CN112121166A - 特异性卟啉自运输纳米载体材料及其制备方法 - Google Patents
特异性卟啉自运输纳米载体材料及其制备方法 Download PDFInfo
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明涉及一种特异性卟啉自运输纳米载体材料的制备方法,其将对硝基苯甲醛、乙酸酐和丙酸、吡咯混合反应获得紫色固体四(4‑硝基苯基)卟啉TNPP,然后制备获得四(4‑氨基苯基)卟啉TAPP;将TPGS、琥珀酸酐、DMAP、三乙胺与1,4‑二氧六环在室温下搅拌,透析,获得羧基TPGS;将羧基TPGS、DMAP和PyBOP混合后,加入DMF和TAPP并在室温搅拌22‑26h,透析,透析液冻干,获得产品TAPP‑TPGS。该载体材料中引入了特异性分子TPGS,使得载体具有辅助抗肿瘤药物治疗癌症的效果,进而实现TPGS、PDT与化疗三种方式的联合治疗来提高抗肿瘤效果。
Description
技术领域
本发明属于载药材料制备技术领域,具体涉及一种特异性卟啉自运输纳米载体材料及其制备方法。
背景技术
化疗是目前癌症最重要的治疗手段之一,但药物普遍存在半衰期短、副作用大和耐药性差等缺点。纳米药物传输系统(nano drug delivery system, NDDS)可以在一定程度上克服临床化疗的缺点,能够增加化疗药物的溶解性、延长药物在血液中的循环时间和靶向投递药物等优势。但是,单一的化疗对癌症治效果不如预期,研究者开始探索新的治疗方式,像免疫、光热、基因和光动力学治疗等。经研究化疗与新的治疗方式联合治疗的效果要优于单一治疗。
光动力学治疗(photodynamic therapy, PDT)是光敏剂通过特定波长的激照射激发后产生活性氧(ROS)物质,来诱导癌细胞坏死或凋亡,而发挥诊断和治疗肿瘤的作用。由于二氢卟吩(Ce6),吲哚菁绿(ICG),卟啉等具有较高的ROS产率,通常被用作光敏剂(PS)。由于大部分PS水溶性差,所以在生物应用之前必须进行水溶性或功能性修饰,自组装成纳米载体。因此,载体材料的合理设计是PDT有理想治疗效果的关键。然而,载体材料的设计通常存在合成困难和成本高等一系列问题,更重要的是,在载体材料中引入复杂化合物使得载体材料存在较大的潜在代谢毒性。因此,近年来,为了避免由于载体材料或其降解产物引起的毒副作用,FDA和EMA批准的赋剂越来越受到研究者们的关注。
发明内容
基于以上问题,本发明提供了一种特异性的卟啉自运输纳米载体材料,并在其疏水的内核负载抗肿瘤药物。此载体材料中引入了特异性的分子TPGS,使得载体具有辅助抗肿瘤药物治疗癌症的效果,进而实现TPGS、PDT与化疗三种方式的联合治疗来提高抗肿瘤效果。卟啉修饰材料被FDA批准,因此减少了因载体降解所产生的毒副作用。另外,此运载体系具有荧光可以实现对药物和材料的示踪。
为实现上述目的,本发明采用如下技术方案:
一种特异性卟啉自运输纳米载体材料的制备方法,其包括如下步骤:
1)将对硝基苯甲醛、乙酸酐和丙酸混合后,加热至75-85℃,然后滴加吡咯和丙酸的混合物,滴加结束后升温至130-140℃回流反应20-40min,冷却至室温,然后于0-6℃静置析出沉淀,过滤,滤饼经洗涤、干燥获得粗品;粗品溶解于吡啶中并回流1-1.5h,冷却至室温,然后放置于冰水浴中20-30h,收集固体并用丙酮洗涤至洗涤液无色,经真空干燥,得紫色固体四(4-硝基苯基)卟啉TNPP;
2)在氮气气氛下,将TNPP溶解于浓盐酸中获得溶液A;在氮气气氛下,将SnCl2·2H2O溶解于浓盐酸中获得溶液B;将溶液A与溶液B混合,并在75-85℃反应20-40min,停止加热并冷却至室温,然后置于冰水浴中继续反应,此时加入氨水以中和浓盐酸,同时有固体出现,过滤,收集滤液;向滤液中加入3-8%NaOH溶液,搅拌,有固体出现,过滤收集固体,经洗涤、真空干燥获得四(4-氨基苯基)卟啉TAPP;
3)将D-α-生育酚聚乙二醇琥珀酸酯(TPGS)、琥珀酸酐、4-二甲氨基吡啶(DMAP)、三乙胺与1,4-二氧六环在室温下搅拌22-26h,然后将反应液装入透析袋(1 kDa)在去离子水中透析45-50 h,然后将透析液冷冻干燥,获得羧基TPGS(C-TPGS);
4)将C-TPGS、DMAP和六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)混合后,加入N,N-二甲基甲酰胺(DMF)和TAPP并在室温搅拌22-26h,然后将反应液移入透析袋(3.5 kDa)在去离子水中透析40-45h,然后将透析液冻干,获得产品TAPP-TPGS。
具体的,步骤1)中,对硝基苯甲醛、乙酸酐和吡咯的摩尔比为1:1.5-2.0:1。
具体的,步骤2)中,紫色固体与SnCl2·2H2O的摩尔比为1:14-15。
进一步的,步骤3)中,TPGS、琥珀酸酐和DMAP的摩尔比为1:2:1;透析袋截留分子量为1 kDa。
进一步的,步骤4)中,C-TPGS和TAPP的摩尔比为4:1;透析袋截留分子量为3.5kDa。
本发明提供了采用上述制备方法制备所得的特异性卟啉自运输纳米载体材料。
本发明还提供了上述特异性卟啉自运输纳米载体材料在制备抗肿瘤纳米药物中的应用,即将其作为药物负载载体用于抗肿瘤药物的负载及肿瘤的联合治疗。
本发明旨在示踪抗肿瘤药物在体内的分布,提高药物在肿瘤部分的富集,减少现在抗肿瘤药物的毒副作用,克服现有载药材料存在的制备过程复杂、生物相容性差等缺点,从而实现多种治疗方式联合抗肿瘤。卟啉具有荧光,且在体内能形成生物相容性的血卟啉而被生物体所吸收,TPGS是FDA批准的赋剂,生物相容性好且有增加细胞摄取和诱导癌细胞凋亡的作用。因此,用TPGS修饰卟啉环制备的纳米载药/材料体系能够示踪药物,提高药物在肿瘤部位的富集,减少药物、载体材料和载体代谢产物对生物体的毒副作用,多种机制联合抗肿瘤提高肿瘤的治疗效果。和现有技术相比,本发明的有益效果如下:
1)所用原料廉价、生物相容性好,制备方法简单易操作,制备载药体系成本低且生物相容性好;
2)特异性分子TPGS的引入使得制备的材料具有一定的抗肿瘤作用;
3)载药体系完成了光敏剂的自运输,其自身有荧光免于有机染料荧光标记且能够对药物示踪,TPGS、光动力和化疗联合抗肿瘤有效提高了抗肿瘤效果。
附图说明
图1为四(4-氨基苯基)卟啉TAPP的质谱图(A)和核磁共振氢谱图(B);
图2为实施例1制备所得TAPP-TPGS 的透射电镜图(A)和粒径分布图(B);
图3 为DPBF检测TAPP-4TPGS纳米胶束ROS产生情况图;
图4为4T1细胞在37 ºC 与TAPP-TPGS共培养4 h 后的激光共聚焦图;
图5为TAPP-TPGS 对HepG2 细胞(A)、MCF-7 细胞(B)和4T1细胞(C)的体外抑制效果。TPGS 和 TAPP-TPGS 对HL-7702 细胞的毒性实验结果(D);
图6为PTX 和 TAPP-TPGS/PTX 对 HepG2 细胞 (A)、MCF-7 细胞 (B) 和 4T1 细胞(C)的体外抑制效果;
图7中,(A)为不同处理组荷瘤小鼠的肿瘤体积变化, (B)为不同处理组荷瘤小鼠的体重变化, (C)为不同处理组荷瘤小鼠的生存率。
具体实施方式
以下结合实施例对本发明的技术方案作进一步地详细介绍,但本发明的保护范围并不局限于此。
实施例1
一种特异性卟啉自运输纳米载体材料(TAPP-TPGS)的制备方法,其包括如下步骤:
1)将对硝基苯甲醛(50 mmol, 7.561 g)、乙酸酐(86 mmol, 8.2 Ml)和丙酸(200 mL)加入两颈烧瓶中,反应体系在油浴中加热到80℃,然后用恒压滴液漏斗缓慢滴入吡咯(50mmol,3.5 mL)和丙酸(10 mL)的混合物。滴加结束,将反应体系的温度升高到135℃回流反应30 min。然后冷却到室温并置于4℃冰箱过夜,翌日有沉淀出现,过滤,滤饼用蒸馏水洗涤(3×80 mL),40℃下真空干燥获得粗品。粗品溶解在80 mL吡啶中并回流1 h,冷却至室温,然后在冰水浴中放置24 h。收集固体并用丙酮洗涤至洗涤液无色。最后将固体在40℃真空干燥,得到紫色固体四(4-硝基苯基)卟啉TNPP(1.093 g),产率约为11%。
2)取步骤1)所得紫色固体产品TNPP(1.3 mmol,1.0069 g)溶解在50 mL的浓盐酸(市售浓度36%~38%的浓盐酸)中获得溶液A,并通20 min氮气。同时,将SnCl2·2H2O(19.1mmol,4.3127 g)溶解在70 mL的浓盐酸中获得溶液B,并通20 min氮气。然后,将上述溶液A和溶液B混合,并在80℃反应30 min,然后停止加热并冷却到室温,然后置于冰水浴中继续反应,此时向反应体系中加入62.5 mL NH3·H2O以中和浓盐酸,同时有固体出现,用砂芯漏斗进行过滤,收集滤液。滤液中加入5% NaOH溶液100 mL,剧烈搅拌,有固体出现,过滤收集固体,滤饼用蒸馏水洗涤三次,40℃下真空干燥,获得粗产物。以丙酮为溶剂,粗产物用索氏提取器抽提,抽提液回收丙酮,得到紫色固体四(4-氨基苯基)卟啉TAPP(168.5 mg, 16.7%。该反应过程主要是用SnCl2将TNPP中的硝基还原为氨基。用1H NMR和MS进行分析,结果见图1。由图1可以看出:8.90 ppm, 7.99 ppm, 7.06 ppm, ‒2.72 ppm 是TAPP的特征峰, 7.99ppm 和 7.06 ppm 是苯环上氢的特征峰, 8.90 ppm 和 ‒2.72 ppm 是吡咯环上氢的特征峰。在质谱图中,675.45是TAPP加一个氢的特征峰。
3)将TPGS (1.5 g, 1 mmol)、琥珀酸酐(200.14 mg, 2 mmol)、DMAP (122.17 mg,1 mmol)和三乙胺 (2 mL)加入到含有45 mL 1,4-二氧六环的圆底烧瓶中,室温磁搅拌24 h后,将反应液移入透析袋(1 kDa)在去离子水中透析48 h,然后将透析液置于冷冻干燥机上冷冻干燥,收集紫色固体,即为羧基TPGS(1.344 g, 84%)。同时用溴甲酚绿对羧基TPGS中的羧基进行了定性分析,证明羧基TPGS 成功制备。
4)将羧基TPGS (320 mg, 0.2 mmol)、DMAP (24.5 mg, 0.2 mmol) 和 PyBOP(104.08 mg, 0.2 mmol)加入到圆底烧瓶中,然后加入DMF(15 mL)以溶解样品;再加入TAPP(33.74 mg, 0.05 mmol)并在室温磁力搅拌24 h,然后将反应液移入透析袋(3.5 kDa)在去离子水中透析42h,然后将透析液置于冷冻干燥机上冷冻干燥24h,获得深绿色产品TAPP-TPGS(254.3mg, 71.9%)。
用核磁共振氢谱对步骤4)所得产物TAPP-TPGS进行了表征,核磁共振氢谱结果显示:8.91 ppm,7.98 ppm和-2.74 ppm 是TAPP的特征峰,3.64 ppm处是乙二醇单体中亚甲基氢的峰,经分析证明TAPP-TPGS成功制备。
通过透射电子显微镜(TEM)对其形貌进行了表征,并统计了其粒径。实验结果见图2。从图2中可以看出:其形貌为近球形的纳米胶束(图2中A),粒径约为20 nm(图2中B)。
载药试验:
将TAPP-TPGS(20 mL)和抗肿瘤药物紫杉醇(PTX)(10 mL)溶解在4 mL丙酮中,在室温搅拌4 h,将此溶液缓慢滴加到去离子水中,滴加结束后在氮气环境中将丙酮除去。残留溶液移入透析袋(3.5 kDa)在去离子水中透析除去残余的丙酮,然后将透析液在冷冻干燥机上冷冻干燥24h,获得产品TAPP-TPGS/PTX,置于4℃存储,载药量8.7%。
采用灵敏的ROS探针1,3-二苯异苯基并呋喃(DPBF)检测胶束产生的单线态产氧。利用荧光探针DPBF的发射光谱对单线态氧进行了鉴定。在TAPP-TPGS (2.7mL, 111 mol /mL)甲醇溶液中加入DPBF (100 mol /mL)。用光功率密度为18 mW/cm2的660 nm激光照射溶液。480nm处DPBF的吸收光谱分别在2、4、6和8min用UV-vis进行记录。测试结果见图3。由图3可以看出:含有纳米胶束的DPBF 溶液的吸光值明显比不含的 DPBF 溶液小,说明TAPP-TPGS纳米胶束在660nm光照条件下可以产生单线态氧。
通过体外和体内实验来研究纳米载体材料的生物性能。通过体外细胞实验来研究载体材料在细胞中的分布,通过细胞毒性试验来研究纳米载体材料的生物相容性和所载药物的体外抗肿瘤效果。
本申请选择小鼠乳腺癌细胞4T1来检测TAPP-TPGS在细胞中的分布,具体如下:
将4T1细胞在DMEM培养基中以5.0 × 10 3的密度在37℃、5%二氧化碳环境中培养24h。然后用含有TAPP-TPGS(4μg/mL)的培养液替换原培养基。在37℃、5%二氧化碳氛围中继续培养4 h。弃去培养液,细胞再用PBS (pH = 7.4)洗涤三次,然后用多聚甲醛(4%)固定10min,接着倒掉多聚甲醛,用PBS洗涤三次,用DAPI染色10 min,再用PBS洗涤。通过激光扫描共聚焦显微镜观察样品,结果见图4。图4结果显示:载体材料TAPP-TPGS能够通过胞吞作用被两种癌细胞摄取并分布在细胞质中。
用4T1、MCF-7、HepG2和HL-7702细胞研究TAPP-TPGS和TAPP-TPGS/PTX的细胞毒性,具体为:
将四种细胞以每孔5×103个细胞的密度接种于96孔板中,在37℃、5% CO2氛围的DMEM培养基中培养。在96孔板中加入不同浓度的TAPP-TPGS和TAPP-TPGS/PTX(每一浓度分别两组,无激光照射组和激光照射组)。24 h后,将一组细胞用660 nm的激光照射,光功率密度为18 mW/cm2,每孔的照射时间为10 min,然后将细胞放入培养箱孵育。24 h后,分别将50μL的3-(4,5-二甲基噻唑-2)(4,二甲基噻唑-2)2,5-二苯基四氮唑溴盐(MTT,5mg/mL)溶液加到每个孔,并与细胞在37 ºC、 5%二氧化碳培养箱中培养4 h。向每孔中加入100微升DMSO,置于摇床中10 min使晶体完全溶解。用酶标仪测定各孔在570 nm处的吸光度。
通过MTT实验研究了PTX、TAPP-TPGS和TAPP-TPGS/PTX对癌细胞4T1、MCF-7、HepG2和正常细胞HL-7702的毒性。将四种细胞接种于96孔板,每孔的细胞密度是5×103,在37℃、5% CO2湿润环境DMEM培养基中培养。将不同浓度的PTX、TAPP-TPGS、TAPP-TPGS/PTX分别加到接种4T1、MCF-7、HepG2细胞的孔中,分为两组,无激光照射组(PTX、TAPP-TPGS、TAPP-TPGS/PTX)和激光照射组(PTX+Laser、TAPP-TPGS+Laser、TAPP-TPGS/PTX+Laser)。24 h后,将激光照射组细胞用光功率密度为18 mW/cm2 的660 nm激光照射,每孔的照射时间为10min,然后将细胞放入培养箱孵育。无激光照射组正常培养。将不同浓度的TPGS和TAPP-TPGS加入HL-7702细胞的96孔板中。24 h后,50μL MTT溶液添加到每个孔,然后将细胞在37℃、5%二氧化碳培养箱继续培养4 h。在每孔中再加入100 mL DMSO,在摇床上摇10min使晶体完全溶解。用酶标仪测定各孔在570 nm处的吸光度。
测试结果见图5和图6。图5和图6的实验结果显示:所制备的纳米载体材料TAPP-TPGS对正常细胞(HL-7702)没有毒性(图5中D),证明其生物相容性较好。而对各种癌细胞具有一定的抑制作用(图5中A、B和C),证明特异性材料TPGS的引入可以使载药材料TAPP-TPGS具有一定的抗肿瘤作用,从而可以辅助所载药物PTX治疗癌症。载体材料TAPP-TPGS的光照与非光照毒性试验结果显示,在激光照射下载体材料的抗肿瘤效果明显增强,说明制备的材料具有光动力学作用,并能够协同抗肿瘤药物有效地治疗癌症。图6结果显示,PTX组无论光照与否其细胞毒性相似,证明PTX没有光动力学作用;TAPP-TPGS/PTX与PTX处理组相比有较大的肿瘤细胞毒性,说明PTX负载到TAPP-TPGS能够有效地提高PTX的治疗效果。TAPP-TPGS/PTX+Laser对肿瘤细胞毒性与TAPP-TPGS/PTX处理组相比更大,说明负载材料具有理想的光动力学作用,能与抗肿瘤药物PTX协同抑制癌细胞增长。
将4T1荷瘤小鼠随机分为6组,每组6只。两组小鼠分别经尾部注射生理盐水和PTX。另外四组小鼠分别经尾静脉注射TAPP-TPGS和TAPP-TPGS/PTX (PTX剂量= 5 mg/kg),分为TAPP-TPGS+Laser、TAPP-TPGS/PTX+Laser组和TAPP-TPGS、TAPP-TPGS/PTX组。注射24 h后,TAPP-TPGS+Laser、TAPP-TPGS/PTX+Laser处理组小鼠用功率密度为18 mW/cm2的660 nm激光照射5 min,TAPP-TPGS和TAPP-TPGS/PTX用作对照组。每隔一天测量肿瘤大小和体重,直到第十五天,同时观察小鼠的生存数量,直到实验结束,试验结果见图7。
图7中A的结果显示:TAPP-TPGS/PTX+Laser组的治疗效果优于两者任一单一方式治疗效果,TPGS的引入使得两者联合治疗肿瘤效果增强,并且有效延长载药材料在生物体内的循环时间。体重实验研究(图7中B)结果表明:在整个实验过程中载体材料TAPP-TPGS、载药材料TAPP-TPGS/PTX和激光照射载药材料TAPP-TPGS/PTX+Laser组小鼠的体重均没有明显的下降,证明材料具有较好的生物相容性,药物负载在纳米材料中可以减少药物对生物体的毒副作用。生存率实验研究(图7中C)结果表明:当实验进行到45天时,激光照射载药材料TAPP-TPGS/PTX+Laser组的荷瘤小鼠还有三只生存,TAPP-TPGS/PTX组和TAPP-TPGS+Laser组荷瘤小鼠分别有两只和一只生存,其余组小鼠均全部死亡,结果证明三种方式联合治疗能有效延长荷瘤小鼠的生存时间。
Claims (7)
1.一种特异性卟啉自运输纳米载体材料的制备方法,其特征在于,包括如下步骤:
1)将对硝基苯甲醛、乙酸酐和丙酸混合后,加热至75-85℃,然后滴加吡咯和丙酸的混合物,滴加结束后升温至130-140℃回流反应20-40min,冷却至室温,然后于0-6℃静置析出沉淀,过滤,滤饼经洗涤、干燥获得粗品;粗品溶解于吡啶中并回流1-1.5h,冷却至室温,然后放置于冰水浴中20-30h,收集固体并用丙酮洗涤至洗涤液无色,经真空干燥,得紫色固体四(4-硝基苯基)卟啉TNPP;
2)在氮气气氛下,将TNPP溶解于浓盐酸中获得溶液A;在氮气气氛下,将SnCl2·2H2O溶解于浓盐酸中获得溶液B;将溶液A与溶液B混合,并在75-85℃反应20-40min,停止加热并冷却至室温,然后置于冰水浴中继续反应,此时加入氨水以中和浓盐酸,同时有固体出现,过滤,收集滤液;向滤液中加入3-8%NaOH溶液,搅拌,有固体出现,过滤收集固体,经洗涤、真空干燥获得四(4-氨基苯基)卟啉TAPP;
3)将D-α-生育酚聚乙二醇琥珀酸酯TPGS、琥珀酸酐、DMAP、三乙胺与1,4-二氧六环在室温下搅拌22-26h,然后将反应液装入透析袋在去离子水中透析45-50 h,最后将透析液冷冻干燥,获得羧基化TPGS,即C-TPGS;
4)将C-TPGS、DMAP和六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷PyBOP混合后,加入DMF和TAPP在室温搅拌22-26h,然后将反应液移入透析袋在去离子水中透析40-45h,最后将透析液冻干,获得产品TAPP-TPGS。
2.如权利要求1所述特异性卟啉自运输纳米载体材料的制备方法,其特征在于,步骤1)中,对硝基苯甲醛、乙酸酐和吡咯的摩尔比为1:1.5-2.0:1。
3.如权利要求1所述特异性卟啉自运输纳米载体材料的制备方法,其特征在于,步骤2)中,TNPP与SnCl2·2H2O的摩尔比为1:14-15。
4.如权利要求1所述特异性卟啉自运输纳米载体材料的制备方法,其特征在于,步骤3)中,TPGS、琥珀酸酐和DMAP的摩尔比为1:2:1;透析袋截留分子量为1 kDa。
5.如权利要求1所述特异性卟啉自运输纳米载体材料的制备方法,其特征在于,步骤4)中,C-TPGS和TAPP的摩尔比为4:1;透析袋截留分子量为3.5 kDa。
6.采用权利要求1至5任一所述制备方法制备所得的特异性卟啉自运输纳米载体材料。
7.权利要求6所述特异性卟啉自运输纳米载体材料在制备抗肿瘤纳米药物中的应用。
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