CN111848658B - 一种靶向线粒体的氟硼二吡咯类化合物及其脂质体包裹纳米粒子的制备方法和用途 - Google Patents
一种靶向线粒体的氟硼二吡咯类化合物及其脂质体包裹纳米粒子的制备方法和用途 Download PDFInfo
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Abstract
本发明涉及一种靶向线粒体的氟硼二吡咯类化合物及其脂质体包裹纳米粒子的制备方法和用途。所述氟硼二吡咯类化合物含有阳离子基团,其易结合细胞内带负电的线粒体膜,从而靶向于细胞的线粒体胞器。利用脂质体对其进行包裹,在水溶液中制备得到粒径均一的脂质体纳米粒子。该纳米粒子在水溶液中具有高稳定性和良好的生物相容性。纳米粒子被肿瘤细胞摄取后,脂质体结构破坏,释放的氟硼二吡咯类化合物靶向于细胞线粒体。此外,在665nm光照条件下,氟硼二吡咯类化合物产生的活性氧对线粒体内的活性分子产生氧化损伤,从而杀伤肿瘤细胞。该脂质体纳米粒子可作为递送各类光敏剂药物应用于肿瘤光动力治疗。
Description
技术领域
本发明属于药物和药剂学领域,具体涉及一种靶向线粒体的氟硼二吡咯类化合物、及其脂质体包裹纳米粒子的制备方法和用途。
背景技术
光动力治疗(Photodynamic therapy,PDT)是一种治疗肿瘤的非侵入性技术。发挥PDT需要满足三个基本条件,即光敏剂、特定波长的光和基态氧。其治疗机理为将光敏剂静脉注射至人体后,光敏剂在肿瘤区域富集。当用特定波长的光照射该区域时,光敏剂吸收光能而跃迁至激发态,处于激发态的光敏剂将能量传递给肿瘤组织和细胞周围的基态氧,进而生成高氧化性的活性氧。活性氧能与细胞中的各种生物大分子发生氧化反应,导致肿瘤细胞凋亡、血管损伤和引发一些炎症反应,最终达到杀伤肿瘤组织和细胞的目的。通常活性氧的半衰期短(<40ns),作用区域小(<20nm),因此单线态氧对肿瘤组织和细胞造成的氧化性损伤被限制在活性氧产生区域。为了提高活性氧的光毒性,因此,开发具有靶向定位于肿瘤细胞器的光敏剂被广泛研究。
细胞器靶向可分为细胞核靶向、溶酶体靶向、线粒体靶向、内质网靶向等。线粒体作为生物系统中的“动力工厂”,在细胞质中分布广泛,且在三羧酸循环、脂肪酸代谢、氧化磷酸化等生理生化过程中发挥着重要作用。另外,线粒体是介导细胞程序性凋亡的主要细胞器。当线粒体的膜电位下降,其膜通透性增加,线粒体内促凋亡因子(如细胞色素C)释放至细胞质中,激活Caspase9酶并进一步激活Caspase3酶和Caspase7酶,最终导致细胞凋亡。由于线粒体的跨膜电位为负值,故亲脂性阳离子型光敏剂能穿过脂质双分子层的疏水屏障并在线粒体基质中富集。例如,Ge等合成了一种线粒体靶向的酞菁类光敏剂用于肿瘤光动力治疗(Y.Ge,et.al.RSC Advances 2013,3,12839-12846)。由于其所合成的锌酞菁光敏剂结构中含有四个亲脂性阳离子基团,使得锌酞菁光敏剂能够在水溶液中自组装成纳米粒子。该纳米粒子被人宫颈癌(HeLa)细胞中摄入后,能够富集在线粒体,在波长为590nm光照下产生活性氧并促使肿瘤细胞发生凋亡。然而,他们所合成的锌酞菁光敏剂自组装形成纳米粒子后,表面电位为正值,在生理条件和血液循环中极不稳定,易结合蛋白质被肝脾巨噬细胞吞噬,无法到达肿瘤且被肿瘤细胞吞噬。2019年,Ko等报道了一种线粒体靶向的光敏剂纳米粒子用于脑部恶性肿瘤光动力治疗(Y.T.Ko,et.al.Biomaterials Science 2019,7,2812-2825)。但其研究中所制备的纳米粒子表面仍带有正电荷,当纳米粒子静脉注射至小鼠体内后,会造成光敏剂全身分布,对正常组织和细胞产生一定的光毒副作用。因此,将线粒体靶向基团键连光敏剂不能有效区分肿瘤细胞与正常细胞(D.Chen,et.al.Journal ofMaterials Chemistry B 2018,6,4522-4530)。
为了降低亲脂性阳离子光敏剂对正常细胞所造成的损伤,Thomas等将透明质酸钠(HA)作为光敏剂的递送载体(A.P.Thomas,et.al.Chemical Science 2017,8,8351-8356)。由于HA是一种带有负电荷的多糖,因此可通过静电作用与阳离子型光敏剂IR-780静电吸附结合,从而降低光敏剂对正常细胞的副作用。但其实验结果表明,HA与光敏剂结合后所形成的纳米粒子粒径分布不均一,限制纳米粒子在肿瘤部位富集量,产生的光动力效果不明显。
氟硼二吡咯类化合物(BODIPY)是由Treibs和Kreuzer在1968年报道的一类性能优异的荧光染料。Yogo等在2005年发现BODIPY经碘原子修饰后,荧光量子产率明显下降,而单线态氧产率明显提高(T.Yogo,et.al.Journal of the American Chemistry Society2005,127,12162-12163)。这表明BODIPY经溴、碘等重原子修饰后,其产生单线态氧的能力增强。另外,通过Knoevenagel反应在氟硼二吡咯上引入共轭基团,增大共轭体系,其吸收波长红移(650-860nm),有助于治疗深部位肿瘤。因此,研制开发具有线粒体靶向的氟硼二吡咯类化合物,而且能在生理条件和血液中具有较长循环时间,且可在良好的生物溶剂中共组装形成粒径均一的纳米粒子对提高PDT的治疗效果具有理论和临床意义。
发明内容
本发明的目的在于提供一种靶向线粒体的氟硼二吡咯类化合物、及其脂质体包裹纳米粒子的制备方法和用途。由于氟硼二吡咯类化合物分子中含有两个阳离子基团,使得氟硼二吡咯类化合物能够穿过线粒体的脂质双分子层并定位于线粒体中。当脂质体对氟硼二吡咯类化合物进行包裹后,能够在水溶液中形成粒径均一的纳米粒子,平均粒径为113.3±10.1nm。纳米粒子在水溶液中具有高稳定性,被肿瘤细胞摄取后,其结构在溶酶体中破坏,释放的氟硼二吡咯类化合物能够定位于线粒体胞器。在665nm激光照射下,在肿瘤细胞内产生大量活性氧,并杀伤肿瘤细胞。
本发明采用以下技术方案:
一种靶向线粒体的氟硼二吡咯类化合物,其化学结构式如式I所示:
X选自如下化学基团:Cl、Br、I;
n值为0-12。
本发明提供一种包裹氟硼二吡咯类化合物的脂质体纳米粒子,由上述的氟硼二吡咯类化合物和二硬脂酰基磷脂乙醇胺-聚乙二醇-键连基团(DSPE-PEGm-Z)制得。
更进一步地,所述键连基团Z为氢、甲氧基、叶酸、多肽、聚氨基酸、生物素、甘露糖或转铁蛋白。
更进一步地,所述聚氨基酸或多肽,选自线性多肽、环肽、或细胞穿膜肽。
更进一步地,所述线性多肽为RGDV、GRGDSPK、CPLGVRGRGDS、GRGDSPC;所述环肽为c(RGDyC)、c(RGDfK)、c[RGDfK(Biotin)]、c(RGDyE)、c(CRGDKGPDC);所述细胞穿膜肽为YGRKKRRQRRR、GRKKRRQRRRPQ、RRRRRRRR。
更进一步地,聚乙二醇的平均分子量m为200-20000。典型但非限制性的,所述的平均分子量m如为600、1000、2000、3500、5000。
本发明提供所述的脂质体纳米粒子的制备方法,步骤如下:将靶向线粒体的氟硼二吡咯类化合物在有机溶剂中配置成一定浓度的母液待用,另配置一定浓度DSPE-PEGm-Z的有机溶液。将上述母液和DSPE-PEGm-Z的有机溶液充分混合后,在超声作用下滴入特定体积的水溶液中,透析,从而制得脂质体纳米粒子。
更进一步地,具体步骤如下:将权利要求1所合成的氟硼二吡咯化合物溶解在二甲基亚砜中配置成0.1-10mg/mL母液待用,另配置质量浓度为0.1-10mg/mL DSPE-PEGm-Z的二甲基亚砜溶液。将母液与DSPE-PEGm-Z充分混合后,滴入0.1-10mL水溶液中并继续超声2小时,透析,从而制得脂质体纳米粒子。
本发明提供一种所述靶向靶向线粒体的氟硼二吡咯类化合物及其脂质体包裹纳米粒子在制备光动力治疗癌症药剂中的应用。
本发明与现有技术相比,其有益效果为:
1)本发明所制备的氟硼二吡咯类化合物能被脂质体DSPE-PEGm-Z包裹,形成具有核-壳结构的脂质体纳米粒子,且粒径均一,平均粒径为113.3±10.1nm(见附图1)。
2)脂质体包裹氟硼二吡咯类化合物能显著降低其暗毒性(见附图2)。细胞毒性结果显示:氟硼二吡咯类化合物在无光照条件下,对人乳腺癌MDA-MB-231细胞(A)和人正常乳腺上皮细胞MCF-10A(B)均产生化学毒性。而脂质体包裹后,脂质体纳米粒子在无光照条件下对细胞不产生毒性,仅在光照条件下产生毒性。
3)脂质体纳米粒子被MDA-MB-231癌细胞摄入后,氟硼二吡咯类化合物能够靶向富集于细胞线粒体(附图3)。
4)在波长为665nm光照下(33mW/cm2)下,氟硼二吡咯类化合物能在MDA-MB-231癌细胞内产生大量单线态氧(见附图4),导致对MDA-MB-231癌细胞产生明显的杀伤效果(见附图5)。
附图说明
图1为本发明所制备的氟硼二吡咯类化合物能够与DSPE-PEGm-Z形成具有核-壳结构的脂质体纳米粒子的透射电子显微镜图片。
图2为光照或黑暗条件下不同浓度的脂质体纳米粒子和氟硼二吡咯类化合物分别与人乳腺癌MDA-MB-231细胞(A)和人正常乳腺上皮MCF-10A细胞(B)孵育24小时的相对存活率;
图3为激光共聚焦显微镜图片证明,纳米粒子与乳腺癌MDA-MB-231细胞共同孵育24小时后,氟硼二吡咯最终定位在线粒体,A:线粒体染料染色的细胞线粒体显绿色;B:氟硼二吡咯光敏剂荧光成像显红色;C:前两种成像的合并图。
图4为激光共聚焦显微镜图片证明,在波长665nm的光照(33mW/cm2)下,乳腺癌MDA-MB-231细胞内的氟硼二吡咯光敏剂被激发产生细胞毒性的活性氧。细胞内活性氧与2’,7’-二氯荧光素二乙酸盐反应生成绿色荧光产物;A:细胞明场照片;B:光照下的荧光照片;C:前两种成像的合并图。
钙黄绿素乙酰氧基甲酯(Calcein Acetoxymethyl Ester,Calcein-AM),是一种可渗透进入细胞、常用于测定真核细胞活力或线粒体通透性转换孔的绿色荧光探针。Calcein-AM本身并无荧光,进入细胞后被细胞中内源性酯酶水解生成具有强负电荷的不能通透细胞膜的极性分子钙黄绿素(Calcein),从而被滞留在细胞内,而Calcein可被激发出绿色荧光。由于死细胞缺乏酯酶,核酸红色荧光染料碘化丙啶(Propidium Iodide,PI)由于不能穿透活细胞的细胞膜而只能染色细胞膜完整性被破坏的死细胞,所以Calcein-AM常常与PI联合使用,对活细胞和死细胞同时进行双重荧光染色。图5为荧光共聚焦显微镜照片证明,氟硼二吡咯光敏剂与MDA-MB-231细胞孵育3小时后,在665nm波长的激光(33mW/cm2)照射下光照区域内的MDA-MB-231细胞死亡,而非光照区域仍为活细胞。A:染料Calcein-AM染色的活细胞;B:染料PI染色的死细胞;C:前两种成像的合并图。
具体实施方式
下面结合实施例对本发明作进一步的详细描述,但本发明不仅限于此。
本发明通式的一般制备方法:
(1)将溶于溶剂(例如四氢呋喃溶液)中后,向其中分别加入2,4-二甲基吡咯和三氟乙酸,在氮气保护下室温反应过夜;向上述混合液中加入2,3-二氯-5,6-二氰基-1,4-苯醌并继续反应一定时间(例如4小时);之后,向上述混合液(0℃下)中依序加入三乙胺和三氟化硼乙醚,继续反应过夜。经硅胶柱层析分离纯化制备得到
(4)将步骤(3)中的所得化合物溶于氯仿和乙醇后,加入吡啶溶液,反应液在一定温度(例如80℃)下反应过夜,经重结晶后得到具有本发明中通式结构的化合物。
实施例1
合成R1为-OCH3修饰的线粒体靶向的氟硼二吡咯类化合物具体制备过程包括:
(1)将4-甲氧基苯甲醛(409mg,3mmol)溶解于90mL无水四氢呋喃中。在搅拌下,加入2,4-二甲基吡咯(0.63g,6.6mmol)、0.25mL三氟乙酸,反应液在氮气保护下反应14小时。之后,将2,3-二氯-5,6-二氰基-1,4-苯醌(0.68g,3mmol)溶解于120mL无水四氢呋喃中,逐滴加入上述反应液中,继续反应4小时。将上述反应液置于冰水混合物中后,逐滴加入18mL三乙胺。反应液在冰水混合物中反应30分钟后,逐滴加入18mL三氟化硼乙醚,反应液继续在25℃下反应14小时。反应结束后,旋蒸除去溶剂,粗产物用200mL二氯甲烷溶解后,用同等体积饱和食盐水洗涤,用无水硫酸钠干燥有机层。收集有机相,旋蒸除去溶剂,通过硅胶柱层析分离得到纯品:
1H-NMR(400MHz,CDCl3)δ=7.18(d,J=9.0,2H),7.03(d,J=9.0,2H),5.97(s,2H),3.87(s,3H),2.55(s,6H),1.43(s,6H).13C-NMR(100MHz,CDCl3)δ=160.20,155.30,143.20,141.90,131.91,129.26,127.10,121.15,114.58,55.34,14.58.HRMS(tof):m/z calcd forC20H21BF2N2O:354.1715;found:355.1721[M+H]+。
(2)将(1)中所得化合物(180mg,0.51mmol)、N-碘代丁二酰亚胺(345mg,1.53mmol)溶解于20mL二氯甲烷中,25℃下避光反应10小时。反应结束后,加入150mL二氯甲烷,用同等体积的饱和亚硫酸钠水溶液洗涤3次,用无水硫酸钠干燥有机相。收集有机相,旋蒸除去溶剂,通过硅胶柱层析分离得到纯品:
1H-NMR(400MHz,CDCl3)δ=7.13(d,J=6.0,2H),7.02(d,J=9.0,2H),3.89(s,3H),2.64(s,6H),1.44(s,6H).13C-NMR(100MHz,CDCl3)δ=160.56,156.59,145.38,141.60,131.75,129.11,126.71,114.88,55.41,17.17,16.02.HRMS(tof):m/z calcd forC20H19BF2I2N2O:605.9726;found:606.9727[M+H]+。
(3)将(2)中所得化合物(100mg,0.165mmol)、(188mg,0.66mmol)溶解于30mL甲苯中,分别加入乙酸和哌啶各0.5mL,通过分水器除去反应产生的水。反应液回流3小时后,冷却至室温。将甲苯减压蒸出,粗产品溶于二氯甲烷,并用饱和食盐水洗涤3次,有机相用无水硫酸钠干燥。收集有机相,旋蒸除去溶剂。粗产品通过硅胶柱分离纯化得到纯品,其化学结构式为:
1H-NMR(400MHz,CDCl3)δ=8.10(d,J=18.0,2H),7.56(t,J=6.0,6H),7.16(d,J=9.0,2H),7.03(d,J=9.0,2H),6.92(d,J=9.0,4H),4.00(t,J=6.0,4H),3.90(s,3H),3.42(t,J=6.0,4H),1.81(m,8H),1.51(t,J=6.0,14H).13C-NMR(100MHz,CDCl3)δ=160.58,160.25,150.48,145.70,139.10,138.58,133.29,129.69,129.27,127.36,116.76,114.89,67.91,55.43,33.75,32.71,29.07,27.95,25.31,17.69.HRMS(tof):m/z calcd forC46H49BBr2F2I2N2O3:1138.0339;found:1139.0356[M+H]+。
(4)将(3)中所得化合物(82mg,0.07mmol)溶解于3mL氯仿中,向上述混合液中加入6mL吡啶和3mL无水乙醇后,该混合液在80℃下搅拌12小时。反应结束后,冷却至室温,将溶剂减压蒸出。粗产品通过重结晶得到纯品,其化学结构式为:
1H-NMR(400MHz,DMSO-d6)δ=9.18(s,4H),8.62(s,2H),8.18(s,4H),8.04(d,J=16.0,2H),7.56(s,4H),7.40(d,J=12.0,2H),7.31(s,2H),7.15(s,2H),7.03(s,4H),4.66(s,4H),4.02(s,4H),3.85(s,3H),1.97(s,4H),1.73(s,4H),1.36(d,J=32.0,14H).13C-NMR(100MHz,DMSO-d6)δ=160.75,160.53,150.26,145.97,145.26,139.83,139.10,133.43,130.16,129.39,129.02,128.57,126.67,116.51,115.77,115.42,84.84,79.71,68.06,61.19,55.84,49.06,31.71,31.09,28.78,25.62,25.37,22.94,22.54,17.72,14.39,11.69.HRMS(tof):m/z[M-2Br]2+/2calcd for C56H59BBr2F2I2N2O3:569.1369;found:569.1364。
(5)将(4)中的化合物溶解在二甲基亚砜中配置成0.5mg/mL母液待用,另配置质量浓度为1mg/mL DSPE-PEG2000-叶酸(购自湖南华腾制药有限公司,下同)的二甲基亚砜溶液。取0.24mL母液与0.9mL DSPE-PEG2000-叶酸充分混合后,滴入0.1mL水溶液中并继续超声2小时,用分子量为3500的透析袋透析24小时,从而制得表面叶酸修饰的脂质体纳米粒子。如附图1所示,本实施例所制备的氟硼二吡咯类化合物能够与DSPE-PEGm-叶酸在良好的生物溶剂中能形成具有核-壳结构的脂质体纳米粒子,且粒径分布均匀。
实施例2
合成R1为-N(C2H5)2修饰的线粒体靶向的氟硼二吡咯类化合物具体制备过程包括:
(1)将4-(N,N-二乙基)氨基苯甲醛(532mg,3mmol)溶解于90mL无水四氢呋喃中。在搅拌下,加入2,4-二甲基吡咯(0.63g,6.6mmol)、0.25mL三氟乙酸,反应液在氮气保护下反应14小时。之后,将2,3-二氯-5,6-二氰基-1,4-苯醌(0.68g,3mmol)溶解于120mL无水四氢呋喃中,逐滴加入上述反应液中,继续反应4小时。将上述反应液置于冰水混合物中后,逐滴加入18mL三乙胺。反应液在冰水混合物中反应30分钟后,逐滴加入18mL三氟化硼乙醚,反应液继续在25℃下反应14小时。反应结束后,旋蒸除去溶剂,粗产物用200mL二氯甲烷溶解后,用同等体积饱和食盐水洗涤,用无水硫酸钠干燥有机层。收集有机相,旋蒸除去溶剂,通过硅胶柱层析分离得到纯品:
1H-NMR(400MHz,CDCl3)δ=7.02(d,J=8.0,2H),6.74(d,J=8.0,2H),5.97(s,2H),3.42(q,J=4.0,4H),2.54(s,6H),1.26(s,6H),1.12(m,6H).13C-NMR(100MHz,CDCl3)δ=148.0,145.7,143.6,137.7,131.8,128.8,126.0,124.4,113.5,111.7,107.4,47.1,14.1,12.9.HRMS(tof):m/z calcd for C23H28BF2N3:395.2344;found:396.2346[M+H]+。
(2)将(1)中所得化合物(396mg,1.0mmol)、N-碘代丁二酰亚胺(675mg,3.0mmol)溶解于20mL二氯甲烷中,25℃下避光反应10小时。反应结束后,加入150mL二氯甲烷,用同等体积的饱和亚硫酸钠水溶液洗涤3次,用无水硫酸钠干燥有机相。收集有机相,旋蒸除去溶剂,通过硅胶柱层析分离得到纯品:
1H-NMR(400MHz,CDCl3)δ=7.01(d,J=8.0,2H),6.76(d,J=8.0,2H),3.40(q,J=4.0,4H),2.52(s,6H),1.21(s,6H),1.14(m,6H).13C-NMR(100MHz,CDCl3)δ=148.3,145.9,143.8,137.2,131.4,128.5,126.1,124.3,113.7,111.6,107.8,47.5,14.4,12.6.HRMS(tof):m/z calcd for C23H26BF2I2N3:647.0277;found:648.0276[M+H]+。
(3)将(2)中所得化合物(100mg,0.15mmol)、(174mg,0.61mmol)溶解于30mL甲苯中,分别加入乙酸和哌啶各0.5mL,通过分水器除去反应产生的水。反应液回流3小时后,冷却至室温。将甲苯减压蒸出,粗产品溶于二氯甲烷,并用饱和食盐水洗涤3次,有机相用无水硫酸钠干燥。收集有机相,旋蒸除去溶剂。粗产品通过硅胶柱分离纯化得到纯品,其化学结构式为:
1H-NMR(400MHz,CDCl3)δ=1H-NMR(400MHz,CDCl3)δ=8.12(d,J=18.0,2H),7.54(t,J=6.0,6H),7.18(d,J=9.0,2H),7.02(d,J=9.0,2H),6.91(d,J=9.0,4H),4.01(t,J=6.0,4H),3.42(t,J=6.0,8H),1.85(m,8H),1.46(t,J=6.0,14H),1,12(m,6H).13C-NMR(100MHz,CDCl3)δ=161.58,161.25,151.48,143.70,139.15,138.23,132.56,129.67,128.45,127.23,116.06,114.02,67.12,57.44,35.75,33.71,28.07,26.95,24.31,16.69.HRMS(tof):m/z calcd for C49H56BBr2F2I2N3O2:1179.0890;found:1180.0893[M+H]+。
(4)将(3)中所得化合物(85mg,0.07mmol)溶解于3mL氯仿中,向上述混合液中加入6mL吡啶和3mL无水乙醇后,该混合液在80℃下搅拌12小时。反应结束后,冷却至室温,将溶剂减压蒸出。粗产品通过重结晶得到纯品,其化学结构式为:
1H-NMR(400MHz,DMSO-d6)δ=9.15(s,4H),8.23(s,2H),8.15(s,4H),8.04(d,J=16.0,2H),7.52(s,4H),7.41(d,J=12.0,2H),7.26(s,2H),7.11(s,2H),7.02(s,4H),4.64(s,4H),4.05(s,4H),3.42(m,4H),1.95(s,4H),1.76(s,4H),1.34(d,J=32.0,14H),1.13(m,6H).13C-NMR(100MHz,DMSO-d6)δ=162.75,160.35,150.62,145.79,145.34,139.83,139.01,133.34,130.61,129.93,129.20,128.75,126.89,116.15,115.77,115.24,84.48,79.17,68.60,61.91,55.48,52.16,49.60,31.17,31.90,28.87,26.26,24.73,22.49,22.45,17.27,14.92,11.19.HRMS(tof):m/z[M-2Br]2+/2calcd for C56H59BBr2F2I2N2O3:589.6683;found:589.6685。
(5)将(4)中的化合物溶解在二甲基亚砜中配置成0.5mg/mL母液待用,另配置质量浓度为1mg/mL DSPE-PEG2000-叶酸的二甲基亚砜溶液。取0.24mL母液与0.9mL DSPE-PEG2000-叶酸充分混合后,滴入0.1mL水溶液中并继续超声2小时,用分子量为3500的透析袋透析24小时,从而制得表面叶酸修饰的脂质体纳米粒子。
实施例3
(1)将4-(4-吗啉)苯甲醛(574mg,3mmol)溶解于90mL无水四氢呋喃中。在搅拌下,加入2,4-二甲基吡咯(0.63g,6.6mmol)、0.25mL三氟乙酸,反应液在氮气保护下反应14小时。之后,将2,3-二氯-5,6-二氰基-1,4-苯醌(0.68g,3mmol)溶解于120mL无水四氢呋喃中,逐滴加入上述反应液中,继续反应4小时。将上述反应液置于冰水混合物中后,逐滴加入18mL三乙胺。反应液在冰水混合物中反应30分钟后,逐滴加入18mL三氟化硼乙醚,反应液继续在25℃下反应14小时。反应结束后,旋蒸除去溶剂,粗产物用200mL二氯甲烷溶解后,用同等体积饱和食盐水洗涤,用无水硫酸钠干燥有机层。收集有机相,旋蒸除去溶剂,通过硅胶柱层析分离得到纯品:
1H-NMR(400MHz,CDCl3)δ=7.16(d,J=9.0,2H),7.02(d,J=9.0,2H),5.98(s,2H),3.73(t,J=8.0 4H),3.15(t,J=8.0,4H),2.29(s,6H),1.41(s,6H).13C-NMR(100MHz,CDCl3)δ=148.8,148.0,143.6,137.7,131.8,128.8,126.0,124.4,113.5,111.7,107.4,66.3,53.3,19.5,14.2.HRMS(tof):m/z calcd for C23H26BF2N3O:409.2137;found:410.2142[M+H]+。
(2)将(1)中所得化合物(209mg,0.51mmol)、N-碘代丁二酰亚胺(345mg,1.53mmol)溶解于20mL二氯甲烷中,25℃下避光反应10小时。反应结束后,加入150mL二氯甲烷,用同等体积的饱和亚硫酸钠水溶液洗涤3次,用无水硫酸钠干燥有机相。收集有机相,旋蒸除去溶剂,通过硅胶柱层析分离得到纯品:
1H-NMR(400MHz,CDCl3)δ=7.18(d,J=9.0,2H),7.04(d,J=9.0,2H),3.74(t,J=8.04H),3.14(t,J=8.0,4H),2.19(s,6H),1.42(s,6H).13C-NMR(100MHz,CDCl3)δ=148.7,148.2,143.5,137.6,131.4,128.3,126.2,124.1,113.3,111.6,107.5,66.4,53.4,19.5,14.1.HRMS(tof):m/z calcd for C23H24BF2I2N3O:661.0070;found:662.0072[M+H]+。
(3)将(2)中所得化合物(100mg,0.15mmol)、(188mg,0.66mmol)溶解于25mL甲苯中,分别加入乙酸和哌啶各0.5mL,通过分水器除去反应产生的水。反应液回流3小时后,冷却至室温。将甲苯减压蒸出,粗产品溶于二氯甲烷,并用饱和食盐水洗涤3次,有机相用无水硫酸钠干燥。收集有机相,旋蒸除去溶剂。粗产品通过硅胶柱分离纯化得到纯品,其化学结构式为:
1H-NMR(400MHz,CDCl3)δ=8.11(d,J=18.0,2H),7.54(t,J=6.0,6H),7.18(d,J=9.0,2H),7.15(d,J=9.0,2H),7.05(d,J=9.0,2H),6.93(d,J=9.0,4H),4.02(t,J=6.0,4H),3.43(t,J=6.0,4H),3.14(t,J=8.0,4H),2.19(s,6H),2.41(s,8H),1.83(m,8H),1.52(t,J=6.0,14H).13C-NMR(100MHz,CDCl3)δ=162.58,161.25,152.48,143.70,140.17,136.58,134.29,128.69,127.27,125.36,118.76,115.89,68.91,56.43,31.75,30.71,28.07,26.95,24.31,16.69.HRMS(tof):m/z calcd for C49H54BBr2F2I2N3O3:1193.0682;found:1194.0685[M+H]+。
(4)将(3)中所得化合物(85mg,0.07mmol)溶解于3mL氯仿中,向上述混合液中加入6mL吡啶和3mL无水乙醇后,该混合液在80℃下搅拌12小时。反应结束后,冷却至室温,将溶剂减压蒸出。粗产品通过重结晶得到纯品,其化学结构式为:
1H-NMR(400MHz,DMSO-d6)δ=9.17(s,4H),8.65(s,2H),8.15(s,4H),8.02(d,J=16.0,2H),7.54(s,4H),7.36(d,J=12.0,2H),7.27(s,2H),7.18(s,2H),7.01(s,4H),4.65(s,4H),4.05(s,4H),2.36(s,6H),1.96(s,8H),1.75(s,10H),1.34(d,J=32.0,14H).13C-NMR(100MHz,DMSO-d6)δ=163.75,162.53,155.26,144.97,143.26,138.83,137.10,135.43,134.16,128.39,127.02,126.57,124.67,118.51,116.77,114.42,88.84,76.71,67.06,65.19,54.84,48.06,35.71,33.09,29.78,23.62,23.37,22.49,22.45,17.27,17.39,14.69.HRMS(tof):m/z[M-2Br]2+/2calcd for C59H64BBr2F2I2N5O3:596.6580;found:596.6610。
(5)将(4)中的化合物溶解在二甲基亚砜中配置成0.5mg/mL母液待用,另配置质量浓度为1mg/mL DSPE-PEG2000-叶酸的二甲基亚砜溶液。取0.24mL母液与0.9mL DSPE-PEG2000-叶酸充分混合后,滴入0.1mL水溶液中并继续超声2小时,用分子量为3500的透析袋透析24小时,从而制得表面叶酸修饰的脂质体纳米粒子。
实施例4
与实施例1相似,将吡啶换成三乙胺。
实施例5
与实施例2相似,将吡啶换成三乙胺。
实施例6
与实施例3相似,将吡啶换成三乙胺。
实施例7
与实施例1相似,将吡啶换成三苯基膦。
实施例8
与实施例2相似,将吡啶换成三苯基膦。
实施例9
与实施例3相似,将吡啶换成三苯基膦。
实施例10
与实施例1相似,将DSPE-PEG2000-叶酸换成DSPE-PEG2000-RGDV。
实施例11
与实施例2相似,将DSPE-PEG2000-叶酸换成DSPE-PEG2000-RGDV。
实施例12
与实施例3相似,将DSPE-PEG2000-叶酸换成DSPE-PEG2000-RGDV。
实施例13
与实施例1相似,将DSPE-PEG2000-叶酸换成DSPE-PEG2000-c(RGDyC)。
实施例14
与实施例2相似,将DSPE-PEG2000-叶酸换成DSPE-PEG2000-c(RGDyC)。
实施例15
与实施例3相似,将DSPE-PEG2000-叶酸换成DSPE-PEG2000-c(RGDyC)。
实施例16
与实施例1相似,将分子量为2000的聚乙二醇换成分子量为1000的聚乙二醇。
实施例17
与实施例1相似,将分子量为2000的聚乙二醇换成分子量为3500的聚乙二醇。
实施例18
与实施例1相似,将分子量为2000的聚乙二醇换成分子量为5000的聚乙二醇。
实施例19
与实施例2相似,将分子量为2000的聚乙二醇换成分子量为1000的聚乙二醇。
实施例20
与实施例2相似,将分子量为2000的聚乙二醇换成分子量为3500的聚乙二醇。
实施例21
与实施例2相似,将分子量为2000的聚乙二醇换成分子量为5000的聚乙二醇。
实施例22
与实施例3相似,将分子量为2000的聚乙二醇换成分子量为1000的聚乙二醇。
实施例23
与实施例3相似,将分子量为2000的聚乙二醇换成分子量为3500的聚乙二醇。
实施例24
与实施例3相似,将分子量为2000的聚乙二醇换成分子量为5000的聚乙二醇。
实施例25
脂质体纳米粒子制备方法与实施例1相似,将浓度为0.5mg/mL的氟硼二吡咯类化合物换成浓度为1.0mg/mL的氟硼二吡咯化合物。
实施例26
脂质体纳米粒子制备方法与实施例2相似,将浓度为0.5mg/mL的氟硼二吡咯类化合物换成浓度为1.0mg/mL的氟硼二吡咯化合物。
实施例27
脂质体纳米粒子制备方法与实施例3相似,将浓度为0.5mg/mL的氟硼二吡咯类化合物换成浓度为1.0mg/mL的氟硼二吡咯化合物。
实施例28
脂质体纳米粒子制备方法与实施例1相似,将浓度为0.5mg/mL的氟硼二吡咯类化合物换成浓度为0.1mg/mL的氟硼二吡咯化合物。
实施例29
脂质体纳米粒子制备方法与实施例2相似,将浓度为0.5mg/mL的氟硼二吡咯类化合物换成浓度为0.1mg/mL的氟硼二吡咯化合物。
实施例30
脂质体纳米粒子制备方法与实施例3相似,将浓度为0.5mg/mL的氟硼二吡咯类化合物换成浓度为0.1mg/mL的氟硼二吡咯化合物。
实施例31
脂质体纳米粒子制备方法与实施例1相似,将浓度为1.0mg/mL的DSPE-PEG2000-叶酸换成浓度为5.0mg/mL的DSPE-PEG2000-叶酸。
实施例32
脂质体纳米粒子制备方法与实施例2相似,将浓度为1.0mg/mL的DSPE-PEG2000-叶酸换成浓度为5.0mg/mL的DSPE-PEG2000-叶酸。
实施例33
脂质体纳米粒子制备方法与实施例3相似,将浓度为1.0mg/mL的DSPE-PEG2000-叶酸换成浓度为5.0mg/mL的DSPE-PEG2000-叶酸。
实施例34
脂质体纳米粒子制备方法与实施例1相似,将浓度为1.0mg/mL的DSPE-PEG2000-叶酸换成浓度为10.0mg/mL的DSPE-PEG2000-叶酸。
实施例35
脂质体纳米粒子制备方法与实施例2相似,将浓度为1.0mg/mL的DSPE-PEG2000-叶酸换成浓度为10.0mg/mL的DSPE-PEG2000-叶酸。
实施例36
脂质体纳米粒子制备方法与实施例3相似,将浓度为1.0mg/mL的DSPE-PEG2000-叶酸换成浓度为10.0mg/mL的DSPE-PEG2000-叶酸。
实施例37:包裹氟硼二吡咯化合物的脂质体纳米粒子细胞毒性实验
将实施例1所制备的纳米粒子加入到培养液中进行细胞培养实验,然后用MTT测定细胞的相对存活率,具体方法为:分别将人乳腺癌细胞MDA-MB-231和正常乳腺细胞MCF-10A(1×104个细胞/孔)种于96孔板中,在培养箱(37℃,5%CO2)中孵育24小时。将不同浓度(0、3、6、9、12、15μg/mL)的纳米粒子与细胞培养24小时。用磷酸盐缓冲液洗涤3次,加入100μL新鲜培养基,对细胞分别进行光照和黑暗处理。然后向每孔中加入20μL MTT溶液,在培养箱中继续孵育3小时。最后,利用酶标仪测定490nm波长每个孔中溶液的吸光度。如图2所示,在黑暗条件下,未用脂质体包裹的氟硼二吡咯类化合物对癌细胞和正常细胞均表现出较高的毒性,而脂质体纳米粒子与癌细胞MDA-MB-231和正常细胞MCF-10A孵育24小时,细胞相对存活率均在90%以上。在665nm光照条件下,癌细胞存活率随着脂质体纳米粒子的增加而降低,表明脂质体纳米粒子对癌细胞具有高的光毒性
类似地,验证了其它实施例中制备的纳米粒子,结果显示,细胞的存活率较高,显示脂质体纳米粒子对细胞毒性低,而在665nm光照条件下对细胞产生高光毒性。
实施例38:荧光标记法证明氟硼二吡咯化合物靶向线粒体实验
将实施例1中所得的脂质体纳米粒子加入到培养液中进行细胞培养实验,然后用细胞线粒体染色试剂MitoTracker Green对线粒体进行染色。由于光敏剂呈现红色荧光,MitoTracker Green呈现绿色荧光,借助激光共聚焦显微镜观察红色荧光和绿色荧光的重合程度(黄色荧光)判断光敏剂是否被肿瘤细胞摄入后定位于线粒体,激发波长为488/561nm。图3为光敏剂定位于线粒体的共聚焦照片,可以看出,氟硼二吡咯光敏剂靶向定位于线粒体。
类似地,验证了其它实施例中所形成的脂质体纳米粒子,结果显示,它们均可靶向细胞线粒体。
实施例39:荧光标记法证明被乳腺癌细胞内吞的纳米粒子光照条件下产生活性氧实验
将实施例1中所制备的纳米粒子与癌细胞MDA-MB-231孵育24小时后,用665nm激光(33mW/cm2)光照细胞5分钟。由于2',7'-二氯荧光素二乙酸盐(DCFH-DA)与细胞内活性氧反应生成2',7'-二氯荧光素(DCF)的机理,DCF在激发波长488nm下显绿色荧光,从而能够通过激光共聚焦显微镜检测证明。如图4所示,实验结果表明,被癌细胞内吞的纳米粒子能够在光照条件下产生大量活性氧。
类似地,验证了其它实施例中制备的纳米粒子,结果显示,它们均可在光照下产生活性氧。
实施例40:活死荧光染色实验证明乳腺癌细胞内吞纳米粒子后光照区域杀伤癌细胞实验
将实施例1中制得的纳米粒子与MDA-MB-231细胞培养3小时后,利用665nm激光(33mW/cm2)光照细胞5分钟。共聚焦显微镜监测乳腺癌MDA-MB-231细胞,激发波长为488/561nm。图5A为染料Calcein-AM染色的活细胞形态照片,图5B为染料PI染色的死细胞形态照片。图5C为前两种图片的合并图。实验结果表明,被乳腺癌细胞内吞的纳米粒子在光照条件下产生的活性氧能够有效杀伤癌细胞。
类似地,验证了其它实施例中制备的纳米粒子,结果显示,它们均可有效的杀伤癌细胞。
本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,凡依本发明申请发明专利范围所做的变化和改进,皆应属于本发明的涵盖范围。
Claims (7)
1.包裹氟硼二吡咯类化合物的脂质体纳米粒子,其特征在于,由靶向线粒体的氟硼二吡咯类化合物和二硬脂酰基磷脂乙醇胺-聚乙二醇-键连基团DSPE-PEGm-Z制得;
靶向线粒体的氟硼二吡咯类化合物化学结构式如式I所示:
其中,R1选自如下的化学基团:H、-CH3、-C2H5、-OCH3、-N(CH3)2、-N(C2H5)2、
X选自如下化学基团:Cl、Br、I;
n值为0-12;
所述键连基团Z为氢、甲氧基、叶酸、多肽、生物素、甘露糖或转铁蛋白。
2.根据权利要求1所述的脂质体纳米粒子,其特征在于,所述多肽选自线性多肽、环肽、或细胞穿膜肽。
3.根据权利要求2所述的脂质体纳米粒子,其特征在于,所述线性多肽为RGDV、GRGDSPK、CPLGVRGRGDS、GRGDSPC;所述环肽为c(RGDyC)、c(RGDfK)、c[RGDfK(Biotin)]、c(RGDyE)、c(CRGDKGPDC);所述细胞穿膜肽为YGRKKRRQRRR、GRKKRRQRRRPQ、RRRRRRRR。
4.根据权利要求1所述的脂质体纳米粒子,其特征在于,聚乙二醇的平均分子量m为200-20000。
5.权利要求1至4任一项所述的脂质体纳米粒子的制备方法,其特征在于,步骤如下:将所述的氟硼二吡咯类化合物在有机溶剂中配置成一定浓度的母液待用,另配置一定浓度DSPE-PEGm-Z的有机溶液;将上述母液和DSPE-PEGm-Z的有机溶液充分混合后,在超声作用下滴入一定体积的水溶液中,透析,制得脂质体纳米粒子。
6.根据权利要求5所述的脂质体纳米粒子的制备方法,其特征在于,具体步骤如下:所述的有机溶剂为二甲基亚砜,所述的母液浓度为0.1-10mg/mL;所述的DSPE-PEGm-Z的有机溶液浓度为0.1-10mg/mL;将母液与DSPE-PEGm-Z充分混合后,滴入0.1-10mL水溶液中并超声,透析,从而制得脂质体纳米粒子。
7.权利要求1-4任一项所述的脂质体纳米粒子在制备光动力治疗癌症药剂中的应用。
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