CN113773488A - 一种水溶性聚卟啉类无载体纳米药物的制备方法 - Google Patents
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Abstract
本发明属于高分子材料,生物医药材料及光动力、声动力治疗技术领域,提供了一种水溶性聚卟啉类无载体纳米药物的制备方法。本发明的制备方法得到的水溶性聚卟啉类无载体纳米药物的数均分子量范围为5000g/mol‑30000g/mol,在水中溶解度可达50mg/mL,单线态氧产率为四羧基苯基卟啉的1.5‑3倍。该聚合物可直接溶解于水中,在浓度为0.01mg/mL‑10mg/mL的范围内,可以无载体的形式自发形成稳定的纳米粒,粒径范围为100nm‑200nm。与普通的聚卟啉相比,该种聚卟啉具有水溶性,提高了生物相容性。该种聚卟啉可以在水中自组装成纳米颗粒,无需其他载体,具有潜在的体内递送效果。
Description
技术领域
本发明属于高分子材料,生物医药材料及光动力、声动力治疗技术领域,涉及一种水溶性聚卟啉的制备方法及在光声动力治疗肿瘤中的应用。
背景技术
癌症是世界上致病和致死的首要原因之一,传统的肿瘤治疗手段有外科手术切除、放射治疗以及化疗,在一定程度上可以达到治疗肿瘤的效果,但是具有易复发、毒副作用大、选择性差以及有创性等弊端。相比于传统治疗肿瘤的方法,光动力治疗及声动力治疗是新型的、非侵入性治疗肿瘤的方法,具有毒副作用小、可重复性、选择性好以及无创或微创性,被认为是具有潜力的治疗肿瘤的新手段。目前,光动力或声动力治疗已成为多种包括癌症在内的疾病的临床治疗方式。
光动力治疗需要光、氧和光敏剂的共同作用,而声动力同样需要借助声敏剂发挥治疗效果,光敏剂或声敏剂是光动力或声动力治疗的核心。目前已被开发的光、声敏剂主要有卟啉类、酞菁类、稠环类以及氟硼二吡咯类等,其中,卟啉类衍生物在光动力或声动力中的应用最为广泛。四苯基卟啉(Tetraphenylporphyrin,TPP)是典型的第二代光敏剂,能高效的产生单线态氧,并且具有相对较低的暗毒性。但由于TPP是平面刚性共轭结构,由于在水性介质中的强疏水性π-π堆积效应,随着浓度的增加光敏剂趋于聚集,容易聚集引发聚集诱导淬灭(ACQ)效应,ACQ效应可能会大大降低单线态氧量子产率,并削弱光动力的功效。另外,TPP是水溶性差的小分子,不利于给药,且在体内循环时间短,这些问题限制了其体内应用。
为了克服上述问题,我们通过引入柔性链设计合成了一种水溶性的无载体的聚卟啉纳米药物,在水中可以自组装成纳米颗粒,并对其单线态氧产率、纳米粒稳定性以及体外光暗毒性进行了测试。此项发明解决了具有大环平面共轭结构的光敏剂极易发生聚集诱导淬灭的问题以及水溶性差的问题。该发明所设计的聚卟啉结构同样可作为声敏剂用于肿瘤的声动力治疗。
发明内容
本发明的目的是主要提供了一种水溶性的聚卟啉类无载体纳米药物的制备方法。
本发明的技术方案:
一种水溶性的无载体的聚卟啉纳米药物的制备方法,步骤如下:
称取1摩尔当量的四羧基苯基卟啉、4-10摩尔当量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)和4-10摩尔当量的对二甲氨基吡啶(DMAP)溶于氯仿(CHCl3)、四氢呋喃(THF)或N,N-二甲基甲酰胺(DMF)中,控制四羧基苯基卟啉的单体浓度范围为0.1M-5M,加热至40-90℃搅拌10-30min;然后,在溶液中加入1.8-2.2摩尔当量的含有多个醚键的二醇类单体,将反应体系升温至90-140℃继续反应12-144h;待反应结束后,将反应液在正己烷或乙醚中沉降(正己烷或乙醚的体积为混合溶液体积的10-50倍),经离心收集沉淀,真空干燥过夜除去残留的溶剂。将沉淀溶于体积比为1/10-0/10的DMSO和H2O的溶剂中,并用截留分子量(MWCO)为300-3000Da的透析袋在去离子水中透析12-48h后,于冷冻干燥机中冻干,最终产物为紫色固体P-nO,n=1-20。该聚卟啉数均分子量范围为5000g/mol-30000g/mol,在水中溶解度可达50mg/mL,单线态氧产率为四羧基苯基卟啉的1.5-3倍。该聚合物可直接溶解于水中,在浓度为0.01mg/mL-10mg/mL的范围内,可以无载体的形式自发形成稳定的纳米粒,粒径范围为100nm-200nm。
反应路线如下:
本发明的有益效果:
(1)本发明提出了一种水溶性聚卟啉的制备方法,且普适性高,为合成高分子量卟啉聚合物提供了技术参考。
(2)与单体小分子卟啉相比,聚卟啉的单线态氧产率可以提高至2-3倍左右。
(3)与普通的聚卟啉相比,该种聚卟啉具有水溶性,提高了生物相容性。
(4)与普通的聚卟啉相比,该种聚卟啉可以在水中自组装成纳米颗粒,无需其他载体,具有潜在的体内递送效果。
附图说明
图1是聚卟啉P-3O的氢核磁。
图2是聚卟啉P-3O的碳核磁。
图3是聚卟啉P-5O的氢核磁。
图4是聚卟啉P-5O的稀释稳定性表征。
图5是聚卟啉P-5O的冻干稳定性表征。
图6是聚卟啉P-5O的光毒性及声毒性。
具体实施方式
以下结合附图和技术方案,进一步说明本发明的具体实施方式。
实施例1:水溶性聚卟啉P-3O的合成
准确称取四羧基苯基卟啉(25mg,0.032mmol)、EDCI(49mg,0.256mmol)和DMAP(15.6mg,0.128mmol)溶于0.2mL-2mLDMF中,加热至60℃-80℃搅拌10min。然后,加入三缩四乙二醇(12.4mg,0.064mmol)和0.1mL-0.5mLDMF的混合溶液,升温至100℃-140℃继续反应24h-48h。待反应结束后,将反应液在15mL乙醚中沉降,经离心收集沉淀,真空干燥过夜除去残留的乙醚。将沉淀溶于体积比为10:1的H2O和DMSO的混合溶剂中,并用截留分子量(MWCO)为500的透析袋在去离子水中透析24h后,于冷冻干燥机中冻干,最终产物为紫色固体P-3O。所得聚卟啉的氢谱核磁表征如图1所示。所得聚卟啉的碳谱核磁表征如图2所示。
实施例2:水溶性聚卟啉P-5O的合成
准确称取四羧基苯基卟啉(25mg,0.032mmol)、EDCI(49mg,0.256mmol)和DMAP(15.6mg,0.128mmol)溶于0.2mL-2mLDMF中,加热至60℃-80℃搅拌10min。然后,加入六甘醇(17.6mg,0.064mmol)和0.1mL-0.5mLDMF的混合溶液,升温至100℃-140℃继续反应24h-48h。待反应结束后,将反应液在15mL乙醚中沉降,经离心收集沉淀,真空干燥过夜除去残留的乙醚。将沉淀溶于体积比为10:1的H2O和DMSO的混合溶剂中,并用截留分子量(MWCO)为500的透析袋在去离子水中透析24h后,于冷冻干燥机中冻干,最终产物为紫色固体P-5O。所得聚卟啉的氢谱核磁表征如图3所示。
实施例3:水溶性聚卟啉P-11O的合成
准确称取四羧基苯基卟啉(25mg,0.032mmol)、EDCI(49mg,0.256mmol)和DMAP(15.6mg,0.128mmol)溶于1mL氯仿中,加热至60℃-80℃搅拌10min。然后,加入分子量为500Da的聚乙二醇(32mg,0.064mmol)和1mL氯仿的混合溶液,升温至70℃继续反应24h-48h。待反应结束后,将反应液在30mL正己烷中沉降,经离心收集沉淀,真空干燥过夜除去残留的正己烷。将沉淀溶于1mL H2O中,并用截留分子量(MWCO)为1000的透析袋在去离子水中透析24h后,于冷冻干燥机中冻干,最终产物为紫色固体P-11O。
实施例4:水溶性聚卟啉P-18O的合成
准确称取四羧基苯基卟啉(25mg,0.032mmol)、EDCI(49mg,0.256mmol)和DMAP(15.6mg,0.128mmol)溶于1mL四氢呋喃中,加热至60℃-80℃搅拌10min。然后,加入分子量为800Da的聚乙二醇(51.2mg,0.064mmol)和1mL四氢呋喃的混合溶液,升温至70℃继续反应24h-48h。待反应结束后,将反应液在30mL正己烷中沉降,经离心收集沉淀,真空干燥过夜除去残留的正己烷。将沉淀溶于1mLH2O中,并用截留分子量(MWCO)为1000的透析袋在去离子水中透析24h后,于冷冻干燥机中冻干,最终产物为紫色固体P-11O。
实施例5:水溶性聚卟啉纳米颗粒的稳定性表征
首先将聚卟啉P-5O直接溶于去离子水中制成母液,浓度为0.5mg/mL,在37℃静置1h,自组装成纳米粒。
对稀释液的稳定性:在室温下将纳米粒用血清缓冲液(FBS:10%)稀释成5倍,10倍,20倍,50倍,100倍,随即通过DLS测量粒径变化,所得粒径如图4所示。
冻干稳定性:将纳米粒溶于PBS缓冲液中进行冻干前后DLS测量粒径,所得粒径如图5所示。
实施例6:水溶性聚卟啉的光声毒性表征
光毒性:采用MTT法测定聚卟啉纳米粒对小鼠肝癌细胞(Hep1-6)的细胞毒性。将Hep1-6细胞以1×104细胞/孔的密度接种在96孔板中,孵育在含10%FBS的细胞培养皿中。更换培养基,加入50μM的纳米粒。培养24小时后,用650nm激光(10mW/cm2)照射孔板30分钟。细胞再孵育20小时后进行MTT检测,以评估细胞存活率。
声毒性:采用MTT法测定聚卟啉纳米粒对小鼠肝癌细胞(Hep1-6)的细胞毒性。将Hep1-6细胞以1×104细胞/孔的密度接种在96孔板中,孵育在含10%FBS的细胞培养皿中。更换培养基,加入50μM的纳米粒。培养24小时后,用超声治疗仪对细胞进行超声(1MHz,700mW/cm2)10分钟处理,细胞再孵育20小时后进行MTT检测,以评估细胞存活率。
Claims (1)
1.一种水溶性的无载体的聚卟啉纳米药物的制备方法,其特征在于,步骤如下:
称取1摩尔当量的四羧基苯基卟啉、4-10摩尔当量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和4-10摩尔当量的对二甲氨基吡啶溶于氯仿、四氢呋喃或N,N-二甲基甲酰胺中,控制四羧基苯基卟啉的单体浓度范围为0.1M-5M,加热至40-90℃搅拌10-30min;然后,在溶液中加入1.8-2.2摩尔当量的含有多个醚键的二醇类单体,将反应体系升温至90-140℃继续反应12-144h;待反应结束后,将反应液在正己烷或乙醚中沉降(正己烷或乙醚的体积为混合溶液体积的10-50倍),经离心收集沉淀,真空干燥过夜除去残留的溶剂;将沉淀溶于体积比为1/10-0/10的DMSO和H2O的溶剂中,并用截留分子量为300-3000Da的透析袋在去离子水中透析12-48h后,于冷冻干燥机中冻干,最终产物为紫色固体P-nO,n=1-20;该聚卟啉数均分子量范围为5000g/mol-30000g/mol,在水中溶解度可达50mg/mL,单线态氧产率为四羧基苯基卟啉的1.5-3倍;该聚合物可直接溶解于水中,在浓度为0.01mg/mL-10mg/mL的范围内,可无载体的形式自发形成稳定的纳米粒,粒径范围为100nm-200nm。
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CN103536919A (zh) * | 2013-10-24 | 2014-01-29 | 天津市肿瘤研究所 | 肿瘤靶向的光动力载药纳米粒子及其制备方法和用途 |
CN110790922A (zh) * | 2019-11-06 | 2020-02-14 | 大连理工大学 | 一种聚卟啉类化合物的制备方法及应用 |
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CN116041319A (zh) * | 2022-11-08 | 2023-05-02 | 江苏中利集团股份有限公司 | 一种吲哚基四氢异喹啉衍生物的制备方法 |
CN116041319B (zh) * | 2022-11-08 | 2023-08-25 | 江苏中利集团股份有限公司 | 一种吲哚基四氢异喹啉衍生物的制备方法 |
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