG2细胞的毒性结果;
图9为实施例11中ICG@UA/PTX纳米对HeLa细胞的毒性实验结果;
图10为实施12中ICG@UA/PTX纳米在体内荧光成像的结果;
图11为实施13中ICG@UA/PTX纳米药物在体内的抗肿瘤效果。
具体实施方式
根据下述实施例,可以更好地理解本发明,下面结合具体实施方式对本发明所述的技术方案作进一步的说明,但是本发明不仅限于此。
实施例1
熊果酸纳米胶束的制备方法
准确称取0.00456g UA粉末,溶于1ml甲醇中,超声溶解,配置成10 mM的溶液;取不同体积的甲醇溶液,在搅拌过程中逐滴滴加到含有2 mL二次水(重蒸水)中(注:滴加过程中高速搅拌,滴加时间为30s),此时UA在溶液中的浓度为31.25μM-1000μM,然后搅拌5 min,即得UA纳米胶束;
本实施例制备的不同浓度的UA纳米胶束平均粒径及PID如表1所示。
表1
实施例2
准确称取0.00853g PTX粉末,溶于1ml甲醇中,超声溶解,配置成10mM的溶液;取不同体积的甲醇溶液,在搅拌过程中逐滴滴加到含有2ml二次水中(注:滴加过程中高速搅拌,滴加时间为30s),此时PTX在溶液中的浓度为31.25μM-1000μM,然后搅拌5min,即得PTX纳米胶束;
本实施例制备的不同浓度的PTX纳米胶束平均粒径及PID如表2所示。
表2
实施例3
准确称取0.00456g UA粉末和0.00853gPTX粉末,分别溶于1ml甲醇中,配置成10mM的溶液,超声溶解;取不同体积的熊果酸甲醇溶液,和不同体积的紫杉醇甲醇溶液,混合,在搅拌过程中逐滴滴加到含有2ml二次水中(注:滴加过程中高速搅拌,滴加时间为30s),然后搅拌5min,制得不同分子摩尔比的UA/PTX纳米胶束;
本实施例制备的不同分子摩尔比形成的UA/PTX纳米胶束平均粒径、PID及电势如表3所示。
表3
实施例4
准确称取0.00456g UA粉末和0.00853g PTX粉末,分别溶于1ml甲醇中,配置成10mM的溶液,超声溶解;取100µL熊果酸的甲醇溶液和100µL的紫杉醇甲醇溶液,混合,在搅拌过程中逐滴滴加到含有2ml二次水中(注:滴加过程中高速搅拌,滴加时间为30s),此时UA在溶液中的浓度为500μM,PTX在溶液中的浓度为500μM,然后往混合溶液中加入不同浓度的ICG,之后搅拌3h,即得不同ICG浓度的ICG@UA/PTX纳米药物,并用透析和沉淀的方法测定纳米药物中三种分子的包封率(EE)。
本实施例制备的不同ICG浓度形成的ICG@UA/PTX纳米胶束平均粒径、PDI、电势和EE如表4所示。
表4
实施例5
准确称取0.00456g UA粉末和0.00853g PTX粉末,分别溶于1ml甲醇中,配置成10mM的溶液,超声溶解;取100µL熊果酸的甲醇溶液和100µL的紫杉醇甲醇溶液,混合,在搅拌过程中逐滴滴加到含有2ml二次水中(注:滴加过程中高速搅拌,滴加时间为30s),此时UA在溶液中的浓度为500μM,PTX在溶液中的浓度为500μM,然后往混合溶液中加入浓度为250μM 的ICG溶液,之后搅拌3h,即得近红外荧光成像小分子抗癌纳米药物ICG@UA/PTX;
本实施例制备的ICG@UA/PTX纳米胶束粒径130.8nm及电势-30.8mv如图1和图2所示。
实施例6
将实施例5制备的ICG@UA/PTX纳米药物、实施例3制备的UA/PTX以及相同浓度的UA甲醇溶液、PTX甲醇溶液和ICG溶液通过紫外吸收检测器进行紫外检测,结果显示本发明制备的纳米抗癌药物有红移现象出现,如图3所示,证明本发明UA、PTX和ICG三者共组装形成了ICG@UA/PTX NPs 抗癌纳米药物。
实施例7
将实施例5制备的ICG@UA/PTX、实施例3制备的UA/PTX以及相同浓度的ICG溶液通过荧光检测器进行荧光检测,结果显示本发明制备的纳米药物荧光强度低于相同浓度ICG的荧光强度如图4所示,证明了UA、PTX和ICG三者共组装形成了ICG@UA/PTX NPs 抗癌纳米药物。
实施例8
将实施例5制备的ICG@UA/PTX纳米药物各取500µL两份于小瓶子中,然后分别加入pH为5.5和7.4的PBS溶液1500µL,观察现象,结果显示本发明制备的近红外荧光成像小分子抗癌纳米药物在pH5.5有浑浊出现,在pH7.4澄清状态,如图5所示,证明了本发明制备的近红外荧光成像小分子抗癌纳米药物具有pH响应的特征。
实施例9
将实施例5制备的ICG@UA/PTX NPs 放置不同的溶液中观察粒径变化和电势变化,结果显示本发明制备的纳米药物在不同溶液中粒径和电势保持一致性,如图6和图7所示,证明了本发明制备的近红外荧光成像小分子抗癌纳米药物具有良好的稳定性。
实施例10
ICG@UA/PTX纳米药物的抗癌活性,通过细胞毒性来实现,采用标准MTT法测定ICG@UA/PTX、 UA、PTX、ICG、UA+PTX、ICG+NIR、ICG@UA/PTX+NIR对HepG2 细胞的增殖抑制活性,具体步骤为:
(1)取处于对数生长期状态良好的HepG2 细胞,经胰蛋白酶消化后,计数并调整细胞密度为0.8×,104个/mL,配成细胞悬液。于每孔100 µl接种到96孔板中,周围用NaCl封板,置于37 ℃, 5% CO2培养箱中培养24 h。
(2)去除旧的培养基,每孔加入100 µL不同浓度梯度的含样品的培养基,另设空白对照组,每组设置5个复孔,于培养箱中继续孵育24h。
(3)移除培养基,于每孔中加入100 µL MTT溶液(无血清、无酚红的RMPI1640培养基:MTT母液=9:1,V:V),继续孵育4 h。
(4)取出96孔板终止培养,用移液枪轻轻吸去96孔板中的上清液,每孔加入DMSO溶液 100 µl,振荡摇匀10 min,使蓝紫色结晶全部溶解,用酶标仪于490nm波长处测定每孔的OD值,使用GraphPad Prism 5处理实验结果显示与其他各组相比,本发明制备的纳米抗癌药物在808nm激光照射下对HepG2细胞有最高的抑制率,如图8所示,本发明制备的近红外荧光成像小分子抗癌纳米药物能抑制癌细胞的活性。
实施例 11
ICG@UA/PTX纳米药物的抗癌活性,通过细胞毒性来实现,采用标准MTT法测定ICG@UA/PTX、UA、PTX、ICG和UA+PTX对HeLa细胞的增殖抑制活性,具体步骤见实施例10,其结果显示本发明制备的纳米抗癌药物对HeLa细胞具有显著的杀伤作用,如图9所示,表明本研究所制备的ICG@UA/PTX+NIR纳米抗癌药物能增加对癌细胞的毒性作用。
实施例12
实施例5制备的ICG@UA/PTX纳米药物在体内具有荧光成像功能,具体步骤为:
(1)H22细胞的复苏:把被冻存在液氮罐中的H22细胞取出,放在37℃的恒温水浴中使其融化,待细胞溶化后立即把细胞拿到超净工作台中,随后对细胞进行离心处理,收集细胞沉淀,用的新的培养基重悬细胞于培养瓶中,置于置于37 ℃, 5% CO2培养箱中培养;
(2)继续培养H22细胞数天后使其长满整个培养瓶,收集细胞沉淀,用NaCl重悬细胞,腹腔注入KM小鼠体内;
(3)细胞在小鼠腹腔长一周后,把小鼠处死取腹水,立即加入红细胞裂解液裂解5min后,离心,再用NaCl溶液重悬细胞,再离心,重复3次后,最后细胞浓度2×107/mL,每只小鼠在右后肢皮下注入200µL;
(4)当肿瘤达到150mm3左右时,种瘤的小鼠静脉(i.v.)注射ICG@UA/PTX纳米或ICG(10mg ICG / kg)。然后通过SI Imaging Amix(USA)在0.5,1,3,6,12和24小时(曝光时间:2s)下使用740nm激发波长和790nm发射波长获取FL图像; 24小时成像后,将小鼠安乐死。其结果显示本发明制备的纳米抗癌药物与相同浓度的ICG相比,经过24小时后在肿瘤部位蓄积的更多,如图10所示证明了本发明制备的近红外荧光成像小分子抗癌纳米药物具有在肿瘤部位聚集的能力。
实施例13
本发明制备的ICG@UA/PTX纳米药物在体内具有很好的抗肿瘤的作用,其具体步骤如下:
(1)H22细胞的复苏:把被冻存在液氮罐中的H22细胞取出,放在37℃的恒温水浴中使其融化,待细胞溶化后立即把细胞拿到超净工作台中,随后对细胞进行离心处理,收集细胞沉淀,用的新的培养基重悬细胞于培养瓶中,置于置于37 ℃, 5% CO2培养箱中培养;
(2)继续培养H22细胞数天后使其长满整个培养瓶,收集细胞沉淀,用NaCl重悬细胞,腹腔注入KM小鼠体内;
(3)细胞在小鼠腹腔长一周后,把小鼠处死取腹水,立即加入红细胞裂解液裂解5min后,离心,再用NaCl溶液重悬细胞,再离心,重复3次后,最后细胞浓度2×107/mL,每只小鼠在右后肢皮下注入200µL;
(4)当小鼠肿瘤体积达到约100mm3时,将小鼠随机分为PBS组,UA(2.67mg / kg),PTX(5.0mg / kg),UA + PTX混合物(2.67mgUA+5.0mgPTX)不含NIR激光照射的Free ICG(2mg / kg),具有NIR激光照射的Free ICG(2.0mg / kg),ICG@UA/PTX(UA 2.67mg / kg,PTX 5.0mg / kg和ICG 2.0 mg / kg)分为无NIR激光照射和有NIR激光照射。将所有的组尾静脉注射上述剂量的药物。在静脉注射12小时后,使用808nm激光(1W / cm2,5分钟)照射Free ICG和ICG@UA/PTX纳米组。在肿瘤照射前第7天,第16天和第21天获得携带H22荷瘤小鼠的肿瘤图像。其结果显示与PBS组相比,本发明制备的纳米抗癌药物在808nm激光照射下能显著抑制H22荷瘤小鼠的肿瘤生长,如图11所示,证明了本发明制备的近红外荧光小分子抗癌纳米药物具有显著地抗肿瘤效果。
Claims (2)
1.一种近红外荧光成像小分子抗癌纳米药物的制备方法,其特征在于:包括以下步骤:
1)将熊果酸和紫杉醇分别溶于良性溶剂Ⅰ中,按一定的摩尔比混合后,在搅拌的状态下缓慢滴加到不良性溶剂中,再于室温下搅拌一定的时间,得到混合溶液,此时熊果酸的浓度为500µM,紫杉醇的浓度为500µM;
2)将吲哚菁绿溶于良性溶剂Ⅱ中,然后往混合溶液中加入终浓度为250µM的吲哚菁绿溶液,继续搅拌一段时间后即得吲哚菁绿@熊果酸/紫杉醇纳米药物;
步骤1)中所述的良性溶剂Ⅰ为二氯甲烷、氯仿、乙醚、乙酸乙酯、乙酸甲酯、丙酮、正丙醇、甲醇、吡啶、乙酸、二甲基亚砜中一种或多种的混合;
步骤1)中所述的不良性溶剂为磷酸盐缓冲液、水、生理盐水、葡萄糖溶液中一种或多种的混合;
步骤2)中所述的良性溶剂Ⅱ为水或甲醇。
2.根据权利要求1所述的一种近红外荧光成像小分子抗癌纳米药物的制备方法,其特征在于:制得的吲哚菁绿@熊果酸/紫杉醇纳米药物的粒径为100-200nm。
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Granted publication date: 20200612 |